Itaconate transport across the plasma membrane and Salmonella-containing vacuole via MCT1/4 modulates macrophage antibacterial activity

Domain: physics
Published: 2025-11-26
Editorial outcome: major_revision

Abstract
Itaconate accumulates in macrophages upon bacterial infection, and manifests antibacterial activity. Convincing evidence substantiates that itaconate is transported across the plasma membrane and vacuolar membrane, but the molecular bases underlying bidirectional transport of itaconate across membranes and its effects on intracellular bacterial replication are less known. Here, we identify MCT1 and MCT4 as bidirectional transporters of itaconate. In addition to modulating itaconate concentration as transporters at the plasma membrane, MCT1 and MCT4 function as itaconate transporters at Salmonella-containing vacuole (SCV). Upon Salmonella infection, MCT1 and MCT4 transport itaconate into SCV facilitated by RAB32. Itaconate is also secreted out of cell through MCT1 and MCT4 as the infection persists. The suppression of MCT1 and MCT4-dependent itaconate secretion increases the overall concentration of itaconate and the proportion of itaconate-targeted Salmonella intracellularly, consequently inhibiting Salmonella replication. Our study thus offers valuable insights into itaconate transport during bacterial infection and provides proof of principle for the development of itaconate-dependent therapeutic strategies.

Introduction
The bacterial infection leads to a dramatic metabolic shift in cells of hematopoietic origin, which is a crucial event of cell-autonomous defenses1–3. The activation of macrophages by bacteria undergoes enhanced glycolysis and disturbed tricarboxylic acid (TCA) cycle. The break at isocitrate dehydrogenase 1 (IDH1) of the TCA cycle results in increasing cis-aconitate4. Subsequently, the highly expressed immune-responsive gene 1 (IRG1, also known as aconitate decarboxylase 1, ACOD1) catalyzes the decarboxylation of cis-aconitate into itaconate2,5. Itaconate has been reported to regulate immune responses by modifying proteins known as “itaconation”6 as well as “itaconylation”7,8. Alternatively, itaconate works as a well-established antibacterial metabolite9–12, coordinated decently by the RAB32-LRRK2 complex as illuminated in our previous study13,14. In the context of antibacterial immunity and immunoregulation, maintaining optimal intracellular itaconate concentration is crucial15,16. And the potential transport of itaconate through plasma membrane or itaconate delivery into bacteria-containing vacuole has been mentioned in various scenarios13,17–20. The bidirectional transporters for itaconate likely exist, given its limited cell permeability16,21. More recently, ABCG2 has been recognized as an exporter of itaconate at plasma membrane22. But the absence of ABCG2 did not completely block the secretion of itaconate from macrophages22, which prompts us to study more about itaconate transport. Overall, currently, the bidirectional transporter(s) at the plasma membrane or bacteria-containing vacuole remain unknown21. Solute carrier (SLC) proteins belong to a major transporter superfamily23, which has been demonstrated as metabolic gatekeepers at the plasma membrane or vacuoles in various cell types, including macrophages24,25. For instance, MCT1 (SLC16A1) facilitates succinate release from muscle tissue26. SLC13A5 is a well-established citrate transporter27. SLC7A11 primarily functions as a cystine/glutamate exchanger, focusing on transporting cystine and glutamate28. SLC1A5 primarily transports neutral amino acids, especially glutamine29. SLC39A7 facilitates the uptake of zinc ions into cells, playing a critical role in zinc homeostasis. SLC22A1, also known as OCT1 (Organic Cation Transporter 1), is involved in the transport of organic cations30. However, little is known about the role of SLC proteins in transporting antibacterial metabolites during bacterial infection.

In this study, we identify MCT1 (SLC16A1) and MCT4 (SLC16A3) as key bidirectional itaconate transporters localized at both the plasma membrane and the bacteria-containing vacuole (BCV), where they regulate itaconate trafficking across cellular membranes. We demonstrate that acidic pH facilitates itaconate transport by promoting its conversion to the monocarboxylic form. Furthermore, we show that RAB32 directs MCT1/4 localization to the Salmonella-containing vacuole (SCV), thereby enhancing itaconate import into this compartment. Our findings connect the metabolic transporters MCT1/4 and anti-bacterial defense by dissecting their important roles in controlling intracellular itaconate distribution.

Results
The inhibition of MCT1 and MCT4 transport activity resists Salmonella infection
We first verified itaconate secretion in bacteria-infected macrophages. Itaconate accumulated in the cytosol (~0.2–3 nmol/106 RAW264.7 cells) and was subsequently released into the cell supernatant (~2–30 nmol/106 RAW264.7 cells) over time following infection with the gram-negative bacteria Salmonella (Fig. 1a, b). A larger proportion of itaconate was secreted into the extracellular space during infection (Fig. 1c), analogous to that observed in LPS-treated macrophages20,31. Based on this point, we reasoned that if itaconate transport across the plasma membrane has been dampened, the concentration of intracellular itaconate would be altered pronouncedly. As a result, it may influence the bacteria-eliminating effects of itaconate. Thus, to search for itaconate transporter(s), we first determined whether some available inhibitors of SLC transporters modulated the bacterial survival in cells. The concentrations of SLC inhibitors (for MCT1, MCT4, SLC13A5, SLC7A11, etc.) were established based on documented methodologies28,29,32–40. The survival of Salmonella enterica serovar Typhi (Salmonella Typhi) was remarkably inhibited in the presence of Su3118 (Syrosingopine, a dual inhibitor for MCT1 and MCT4)41 in RAW264.7 cells (Fig. 1d, e) and bone marrow-derived dendritic cells (Supplementary Fig. 1a). We further found that Su3118 attenuated the replication of the broad-host-range serotype Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) (Fig. 1f). VB12442 (a MCT4 inhibitor) in combination with AZD396543 (a MCT1 inhibitor) also had a strong inhibitory effect on Salmonella proliferation in RAW264.7 cells (Fig. 1g). Importantly, a lower bacterial load was detected in human monocyte-derived macrophages (hMDM) with MCT1 and MCT4 impairments (Fig. 1h). And these inhibitors themselves had no significant influence on Salmonella growth in a mammalian-cell-free system (Supplementary Fig. 1b–d). We also examined whether MCT1 or MCT4 (MCT1/4) inhibitors inhibited the phagocytic capacity of cells, which probably led to the detection of a lower bacterial load in cells. We found that long-term inhibition of MCT1 and MCT4 transport activity did not alter the entry of Salmonella into bone marrow-derived macrophages (BMDM), RAW264.7 cells, or DC2.4 cells (Supplementary Fig. 1e–h). It indicates that blocking MCT1/4 does not affect macrophage phagocytosis. We then constructed MCT1/4 depletion cell line by CRISPR/Cas9 (Supplementary Fig. 1i). It is worth noting that Mct4−/− RAW264.7 cells treated with the MCT1 inhibitor (AZD3965) sustained less bacterial replication (Fig. 1i). And the MCT4 inhibitor VB124 dramatically reduced bacterial load in Mct1−/− RAW264.7 cells (Fig. 1j). These data highlighted that the combinational defect in MCT1 and MCT4 enhances the antibacterial activity in macrophages.

However, the bactericidal effect of Su3118/(AZD3965+VB124) disappeared or was reduced in Irg1-deficient RAW264.7 cells or HeLa cells, which lack endogenous itaconate generation (Fig. 1k, l and Supplementary Fig. 1j, k). IRG1 expression in HeLa cells rescued the bacterial-killing properties of Su3118/(AZD3965+VB124) (Fig. 1m). Consistently, the ablation of Irg1 in mice led to the inefficiency of Su3118 in reducing Salmonella load (Fig. 1n). Besides, we observed that the protein level of IRG1 and MCT4 concurrently increased in RAW264.7 cells, spleen, and liver after bacterial infection with a modest change in mRNA level of MCT1/4 (Fig. 1o, p and Supplementary Fig. 1l–o). Taken together, these data underlined the critical roles of MCT1 and MCT4 transport activity in itaconate-dependent immune responses against bacterial infection, which predicted that the solute carriers MCT1 and MCT4 were itaconate transporters.

