Kynurenine mediates the chemotherapy-induced intestinal toxicity through modulation of gut microbiota

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: minor_revision

Abstract
Chemotherapy-induced intestinal toxicity is a major dose-limiting complication, but the underlying mechanisms linking systemic metabolism to localized gut damage are poorly understood. Here we show that serum L-kynurenine, a tryptophan metabolite, is elevated in patients with severe oxaliplatin-induced intestinal toxicity. Accumulation of L-kynurenine is driven by IFNγ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) in myeloid cells. Using scRNA-seq and myeloid cell-specific knockout models, we confirm that myeloid cell-derived L-kynurenine exacerbates toxicity. Critically, L-kynurenine accumulation drives gut dysbiosis, characterized by the loss of Lactobacillus johnsonii, and subsequently activates the TNFα/JNK pathway, leading to intestinal epithelial apoptosis. Pharmacological inhibition or engineered reduction of L-kynurenine mitigates chemotherapy-induced intestinal injury. Our findings reveal an important role of L-kynurenine from myeloid cells in chemotherapy tolerance and propose its targeting as a potential therapeutic strategy.

Introduction
Cytotoxic chemotherapeutics are crucial for cancer treatment because they primarily kill tumor cells1. However, the occurrence of chemotherapy-induced side effects restricts the ability of cancer patients to maintain appropriate therapeutic dosing and duration, in turn limiting treatment efficacy2. One of the most common side effects of these drugs, affecting 40–80% of patients, is the onset of gastrointestinal toxicity3. Oxaliplatin, one of the common drugs used to treat colorectal cancer (CRC), is associated with neural and intestinal toxicity4. Therefore, a better understanding of intestinal toxicity in response to oxaliplatin is desperately needed, so that new concepts can be developed for future therapies.

Metabolic reprogramming plays a critical role in cancer therapy5. An increasing number of studies have further emphasized the significance of tryptophan metabolism in the chemotherapeutic response. A higher level of 3-indoleacetic acid (3-IAA) in serum has been demonstrated to be associated with increased treatment sensitivity in pancreatic cancer6. Indole-3-propionic acid (IPA) has also been shown to improve immune checkpoint blockade responsiveness by modulating the stemness program of CD8+ T cells7. In addition, a recent study revealed an association between altered metabolism and the severity of chemotherapy-induced gastrointestinal and hematologic toxicity8. Although evidence have indicated that some metabolites such as short-chain fatty acids are associated with chemotherapy-induced side effects, the role of tryptophan metabolism in chemotherapy-induced side effects is unclear.

Chemotherapy-induced side effects, especially intestinal injury, are also associated with alterations in the gut microbiota9. A previous study revealed an association between the microbiome and chemotherapy-induced gastrointestinal toxicity in children with acute lymphoblastic leukemia10. Enrichment of bacterial taxa Lachnospiraceae and Enterococcaceae was associated with postradiation restoration of gastrointestinal toxicity11. The supplementation of probiotics has also been shown to alleviate chemotherapy-induced toxicity8,12,13. However, the underlying mechanism through which the microbiota is modulated during chemotherapy-induced intestinal toxicity remains poorly understood.

Here, we show that specific tryptophan metabolites are associated with chemotherapy-induced side effects through targeted metabolomics analysis of CRC patient serum. Using distinct CRC mouse models, we demonstrate the contribution of specific metabolite to chemotherapy-induced intestinal toxicity and characterize the associated alterations in microbial components. Furthermore, we elucidate the underlying mechanism, suggesting that modulating tryptophan metabolic pathway offers a therapeutic strategy to improve tolerance to chemotherapy.

Results
Chemotherapy-induced intestinal toxicity is associated with the accumulation of L-kynurenine
To assess the role of tryptophan metabolism in chemotherapy-induced side effects, we established a clinical cohort and collected plasma from sixty-four patients who received oxaliplatin-based chemotherapy for targeted metabolomics of tryptophan metabolism (Fig. 1A). On the basis of the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI-CTCAE v5.0), these patients were classified into a high-toxicity group (n=30) and a low-toxicity group (n=34). Although similar baseline characteristics were found in these two groups, significantly higher levels of C-reactive protein (CRP) and lower level of white blood cells (WBCs) and neutrophils were detected in patients from higher toxicity group (Table S1). More importantly, a distinct pattern of tryptophan metabolites was observed between the high- and low-toxicity groups (Fig. 1B). Alteration of these tryptophan metabolites were closely correlated with different clinical parameters (Fig. 1C). Specifically, only four metabolites, L-kynurenine (L-Kyn), picolinic acid (PA), nicotinic acid (NAcid), and 3-hydroxyanthranilic acid (3-HAA), were significantly upregulated in plasma from the high-toxicity group (Fig. 1D–G). The increase of L-kynurenine in plasma from higher toxicity group was the most obvious metabolite in tryptophan metabolic pathway (Fig. 1B). A higher concentration of L-kynurenine in the plasma was associated with the exacerbation of toxicity, higher levels of CRP, and lower WBC and neutrophil counts (Fig. 1C). Collectively, these data indicated that increased levels of L-kynurenine were associated with chemotherapy-induced side effects.