The inhibition of MCT1 and MCT4 transport activity promotes the prevalence of itaconate-targeted Salmonella
We next continued to explore the roles of MCT1 and MCT4 in regulating itaconate-based bacterial killing. Markedly, MCT1 and MCT4 were detected to be located at the plasma membrane in macrophages (Fig. 2a and Supplementary Fig. 2a, b). The transport activity of MCT1 and MCT4 at the plasma membrane may modify the level of itaconate within cells. To better understand whether the MCT1 and MCT4-related restriction of bacteria growth is metabolites-related, we profiled the metabolic changes of BMDMs under various conditions. We have observed an accumulation of itaconate in BMDMs with disrupted MCT1 and MCT4 transport activity through untargeted metabolomics (Fig. 2b). Spontaneously, succinate, lactate and pyruvate, which are also substrates of MCT1 or MCT4, accumulated (Fig. 2b). The inhibition of MCT1 and MCT4 also increased the concentration of itaconate in RAW264.7 cells at the late stage of infection (Fig. 2c). We also scrutinized whether a higher concentration of itaconate resulted in a greater number of itaconate-targeted Salmonella Typhi. To do that, we took advantage of the nanoluciferase-biosensor of itaconate located in bacteria (Supplementary Fig. 2c)13. We found that more bacteria were targeted by itaconate in RAW264.7 cells when MCT1 and MCT4 activity was suppressed at the later phase of infection (Fig. 2d and Supplementary Fig. 2d, e). Importantly, we also found that more Salmonella were targeted by itaconate in hMDM with the treatment of Su3118, detected by using itaconate biosensor (Fig. 2e). Furthermore, administration of Su3118 in WT mice or administration of AZD3965 in Mct4−/− mice significantly promoted the accumulation of intracellular itaconate in the spleen, leading to the detection of higher levels of itaconate by itaconate biosensor in Salmonella (Fig. 2f, g). As a result, a lower bacterial load was detected in spleen from mice with MCT1 and MCT4 impairments shown in Fig. 1n. However, in macrophages infected with Salmonella Typhi for a shorter period (~7 h), Su3118 failed to modulate the level of the intracellular itaconate (Fig. 2h). This may explain why the percentage of itaconate-targeted Salmonella Typhi at around 7 h changed modestly in RAW264.7 cells with MCT1 and MCT4 deficiency (Fig. 2i). In consequence, the replication of Salmonella in RAW264.7 cells with the weak activity of MCT1 and MCT4 was strongly inhibited at late but not early (~7 h) time-point of infection (Fig. 1 and Supplementary Fig. 2f, g). Considering a lower proportion of itaconate was secreted at 7–8 h post-infection (Fig. 1c), it is predictable that the effect of the inhibitors on itaconate concentration or Salmonella killing in the initial phase of infection is rather limited. With the extension of infection, the proportion of secreted itaconate is larger (Fig. 1c). So, in the later period, MCT1 and MCT4 inhibitors effectively control the secretion of itaconate, leading to increased cellular itaconate and also the attenuated growth of bacteria. To see the influence of MCT1 and MCT4 deficiency on the protein level of IRG1, we analyzed whether the protein level of IRG1 was upregulated by the inhibitors. The results have shown that these inhibitors did not change IRG1 protein level (Fig. 2j and Supplementary Fig. 2h). The double deficiency of MCT1 and MCT4 also did not raise the IRG1 protein level (Fig. 2k).

Given itaconate also has a role in regulating lysosomal biogenesis8, we measured that whether blocking MCT1 and MCT4 affected lysosomal gene expression. LPS activated the expression of lysosomal genes (Supplementary Fig. 2i). Su3118, the MCT1 and MCT4 inhibitor, hardly regulated the expression of the lysosomal genes (Fig. 2l). Su3118 also did not affected the protein level of Lamp1 (Supplementary Fig. 2j). Overall, blocking MCT1/4 probably did not boost the expression of lysosomal genes.

It is possible that macrophage polarization participate in itaconate-based antibacterial effect8. Blocking MCT1 and MCT4 had minimal impact on the polarization of macrophages under during bacterial infection (Fig. 2m). Representative flow plot from BMDMs were shown (Supplementary Fig. 2k). Furthermore, the polarization of the majority of macrophages under LPS/IL4 stimulation were not influenced by the inhibition of MCT1 and MCT4 (Fig. 2n). Su3118 also did not significantly change the mRNA level of Cd206 or Cd86 in macrophages in response to bacterial infection or LPS/IL4 treatment (Supplementary Fig. 2l). These data suggest that the inhibition of MCT1/4 does not change M1/M2 polarization of macrophages.

We next also ruled out that the antibacterial immunity was attributed to the effect of lactate, pyruvate, or succinate, which were reported to be transported by MCT1/4. These metabolites showed no antibacterial activity (Supplementary Fig. 3a–c) as documented44–46. Therefore, our findings indicate that the inhibition of MCT1 and MCT4 increases the general amount of itaconate intracellularly, and naturally enhances the antibacterial efficacy of itaconate by promoting the prevalence of itaconate-targeted Salmonella.

MCT1 and MCT4 transport intracellular itaconate out of cells
To see whether MCT1 and MCT4 transport itaconate out of cells, we have developed an efficient assay to examine itaconate secretion (Fig. 3a and Supplementary Fig. 2c), based on an itaconate biosensor as delineated13. We have proved that the eGFP-biosensor of itaconate showed competence in detecting itaconate secretion of LPS-activated RAW264.7 cells (Supplementary Fig. 4a, b), which also indicated that itaconate was released from macrophages in a time- or dose-dependent manner. The data from the LC-MS analysis showed that itaconate was released in a similar pattern (Supplementary Fig. 4c, d). The cell supernatant from same pools of cells was tested separately by LC-MS or itaconate biosensor (Supplementary Fig. 4e). The comparable results underscored the reliability and accuracy of our assays (Supplementary Fig. 4e). And according to our assay, when the simultaneous inhibition of MCT1 and MCT4 activity occurred, the amount of secreted itaconate decreased robustly (Fig. 3b and Supplementary Fig. 4f). Su3118, compared to other MCT1/4 inhibitors AZD3965, VB124, AZD0095 or AR-C155858, showed a clear dose-dependent inhibition on the secretion of itaconate (Fig. 3c and Supplementary Fig. 4g–j). We then also evaluated the MCT1 and MCT4-related effect on itaconate concentration by LC-MS. In HEK 293T cells stably expressing IRG1, overexpression of MCT1 and MCT4 synergistically lowered intracellular itaconate but increased the amount of itaconate in cell supernatant (Fig. 3d and Supplementary Fig. 4k). The inhibition of MCT1 and MCT4 by Su3118 impaired the release of itaconate in RAW264.7 cells (Fig. 3e). Spontaneously, this inhibition led to increasing itaconate in RAW264.7 cells (Fig. 3f). Similarly, in hMDM, itaconate transport is tightly related to MCT1 and MCT4 transport activity (Fig. 3g, h). In Mct1−/−Mct4−/− RAW264.7 cells, the amount of itaconate also increased intracellularly and decreased in extracellular space (Fig. 3i, j and Supplementary Fig. 4l). Importantly, similar results were obtained when treating Mct4−/− BMDMs with AZD3965 or WT BMDMs with Su3118 (Fig. 3k, l and Supplementary Fig. 4m). The administration of Su3118 in mice also attenuated the secretion of itaconate into the serum or peritoneal lavage supernatant (Supplementary Fig. 4n, o). These data suggested the important roles of MCT1 and MCT4 in transporting itaconate. These data also indicated that the elevated itaconate levels in cells did not lead to increased extracellular itaconate (Fig. 3e, j, k). If the increase in intracellular itaconate results from an upregulated itaconate production rather than a diminished itaconate secretion, more itaconate should have been detected extracellularly. But less itaconate was identified in the supernatant of cells with the compromised MCT1 and MCT4 activity. We further found that the defect in MCT1 and MCT4 still maintained the level of intracellular itaconate-targeted bacteria even when the production of itaconate was damaged by an IRG1 inhibitor (IRG1-IN-1) (Fig. 3m). These data along with Fig. 2i, j indicated that the high level of intracellular itaconate originates from impaired itaconate secretion caused by deficient MCT1 and MCT4 but not enhanced itaconate production. Given that ABCG2 serves as an exporter of itaconate at plasma membrane, we examined the effect of inhibition of ABCG2, MCT1, and MCT4 on itaconate export. We found that the suppression of MCT1 and MCT4 exerts a more significant influence on itaconate release compared to that of ABCG2 when employing Su3118 or KO143 (an ABCG2 inhibitor) at equivalent concentrations. Importantly, the inhibition of ABCG2, MCT1, and MCT4 synergistically block the release of the majority of itaconate from macrophages (Supplementary Fig. 5a–c). Collectively, these findings have strongly supported that MCT1 and MCT4 are exporters of intracellular itaconate.

MCT1 and MCT4 transport extracellular itaconate into cells
SLC proteins function as bidirectional transporters depending on the concentration gradient of substrates23. We next investigated whether MCT1 and MCT4 also transport extracellular itaconate into cells using itaconate biosensor (Fig. 4a). To avoid the interference of endogenous itaconate, we examined itaconate entry in Irg1−/− cells or HeLa cells, which lack endogenous IRG1 (Supplementary Fig. 1i, 6a). The MCT1 and MCT4 inhibitors Su3118 (Fig. 4b, c) or AZD3965+VB124 (Supplementary Fig. 6b, c) obviously impaired itaconate uptake into Irg1-deficient cell lines. Interestingly, we found that itaconate in the cell supernatant of activated RAW264.7 cells or DC2.4 cells was capable of entering HeLa cells (Supplementary Fig. 6d). And Su3118 or AZD3965+VB124 hindered itaconate uptake into HeLa cells (Supplementary Fig. 6e). The genetical ablation of MCT1 and MCT4 also impaired itaconate uptake into macrophages (Fig. 4d). Furthermore, less itaconate uptake caused by inhibiting MCT1 and MCT4 transport activity attenuated the effect of itaconate on an oxidative-stress regulator NRF2 (Fig. 4e, f). NRF2 has been reported to be upregulated by itaconate16 and has a positive role in resisting bacterial infection47,48. This implies that itaconate imported by MCT1 and MCT4 also participates in NRF2-dependent antibacterial immunity in cells. More specifically, we utilized 13C-labelled itaconate to assess itaconate uptake into BMDMs. We found that Su3118 inhibited the uptake of 13C-itaconate in a dose-dependent manner in BMDM cells derived from WT or Irg1-deficient mice (Fig. 4g). Considering MCT1 and MCT4 are also responsible for transporting lactate, succinate, and pyruvate, we scrutinized the impact of these substrates on itaconate transport. We observed that itaconate inhibited lactate uptake in a dose-dependent manner. Based on this data, around 20 μM itaconate effectively inhibits 50% of 13C-labelled lactate uptake into cells (Fig. 4h). Lactate, succinate, and pyruvate, in contrast to malic acid, also impaired the uptake of itaconate (Fig. 4i). We also constructed plasmids for the inactive mutants of MCT1 and MCT4 as documented for lactate transport43,49,50. The results from LC-MS showed that the overexpression of WT MCT1 or MCT4 in cells promoted itaconate transport (Fig. 4j). Furthermore, compared to the MCT1/4 mutants, WT MCT1/4 facilitated more itaconate entry into cells, which subsequently was detected by LC-MS (Fig. 4k, l) or itaconate biosensor (Fig. 4m, n). Consistently, WT MCT1/4 enhanced lactate secretion compared to the MCT1/4 mutants (Supplementary Fig. 6f–k). This suggests that itaconate shares transport pathways with lactate, succinate, and pyruvate via MCT1 and MCT4. Taken together, these findings suggest that MCT1 and MCT4 are transporters responsible for itaconate translocation into cells.

Itaconate transport through MCT1/4 is pH-dependent
Itaconate is a dicarboxylate with two different ionization status, HITA- and ITA2- (pK 3.85 and 5.55)51. It will be important to validate whether the variation in protonation states affects itaconate transport. Based on the Henderson-Hasselbalch equation, the percentage of monocarboxylic itaconate increases during intracellular acidification (Supplementary Fig. 6l). When stimulating primary macrophages with LPS, we observed a decreased intracellular pH (Supplementary Fig. 6m), which was consistent with the previous report52. It indicates that bacterial infection causes intracellular acidification and promotes itaconate transfer to its monocarboxylate form. To further explore whether the ionization status of itaconate impacts its transport, we evaluated itaconate uptake and release under different pH conditions. A culture medium containing pH-adjusted itaconic acid was added to the BMDMs. We observed that more itaconate was transported intracellularly at acidic pH (Fig. 4o). We also examined the effect of pH on endogenous itaconate release by adjusting intracellular pH with EIPA. As intracellular pH increased from 5.2 to 8.2, there was a corresponding decrease in the amount of secreted itaconate (Fig. 4p). Consistently, endogenous itaconate accumulated more intracellularly as pH increased (Fig. 4q). Taken together, these data support that itaconate is mainly transported in its monocarboxylate form through MCT1/4.

Validation of itaconate transport by MCT1/4 in xenopus oocytes and liposomes
To gain more convincing data to support that MCT1 or MCT4 directly transport itaconate, we have set up a Xenopus laevis oocytes-dependent transport assay (Fig. 5a, b), which is widely employed to investigate monocarboxylate transport26. Human cRNA (complementary RNA) of MCT1 or MCT4 was injected into Xenopus laevis oocytes and incubated for three days. In the itaconate transport assay, we observed that the oocytes expressing recombinant MCT1 or MCT4 demonstrated enhanced uptake of itaconate (Fig. 5c). This result indicates that MCT1 or MCT4 transports itaconate directly. We next also reconstructed MCT1/4 proteoliposomes by inserting purified human MCT1 or MCT4 protein (Fig. 5d) as described in refs. 22,53. The proteoliposomes were imaged by cryo-TEM or ODT-microscopy (Fig. 5e). Elevated levels of itaconate are efficiently transported into liposomes incorporating WT MCT1 or MCT4, but not those with mutant forms (Fig. 5f, g). These data suggest that MCT1 and MCT4 directly transport itaconate.

MCT1 and MCT4 transport itaconate into Salmonella-containing vacuole
Given that Salmonella-containing vacuole (SCV) is developed from the plasma membrane54, it is possible that MCT1 and MCT4 are also located at SCV and transport itaconate into SCV. To investigate this hypothesis, we first examined whether MCT1 and MCT4 are located at Salmonella-containing vacuole. We infected RAW264.7 cells by using Salmonella stably expressing mScarlet. MCT1 and MCT4 were detected to be recruited to Salmonella (Fig. 6a, b and Supplementary Fig. 7a, b). Moreover, MCT1 and MCT4 bound to intravacuolar Salmonella manifested by a SCV-reporter strain (Supplementary Fig. 7c, d)55, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To validate the necessity of the tight association between MCT1/4 and Salmonella in itaconate delivery, we analyzed whether MCT1/4-surrounded Salmonella was prone to be targeted by itaconate. We have imaged a random sample of RAW264.7 cells infected by S. Typhi carrying itaconate eGFP-biosensor and expressing mScarlet (Fig. 6e, f). Intriguingly, most of bacteria were simultaneously targeted by MCT1/4 and itaconate, which aligns with our prior observation that itaconate is delivered into SCV13. These observations suggested that the association of MCT1/4 and Salmonella may have a strong link to itaconate transport at SCV. We then continued to investigate the hypothesis that MCT1 and MCT4 transported itaconate into Salmonella-containing vacuole. In this study, our earlier data have shown that itaconate concentration as well as the ratio of itaconate-targeted Salmonella were intracellularly improved in macrophages following Su3118 treatment (Figs. 2 and 3). So, we next tried to assess the impact of concentrated itaconate within cells on the proportion of bacteria decorated by MCT1/4. According to our random sampling and statistical analysis, Su3118 increased the percentage of MCT1/4-targeted Salmonella (Fig. 6a–d). To

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns.", "reviewer_4": "The revised manuscript from Meng et al. made a significant effort in responding to the comments of reviewer 3. Overall, the comments have been addressed but some points need to be clarified or further assessed.\n1/ For R#3, question 1: Authors now show that the human transporters MCT1 and MCT4 can directly mediate itaconate uptake using expression of recombinant MCT1/4 in Xenopus laevis oocytes as well as in liposomes (new Figure 5). However, for experiment with reconstructed MCT1/MCT4 liposomes (figure 5f, 5g, 5h), how authors can manage the orientation of the transporters (in/out), and consequently determine whether they will assess uptake or export of itaconate ?\n2/ For R#3, question 2: Authors now assessed lactate and itaconate transport in 293T and U20S cells overexpression WT or dead mutants of MCT1/MCT4 in a new Figure 4j-n and extended Figure 6f-i.\nRegarding itaconate transport, they showed that inactive mutants of MCT1/MCT4 inhibited itaconate uptake by the cells (Figure 4j-n). However, regarding lactate transport, they concluded that WT MCT1/MCT4 enhanced lactate secretion compared to mutants (line 238-239), but authors rather showed that inactive mutants of MCT1/MCT4 increased intracellular lactate compared to WT (extended Figure 6f-i). To conclude regarding lactate secretion, authors should measure and show extracellular lactate levels. Indeed, decrease intracellular levels does not necessarily means increased export, as lactate could be metabolized (conversion into pyruvate by lactate deshydrogenase).\n3/ For R#3, question 3: To exclude a secondary pathway involved, authors validated their conclusions into new models of transport, Xenopus oocytes and proteoliposomes, with medium containing only itaconate (and not other metabolites sensed by MCT1/MCT4). This demonstration is valid regarding itaconate transport.\nRegarding the impact of bacterial growth, the effect could be not direct, but indirect through modulation of immune response that subsequently impact bacterial growth. Blocking MCT1/MCT4 could affect the functionality of macrophage, that in turn inhibit bacterial growth. See point 6/ below for further suggestion to address this potential secondary pathway.\n4/ I think that the title is not sustained by the experimental work. The title states that “the transport of itaconate [….] via MCT1/4 modulates antibacterial immune responses”. In this work, authors just show an effect on inhibition of bacterial growth, but not at all on antibacterial immune responses. This point needs to be further addressed, by investigating for example the lysosomal biogenesis, bacteriolysis activity of macrophages, or other functional features related to antibacterial immune responses in their in vivo model.\n5/ This work has been entirely done with tumoral cell lines [RAW264 (murine macrophage line), HeLa (human cervical cancer line), HEK293T (adenovirus-immortalized human embryonic kidney cells), DC2.4 (mouse DC cell line)] or murine BMDC or BMDM cells. MCT1/MCT4 expression levels, sensibility to inhibitor, as well as ability to sense metabolites could be dependent on cell type and species. It is well documented that MCT1/MCT4 expression levels and affinity for metabolites are different on tumor cells compared to immune cells. This study could gain interest if authors perform some key findings in vitro with human primary macrophages, or in vivo with humanized mice (bearing a human immune system), as the finality is to develop therapeutic strategies in humans targeting itaconate/MCT pathway, able to reinvigorate antibacterial immunity. Therefore, the use of human primary immune cells is crucial to draw such conclusion.\n6/ Another key point is to assess how itaconate / MCT inhibition affect macrophage M1/M2 polarization, toward pro-inflammatory or anti-inflammatory state. Itaconate has been shown to be rather anti-inflammatory (https://doi.org/10.1016/j.cmet.2025.03.004). Authors should investigate the impact of MCT1/MCT4 blocking and/or itaconate level on the immunomodulatory role of macrophage, at phenotypic and functional levels. For example, investigate some surface markers (such as CD86, CD206) or cytokine secretion (such as TNF-α, IL-1β, IL-6, MCP-1, IL-10, TGF-β), or other molecules (arginase 1) that could distinguish M1 versus M2 polarization of macrophages. It will allow to link the inhibition of bacterial growth with some markers of immune response.\n7/ It would be interesting to decipher differences between MCT1 and MCT4 regarding itaconate transport. How do they compare regarding uptake / export, threshold for sensing itaconate, and competition for lactate transport ?\n8/ Line 66, it is mentioned “anti-bacterial infection”. This formulation is weird. It should be either “anti-bacterial defense” or “bacterial infection”."}, "author_response": "We sincerely thank the Editors and Reviewers for their positive feedback and valuable suggestions. We have carefully and thoroughly considered the suggestions and concerns raised by the Reviewer#4. We believe we have addressed all comments thoroughly and really hope the Editors and Reviewer#4 will be pleased with our revised manuscript. To facilitate the review process, we have showed all changes in the manuscript text file with color highlighting. Thank you so much for your understanding.\nResponse to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my concerns.\nWe sincerely thank Reviewer #1 and #2 for the support of the publication.\nReviewer #4 (Remarks to the Author):\nThe revised manuscript from Meng et al. made a significant effort in responding to the comments of reviewer 3. Overall, the comments have been addressed but some points need to be clarified or further assessed.\n#1．For R#3, question 1: Authors now show that the human transporters MCT1 and MCT4 can directly mediate itaconate uptake using expression of recombinant MCT1/4 in Xenopus laevis oocytes as well as in liposomes (new Figure 5). However, for experiment with reconstructed MCT1/MCT4 liposomes (figure 5f, 5g, 5h), how authors can manage the orientation of the transporters (in/out), and consequently determine whether they will assess uptake or export of itaconate?\nTo Reviewer# Figure 1. MCT1/4 transports itaconate into liposomes.\nAnswer: We appreciate the Reviewer’s valuable comments. Physiologically, many SLC proteins are highly dynamic (Cell 2021, 184(2): 370–383), which is challenging to fully recapitulate in vitro. To make our results reliable, we performed the liposome-based transport assay by following well-established protocols, as described in the manuscript (Cell, 2021, 184(2): 370-383; Cell Metabolism, 36(3), 498-510; Nature protocols, 3(2), 256-266).\nImportantly, two key points assure that itaconate uptake was measured in our assay. First, the transport direction of MCT1/4 is mainly determined by the concentration gradient of substrates (Cell, 2021, 184(2): 370-383; Physiological Reports, 2022, 10(17)). The MCT1/4 liposomes contain no itaconate inside. When liposomes were incubated in itaconate-containing buffer, the concentration of itaconate is much higher out of liposomes. MCT1/4 is prone to transport itaconate into liposomes (To Reviewer# Figure 1). Second, only itaconate in the liposomes were collected. We washed the MCT1/4 liposomes after the incubation with itaconate. Only itaconate in the liposomes was extracted and analyzed. Besides, we took advantage of a Xenopus oocytes-dependent assay and other experiments (Figure 3 and 4 in the manuscript) to examine itaconate transport, which support MCT1 and MCT4 transport itaconate.\n#2．For R#3, question 2: Authors now assessed lactate and itaconate transport in 293T and U2OS cells overexpression WT or dead mutants of MCT1/MCT4 in a new Figure 4j-n and extended Figure 6f-i.\nRegarding itaconate transport, they showed that inactive mutants of MCT1/MCT4 inhibited itaconate uptake by the cells (Figure 4j-n). However, regarding lactate transport, they concluded that WT MCT1/MCT4 enhanced lactate secretion compared to mutants (line 238-239), but authors rather showed that inactive mutants of MCT1/MCT4 increased intracellular lactate compared to WT (extended Figure 6f-i). To conclude regarding lactate secretion, authors should measure and show extracellular lactate levels. Indeed, decrease intracellular levels does not necessarily means increased export, as lactate could be metabolized (conversion into pyruvate by lactate deshydrogenase).\nTo Reviewer# Figure 2. (A, B) U2OS cells were transfected with MCT1 (A), MCT4 (B) or their mutants. 24 h later, the media was replaced with fresh media, and extracellular lactate levels were measured at 1, 3, and 6 h using the Lactate-Glo™ Assay.\nAnswer: As suggested by the Reviewer, we examined the extracellular lactate level. Compared to the cells transfected with wild-type MCT1/MCT4, the cells transfected with the mutants show reduced lactate export (To Reviewer# Figure 2A, 2B, refer to new Supplementary Data Fig. 6j, 6k). These findings along with the results of Supplementary Figure 6h, 6i in the manuscript support that MCT1/4 mutations attenuated lactate transport.\n#3．For R#3, question 3: To exclude a secondary pathway involved, authors validated their conclusions into new models of transport, Xenopus oocytes and proteoliposomes, with medium containing only itaconate (and not other metabolites sensed by MCT1/MCT4). This demonstration is valid regarding itaconate transport. Regarding the impact of bacterial growth, the effect could be not direct, but indirect through modulation of immune response that subsequently impact bacterial growth. Blocking MCT1/MCT4 could affect the functionality of macrophage, that in turn inhibit bacterial growth. See point 6/ below for further suggestion to address this potential secondary pathway. #6．Another key point is to assess how itaconate / MCT inhibition affect macrophage M1/M2 polarization, toward pro-inflammatory or anti-inflammatory state. Itaconate has been shown to be rather anti-inflammatory (https://doi.org/10.1016/j.cmet.2025.03.004). Authors should investigate the impact of MCT1/MCT4 blocking and/or itaconate level on the immunomodulatory role of macrophage, at phenotypic and functional levels. For example, investigate some surface markers (such as CD86, CD206) or cytokine secretion (such as TNF-α, IL-1β, IL-6, MCP-1, IL-10, TGF-β), or other molecules (arginase 1) that could distinguish M1 versus M2 polarization of macrophages. It will allow to link the inhibition of bacterial growth with some markers of immune response.\nTo Reviewer# Figure 3. (A, B) BMDMs (bone marrow-derived macrophages) were treated with DMSO/Su3118 (5 µM) and infected with or without Salmonella Typhi (24 h). The cells were stained with anti-CD11b, anti-CD86 and anti-CD206. The expression of M1 surface marker CD86 or M2 surface markers CD206 of cells were measured by Flow cytometry.\nAnswer: We sincerely appreciate these suggestions from the Reviewer. In our study, we have examined the antibacterial role of MCT1 and MCT4 inhibition in bacteria-infecting macrophages. On the basis of this, to assess whether the antibacterial effect involves MCT1/4-mediated macrophage polarization, we analyzed macrophage polarization during bacterial infection.\nFlow cytometry results confirmed that inhibiting MCT1 and MCT4 by Su3118 did not significantly alter M1/M2 marker protein levels in bacteria-infected BMDMs (To Reviewer# Figure 3A, 3B). These results rule out an effect of MCT1 and MCT4 on macrophage polarization during bacterial infection.\n#4．I think that the title is not sustained by the experimental work. The title states that “the transport of itaconate [….] via MCT1/4 modulates antibacterial immune responses”. In this work, authors just show an effect on inhibition of bacterial growth, but not at all on antibacterial immune responses. This point needs to be further addressed, by investigating for example the lysosomal biogenesis, bacteriolysis activity of macrophages, or other functional features related to antibacterial immune responses in their in vivo model.\nAnswer: We thank the Reviewer for the helpful reminder. To address the Reviewer’ s concern, the title of our article has been modified as “MCT1/4-dependent itaconate transport across the plasma membrane and Salmonella-containing vacuole modulates antibacterial efficacy”.\nThe previous one：Transport of itaconate across the plasma membrane and Salmonella-containing vacuole via MCT1/4 modulates antibacterial immune responses.\n#5．This work has been entirely done with tumoral cell lines [RAW264 (murine macrophage line), HeLa (human cervical cancer line), HEK293T (adenovirus-immortalized human embryonic kidney cells), DC2.4 (mouse DC cell line)] or murine BMDC or BMDM cells. MCT1/MCT4 expression levels, sensibility to inhibitor, as well as ability to sense metabolites could be dependent on cell type and species. It is well documented that MCT1/MCT4 expression levels and affinity for metabolites are different on tumor cells compared to immune cells. This study could gain interest if authors perform some key findings in vitro with human primary macrophages, or in vivo with humanized mice (bearing a human immune system), as the finality is to develop therapeutic strategies in humans targeting itaconate/MCT pathway, able to reinvigorate antibacterial immunity. Therefore, the use of human primary immune cells is crucial to draw such conclusion.\nTo Reviewer# Figure 4. (A) Human monocyte-derived macrophages (hMDM) treated with DMSO/Su3118 (5 µM) were infected with Salmonella Typhi for 22 h. The number of bacteria in cells was examined. (B, C) The level of itaconate from hMDM treated with Su3118 (5 µM) and infected with Salmonella Typhi for around 22 hours (LC-MS). (D) Intracellular itaconate from hMDM treated with Su3118 (5 µM), and infected by Salmonella Typhi for 22 h was determined by itaconate biosensor. The levels of luciferase were measured.\nAnswer: We agree with the Reviewer that it’s necessary to repeat key experiments using human monocyte-derived macrophages (hMDM), obtained as described in the methods in the manuscript. With the limited primary human cells, we then have tried to examine itaconate transport and the related antibacterial effect.\n(1) CFU assay: A lower bacterial load was detected in hMDM cells with MCT1 and MCT4 impairments (To Reviewer# Figure 4A, refer to Fig. 1h in the new manuscript).\n(2) LC-MS analysis: The inhibition of MCT1 and MCT4 by Su3118 impaired the release of itaconate (To Reviewer# Figure 4B, refer to Fig. 3g, 3h in the new manuscript). Spontaneously, this inhibition led to increasing itaconate intracellularly in hMDM (To Reviewer# Figure 4C).\n(3) Itaconate-biosensor assay: We found that more bacteria were targeted by itaconate in hMDM when MCT1 and MCT4 activity was suppressed, detected by using itaconate biosensor (To Reviewer# Figure 4D, refer to Fig. 2e in the new manuscript). These finding implied that itaconate exerted its antibacterial effect in a MCT1/4-dependent manner (To Reviewer# Figure 4A).\nThese findings are consistent with our previous observations in primary macrophages from mice or RAW264.7 cells, which support our important conclusions.\n#7．It would be interesting to decipher differences between MCT1 and MCT4 regarding itaconate transport. How do they compare regarding uptake / export, threshold for sensing itaconate, and competition for lactate transport?\nAnswer: We highly agree with the Reviewer and the Editors that it would be interesting to learn more about the different roles of MCT1 and MCT4 in transporting itaconate. As kindly noted by the Editors, we have expanded the relevant discussion in the revised manuscript. We hope this revised discussion satisfactorily acknowledges the importance of the Reviewer’s suggestion.\nLine 373 to 387 in the revised manuscript:\nAdditionally, MCT1 is a widely expressed protein, found in the macrophages as well as various mouse tissues such as spleen, liver, brain, and kidney, while MCT4 is an inducible protein in the macrophages, spleen and liver, but not expressed in the brain and kidney (Fig. 1o, 1p, and Supplementary Fig. 1o). These observations suggest that MCT1 and MCT4 may function differently in the uptake of itaconate across different tissues. Given that both transporters are also known to carry lactate or other related substrates, their efficiency and specificity for itaconate transport may be influenced by substrate competition, depending on the cellular context and metabolic demands. Their transport activity is probably not fixed but dynamically regulated by the availability of competing molecules. We found that the presence of itaconate attenuated lactate uptake (Figure 4h). And lactate also reduced itaconate uptake (Figure 4i). Since MCT1 has a higher affinity for lactate compared to MCT4 (Cell reports, 2018, 25(11): 3047-3058), it is possible that lactate competition has a greater impact on itaconate transport through MCT1. Further investigations along these lines would be valuable in the future.\n#8．Line 66, it is mentioned “anti-bacterial infection”. This formulation is weird. It should be either “anti-bacterial defense” or “bacterial infection”.\nAnswer: We are really sorry for this mistake. We have corrected the mentioned words (Line 64) as suggested by the Reviewer and highlighted them in yellow in the revised manuscript.\nResponse to Reviewers’ Comments\nOnce again, we extend our sincere thanks to Reviewer #4 for the valuable comments that have helped us further improve the manuscript. We have thoroughly considered the suggestions raised by Reviewer#4 and tried our best to address the concerns. A total of approximately 32 new panels, have been added to clearly illustrate our findings in the manuscript or the response letter. To facilitate the review, we have marked the changes in the manuscript with color highlighting."}, {"round": 2, "reviewer_comments": {"reviewer_4": "The revised manuscript from Meng et al. addressed most of the concerns. They provide explanation regarding the orientation of the transporters, further addressed lactate transport by MCT1/4, provided data with primary human macrophages, and more deeply discussed differences between MCT1 and MCT4.\nThere are two remaining concerns:\n1/ Regarding the modulation of immune response that could subsequently impact bacterial growth, I think the investigations are not sufficient. The authors should assess whether blocking MCT1/MCT4 could affect the functionality of macrophage, such as cytokine secretion profile or capacity of phagocytosis. These data need to be added in the paper. We need to clearly know if the effect of MCT1/MCT4 blocking is direct (direct impact on bacterial growth) or indirect through modulation of the functionality of macrophages, or both.\nTo assess M1/M2 macrophages polarization, authors performed CD86 and CD206 surface staining on BMDMs treated with Su3118 and infected with bacteria. We don’t known the timing, and in which population expression is defined (% within which cell population ?). To really assess how itaconate / MCT inhibition affect macrophage M1/M2 polarization toward pro-inflammatory or anti-inflammatory state, authors should investigate the impact of MCT1/MCT4 blocking and/or itaconate level on the immunomodulatory role of macrophage, at functional levels: investigate cytokine secretion (such as TNF-α, IL-1β, IL-6, MCP-1, IL-10, TGF-β), or other molecules (arginase 1) that could distinguish M1 versus M2 polarization of macrophages. They should use positive control of polarization, such as culture with LPS and IL4.\nFunctionality of macrophages could also be assessed through investigation of lysosomal biogenesis, and bacteriolysis activity of macrophages.\n2/ The new title is still ambiguous. Authors changed “antibacterial immune responses” by “antibacterial efficacy”.\nIndeed, authors showed an effect on inhibition of bacterial growth, but not at all on antibacterial immune responses, or on the efficacy of macrophages to clear infection. I think it worth assessing some functionality of macrophages to really conclude on this point and keep the proposed title."}, "author_response": "Response to Reviewers’ Comments\nOnce again, we extend our sincere thanks to Reviewer #4 for the valuable comments that have helped us further improve the manuscript. We have thoroughly considered the suggestions raised by Reviewer#4 and tried our best to address the concerns. A total of approximately 32 new panels, have been added to clearly illustrate our findings in the manuscript or the response letter. To facilitate the review, we have marked the changes in the manuscript with color highlighting.\nReviewer #4 (Remarks to the Author):\nThe revised manuscript from Meng et al. addressed most of the concerns. They\nprovide explanation regarding the orientation of the transporters, further addressed\nlactate transport by MCT1/4, provided data with primary human macrophages, and\nmore deeply discussed differences between MCT1 and MCT4.\nWe deeply thank the reviewer for the positive feedback regarding our efforts in\nthe previous version of the manuscript.\nThere are two remaining concerns:\n1# Regarding the modulation of immune response that could subsequently impact\nbacterial growth, I think the investigations are not sufficient. The authors should assess\nwhether blocking MCT1/MCT4 could affect the functionality of macrophage, such as\ncytokine secretion profile or capacity of phagocytosis. These data need to be added in\nthe paper. We need to clearly know if the effect of MCT1/MCT4 blocking is direct (direct\nimpact on bacterial growth) or indirect through modulation of the functionality of\nmacrophages, or both.\nTo assess M1/M2 macrophages polarization, authors performed CD86 and CD206\nsurface staining on BMDMs treated with Su3118 and infected with bacteria. We don’t\nknow the timing, and in which population expression is defined (% within which cell\npopulation?). To really assess how itaconate/MCT inhibition affect macrophage M1/M2\npolarization toward pro-inflammatory or anti-inflammatory state, authors should\ninvestigate the impact of MCT1/MCT4 blocking and/or itaconate level on the\nimmunomodulatory role of macrophage, at functional levels: investigate cytokine\nsecretion (such as TNF-α, IL-1β, IL-6, MCP-1, IL-10, TGF-β), or other molecules\n(arginase 1) that could distinguish M1 versus M2 polarization of macrophages. They\nshould use positive control of polarization, such as culture with LPS and IL4.\nFunctionality of macrophages could also be assessed through investigation of\nlysosomal biogenesis, and bacteriolysis activity of macrophages.\nAnswer: We appreciate these detailed suggestions from the Reviewer. We have\ncarefully examined the effect of MCT1 and MCT4 blocking on the functionality of\nmacrophages. The newly generated data have been incorporated into the manuscript.\nTopic one: polarization and cytokine expression\n(1) Flow Cytometry Gating Strategy\nSupplementary Fig. 2k: Representative flow cytometry plots of CD11b⁺ BMDMs showing CD86 and CD206\nexpression following Salmonella (S. Typhi) infection (24 h) with or without Su3118 (5 μM, 22h) treatment.\nIn our previous response, we stated that “Su3118 did not alter M1/M2 marker protein\nlevels in bacteria-infected BMDMs.” We apologize for not including the gating strategy\nat that time, which may have caused confusion. As clarified here, BMDMs were\nproperly differentiated using the protocol described previously. Cells were stained with\nantibodies against CD11b, CD86, and CD206. M1 macrophages were identified as\nCD11b⁺CD86⁺. M2 macrophages were analyzed based on CD11b⁺CD206⁺\npopulations (Supplementary Fig. 2k).\nThe detailed gating strategy has now been included in this letter for clarity, and the full\nmethod has also been added to the \"METHODS\" section of the revised manuscript.\nAll flow cytometry data throughout the study were acquired and analyzed following this\nconsistent approach.\n(2) Blocking MCT1 and MCT4 does not significantly change the polarization of\nmacrophages.\nFig. 2m: Flow cytometry data from Supplementary Figure 2k showing the percentages of CD11b⁺CD86⁺ and\nCD11b⁺CD206⁺ BMDMs. Fig. 2n: Flow cytometry data from BMDMs treated with Su3118 (5 μM, 24 h), LPS (100 ng/ml, 24 h), or IL-4 (20 ng/ml, 24 h), showing the percentages of CD11b⁺CD86⁺ and CD11b⁺CD206⁺ cells. Supplementary\nfig. 2l: Relative mRNA levels of the indicated cytokines in BMDMs treated with Su3118 (5 µM, 22 h) with LPS (100\nng/ml, 18 h)/IL-4 (20ng/ml, 18 h)/S. Typhi infection (24 h) were determined by quantitative real-time PCR.\nTo assess whether the antibacterial effect involves MCT1/4-mediated macrophage\npolarization, we analyzed repeatedly macrophage polarization during bacterial\ninfection or under LPS/IL4 stimulation. Blocking MCT1 and MCT4 had minimal impact\non the polarization of macrophages under during bacterial infection (Fig.\n2m). Representative flow plots from BMDMs were shown (Supplementary Fig. 2k).\nFurthermore, the polarization of the majority of macrophages under LPS/IL4\nstimulation were not influenced by the inhibition of MCT1 and MCT4 (Fig. 2n). Su3118\nalso did not significantly change the mRNA level of Cd206 or Cd86 in BMDMs in\nresponse to bacterial infection or LPS/IL4 treatment (Supplementary Fig. 2l).\nThese data rule out the possibility that blocking MCT1 and MCT4 reduces bacterial\nsurvival by altering the polarization of the majority of macrophages. Therefore, our\nconclusion remains consistent with the previous one: blocking MCT1 and MCT4 did\nnot significantly alter macrophage polarization.\nR-Fig. 1: Relative mRNA levels of the indicated cytokines in RAW264.7 cells treated with Su3118 (5 µM, 24h) with\nLPS (100 ng/ml, 18 h)/IL4 (20ng/ml, 18h) were determined by quantitative real-time PCR. Expression was normalized\nto untreated cells.\nRegarding cytokine functionality, we have further analyzed the expression of cytokines\nand markers that differentiate M1 from M2 macrophage polarization. We found that\ninhibiting MCT1 and MCT4 in macrophages did not remarkably influence the mRNA\nlevel of cytokines, including Arg1, Tgf-b1, Mrc1, Mcp1, Irf5, Tnfa, Il6, Il1b, Il-10, Cd86,\nCd80 and Cd206 (R-Fig. 1). These data suggest that the inhibition of MCT1and MCT4\nneither changes M1/M2 polarization nor alters the expression of pro- or anti-\ninflammatory cytokines or markers in macrophages. Itaconate accumulation caused\nby Su3118 may not be sufficient on its own and may require co-stimulatory signals to\nmodulate these cytokines. It’s also possible that the accumulation of other substrates\nof MCT1 and MCT4 in cells caused by Su3118 is also involved in these results.\nFig. 4e, f: RAW264.7 (e) or HeLa (f) cells were treated with MG132 (10 µM), itaconate (1 mM) or Su3118 (10 µM) as\nindicated. The cell lysates were analyzed by the immunoblotting with anti-NRF2 and anti-GAPDH.\nWe did observe that less itaconate uptake caused by inhibiting MCT1 and MCT4\ntransport activity attenuated the effect of itaconate on an oxidative-stress regulator\nNRF2 (Fig. 4e, f). NRF2 has been reported to be upregulated by itaconate and has a\npositive role in resisting bacterial infection (Cell Reports 37.3 (2021)). This implies that\nitaconate imported by MCT1 and MCT4 also participates in NRF2-dependent\nantibacterial immunity in cells. MCT1 and MCT4 probably affect certain immune\npathways (e.g. NRF2-related pathway) in an itaconate-dependent manner, which in\nturn has a role in regulating bacterial survival intracellularly.\nTopic two: phagocytosis and bacteriolysis\n(1) Blocking MCT1 and MCT4 does not affect macrophage phagocytosis.\nSupplementary Fig. 1e, g, h: RAW264.7 cells (e) pretreated with Su3118 (0, 10 µM, 22 h) were infected with S. Typhi.\nThe number of bacteria in cells was determined at 0.5 h after infection. (g, h) RAW264.7 or BMDMs were pretreated\nwith Su3118 (0 or 10 μM, 22 h) and infected with mScarlet-expressing S. Typhi. At the indicated time, cells were fixed,\nstained with Hoechst, and imaged, quantified using the Opera Phenix Plus system.\nTo evaluate whether blocking MCT1/MCT4 affects the phagocytic capacity of\nmacrophages, we conducted experiments assessing their short-term uptake of\nSalmonella (within one hour of infection).\nA colony-forming unit (CFU) assay was used to assess the bacterial uptake 0.5 h post-\ninfection, which reflects the phagocytosis of macrophages. Even with long-term\ninhibition of MCT1 and MCT4 transport activity via prior treatment with Su3118, it did\nnot alter bacterial uptake in macrophages (Supplementary Fig. 1e).\nAdditionally, a fluorescence-based bacterial uptake assay using a high-content\nimaging system was performed to evaluate phagocytosis in both RAW264.7 and\nprimary macrophages. The results were consistent with the CFU assay, showing that\nSu3118 did not affect Salmonella uptake at either 30 minutes or 1 h post-infection\n(Supplementary Fig. 1g, h).\n(2) Blocking MCT1 and MCT4 does not affect lysosomal biogenesis.\nSupplementary Fig. 2i and Fig. 2l: Relative mRNA levels of lysosomal genes in BMDMs treated with Su3118 (5 μM, 24 h),\nwith or without LPS (100 ng/ml, the indicated times), were measured by RT-PCR. Expression was normalized to\nuntreated cells, and triplicate values were shown as heatmaps. Supplementary Fig.2j: RAW264.7 cells treated with\nLPS (100 ng/ml, 24 h) or IL-4 (20 ng/ml, 24 h). The cell lysates were analyzed by immunoblotting with anti-Lamp1 and\nanti-b-actin.\nThe reviewer also provided suggestions about the investigation of lysosomal\nbiogenesis of macrophages. We appreciate the suggestion from the Reviewer.\nLysosomal biogenesis is essential for macrophage bacteriolytic activity. We found LPS\nactivated five key genes of lysosomal biogenesis in a time-dependent manner\n(Supplementary Fig. 2i), which was the same as described before (Molecular Cell\n82.15 (2022): 2844-2857). When treating cells with Su3118, the MCT1 and MCT4\ninhibitor, it hardly regulated the expression of the lysosomal genes (Fig. 2l). Su3118\nalso did not affected the protein level of Lamp1 (Supplementary Fig. 2j). Itaconate\nalone, as induced by Su3118, may be limited to drive lysosomal biogenesis without\ncomplementary signals. And, besides itaconate, other substrates of MCT1 and MCT4,\nwhich accumulate intracellularly under Su3118 treatment, probably also contribute to\nthis outcome, which requires further study. Overall, blocking MCT1 and MCT4\nprobably did not boost the expression of lysosomal genes.\nIn conclusion, our new findings above excluded that Su3118 suppresses intracellular\nbacterial survival through changing macrophage polarization, phagocytic activity or\nlysosomal biogenesis. Importantly, using an itaconate biosensor to detect\nendogenous itaconate in bacteria-containing vacuole, we have clearly confirmed that\nthe ratio of itaconate-targeting bacteria in vitro or in vivo were regulated by MCT1 and\nMCT4 (Fig. 2d-g), which led to a substantial decrease in bacterial load upon Su3118\ntreatment, dependent on itaconate, observed both in vitro and in vivo (Fig. 1k and 1n).\nIn general, we believe the primary effect of blocking MCT1/MCT4 is a direct impact on\nbacterial growth, particularly considering the influence of itaconate transport across\nthe bacteria-containing vacuole membrane. The use of itaconate biosensor provides\ncompelling evidence for this direct targeting. We fully agree with the reviewer’s\ninsightful point that itaconate may impact multiple pathways (e.g. NRF2 pathway)\nassociated with bacterial growth. This reinforces the importance of exploring these\nregulatory mechanisms in future research.\n2# The new title is still ambiguous. Authors changed “antibacterial immune responses”\nby “antibacterial efficacy”.\nIndeed, authors showed an effect on inhibition of bacterial growth, but not at all on\nantibacterial immune responses, or on the efficacy of macrophages to clear infection.\nI think it worth assessing some functionality of macrophages to really conclude on this\npoint and keep the proposed title.\nAnswer: We sincerely appreciate the reviewer’s thoughtful reminder. To address the\nreviewer’s concern, we have assessed the functionality of macrophages, including\nmacrophage polarization, cytokine profile, capacity of phagocytosis, or lysosomal\nbiogenesis. Our findings suggest that the accumulation of itaconate caused by the\ninhibition of MCT1 and MCT4 probably indirectly suppresses their replication by\nmodulating certain immune responses (e.g. NRF2 pathway) in macrophages.\nThe related discussion was highlighted in the manuscript (Line 374-386): …The\nattenuation of the itaconate-related effect was shown when inhibiting its uptake via\nMCT1 and MCT4, as evidenced by the downregulation of NRF2 expression (Fig. 4e,\nf). Itaconate, imported through MCT1 and MCT4, may act as an important messenger\nto activate antibacterial activity or other immune responses intracellularly. Additional\nresearch is necessary to comprehensively understand the physiological effect of\nitaconate transported by MCT1 and MCT4.\nMoreover, our findings support that MCT1 and MCT4-dependent transport of itaconate\ninto bacteria-containing vacuole is facilitated by RAB32 (Fig. 7a-g). The RAB32-\nmediated cell-autonomous antibacterial defense represents an innate immune\nmechanism that has been well established in multiple previous studies (Science\n369.6502 (2020): 450-455; Science 338.6109 (2012): 960-963; Cell host & microbe\n19.2 (2016): 216-226; iscience 24.1 (2021)). Our data further support the role of MCT1\nand MCT4 in transporting the antimicrobial metabolite itaconate during the RAB32-\nmediated immune response, establishing a link between metabolite transport and\nantimicrobial immunity.\nBased on these points, our findings support our manuscript title. And, as suggested by\nthe reviewer, we have kept the proposed title “Transport of itaconate across the plasma\nmembrane and Salmonella-containing vacuole via MCT1/4 modulates antibacterial\nimmune responses”."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have addressed most of the concerns. Here are my remaining comments:\n1/ I’m not convinced by the gating strategy to identify M1/M2 macrophages (Suppl Figure 2k). Authors gated the BMDMs as CD11b+ CD86+ and CD11b+ CD206+ respectively, but they should gate CD11b+ CD86+CD206- and CD11b+ CD206+CD86-. Indeed, with the current gating strategy, there is 54% of M1 and 82% of M2, so a total largely superior to 100%, meaning that a large majority of cells co-express both markers, and are not clearly M1 or M2. A dot plot pre-gated on CD11b+ and displaying CD86 versus CD206 should help depicting more clearly M1 and M2 macrophages on the same dot plot.\nIn contrast, the quantification of mRNA levels (Suppl Figure 2l) clearly shows that LPS induces CD86 expression (marker of M1), and IL4 triggers CD206 (marker of M2), as expected. In addition, S. Typhi induced CD86 (4-fold induction) but not CD206, sustaining a M1 polarization.\nThe authors should clarify this point, especially the gating strategy for M1/M2 and divergence between flow cytometry and mRNA analyses.\n2/ Regarding the title:\nAuthors shows that transport of itaconate is facilitated by RAB32, yet this component is not specific to immune cells. The NRF2 pathway, induced by itaconate, even if it is involved in antibacterial defense mechanisms, plays also an important role in the regulation of genes that control the expression of proteins critical in the detoxication and elimination of ROS. These two pathways are not specific of immune responses. As the new data demonstrate no effect of itaconate on macrophage polarization, functionality, capacity of phagocytosis or lysosomal biogenesis (which would be pertinent immune response elements), the link between itaconate and antibacterial immune responses is really not evident and not sustain by the content of the paper. A suggestion would be: “Transport of itaconate […] modulates macrophage’ antibacterial activity”."}, "author_response": "Response to Reviewers’ Comments\nWe extend our sincere thanks to Reviewer #4 for the valuable comments that have\nhelped us further improve the manuscript. We have thoroughly considered the\nsuggestions raised by Reviewer#4 and tried our best to address the concerns.\nReviewer #4 (Remarks to the Author):\n1/ I’m not convinced by the gating strategy to identify M1/M2 macrophages (Suppl\nFigure 2k). Authors gated the BMDMs as CD11b+ CD86+ and CD11b+ CD206+\nrespectively, but they should gate CD11b+ CD86+CD206- and CD11b+\nCD206+CD86-. Indeed, with the current gating strategy, there is 54% of M1 and 82%\nof M2, so a total largely superior to 100%, meaning that a large majority of cells co-\nexpress both markers, and are not clearly M1 or M2. A dot plot pre-gated on\nCD11b+ and displaying CD86 versus CD206 should help depicting more clearly M1 and\nM2 macrophages on the same dot plot.\nIn contrast, the quantification of mRNA levels (Suppl Figure 2l) clearly shows that LPS\ninduces CD86 expression (marker of M1), and IL4 triggers CD206 (marker of M2),\nas expected. In addition, S. Typhi induced CD86 (4-fold induction) but not CD206,\nsustaining a M1 polarization.\nThe authors should clarify this point, especially the gating strategy for M1/M2 and\ndivergence between flow cytometry and mRNA analyses.\nAnswer: We sincerely appreciate the reviewer’s thoughtful reminder. Following the\ncomments from the reviewer, we changed the gating strategy (Supplementary Fig.\n2k). The new gating strategy again showed that the inhibition of MCT1 and MCT4 did\nnot affect macrophage polarization (Figure 2m and 2n). The updated dot plots\n(CD11b+CD86+CD206- and CD11b+CD206+CD86-) now clearly show the distinct M1\nand M2 populations. Importantly, the overall trends remain consistent with our mRNA\nanalyses (Supplementary Fig. 2l).\n2/ Regarding the title:\nAuthors shows that transport of itaconate is facilitated by RAB32, yet this component\nis not specific to immune cells. The NRF2 pathway, induced by itaconate, even if it is\ninvolved in antibacterial defense mechanisms, plays also an important role in the\nregulation of genes that control the expression of proteins critical in the detoxication\nand elimination of ROS. These two pathways are not specific of immune responses.\nAs the new data demonstrate no effect of itaconate on macrophage polarization,\nfunctionality, capacity of phagocytosis or lysosomal biogenesis (which would be\npertinent immune response elements), the link between itaconate and antibacterial\nimmune responses is really not evident and not sustain by the content of the paper. A\nsuggestion would be: “Transport of itaconate […] modulates macrophage’ antibacterial\nactivity”.\nAnswer: We sincerely thank the reviewer for their valuable comments. In accordance\nwith the suggestions, we have revised the title in the latest version of the manuscript."}]

Decision letter