To further validate the role of tryptophan metabolites in chemotherapy-induced side effects, we established a chemotherapy-induced toxicity model. C57BL/6J mice were treated with high-dose oxaliplatin (20 mg/kg) or PBS control once a week (Fig. 1H). Compared with control mice, mice treated with high-dose oxaliplatin exhibited significantly greater weight loss, worsened clinical scores, reduced spleen index and shortened colon length (Fig. 1I–L and Fig. S1A). Intestinal toxicity is among the important manifestations of chemotherapy-induced side effects. We found that mice exposed to high-dose oxaliplatin exhibited significantly greater separation between the crypt bases and muscularis mucosa (Fig. 1M), indicating the exacerbation of intestinal edema. Moreover, significantly decreased cellular proliferation and increased apoptosis of crypt cells were observed in mice treated with high-dose oxaliplatin (Fig. 1N, O). Immunofluorescence analysis and transcript detection of intestinal tissues also revealed significant downregulation of MUC2 in mice with increased intestinal toxicity (Fig. 1P). To evaluate the alteration of tryptophan metabolites in a chemotherapy-induced toxicity mouse model, plasma from mice was collected to determine the targeted metabolome of tryptophan metabolism. Consistently, oxaliplatin-induced intestinal toxicity in mice was associated with a significant upregulation of L-kynurenine (Fig. 1Q). Taken together, these data indicated that oxaliplatin-induced intestinal toxicity was associated with alterations of tryptophan metabolites, especially the accumulation of L-kynurenine.

Oxaliplatin-induced toxicity depends on IDO1 expression
To validate the role of L-kynurenine in oxaliplatin-induced toxicity, mice were gavaged with L-kynurenine three times a week, followed by oxaliplatin challenge, as shown in Fig. 2A. The toxicity induced by oxaliplatin, including severe weight loss, worsened clinical scores, decreased survival rate and decreased spleen index, was exacerbated in mice treated with L-kynurenine (Fig. 2B–E and Fig. S1B). In addition, mice treated with oxaliplatin and L-kynurenine exhibited obvious exacerbation of intestinal toxicity, such as shortened colon length, severe intestinal edema, decreased cellular proliferation, increased apoptosis of crypt cells and impairment of the intestinal barrier (Fig. 2F–J). Similar exacerbation of intestinal toxicity induced by oxaliplatin was also observed in a subcutaneous tumor xenograft model with L-kynurenine intervention (Fig. S1C–L). However, the chemotherapeutic potential of oxaliplatin was not affected (Fig. S1M, N). These data indicated that the accumulation of L-kynurenine aggravated oxaliplatin-induced intestinal toxicity in mice.

However, how L-kynurenine accumulates during chemotherapy remains unclear. A previous study demonstrated that L-kynurenine is converted from tryptophan via the key rate-limiting enzyme indoleamine 2,3-dioxygenase 1 (IDO1) (Fig. 2K). Significant upregulation of IDO1 expression was detected in mouse intestinal tissues (Fig. 2L), indicating the important role of IDO1 in chemotherapy-induced toxicity. Thus, we constructed a chemotherapy-induced toxicity model in Ido1 knockout (Ido1-/-) mice. Ido1-/- mice and wild-type (WT) mice were injected with a high dose of oxaliplatin on days 0 and 7 (Fig. 2M). Strikingly, severe systemic toxicity in WT mice treated with oxaliplatin was significantly alleviated in Ido1-/- mice (Fig. 2N–P and Fig. S2A). Compared with those of WT mice, significant improvements in intestinal toxicity were also observed in Ido1-/- mice after treatment with a high dose of oxaliplatin (Fig. 2Q–U). Serum L-kynurenine levels were significantly lower in Ido1-/- mice than in control mice both before and after OXA treatment (Fig. S2B). Collectively, these data indicated that oxaliplatin-induced toxicity depended on the expression of IDO1. Targeted intervention of IDO1 may be an important therapeutic strategy to alleviate the intestinal toxicity induced by oxaliplatin.

IFNγ-mediated upregulation of IDO1 exacerbates chemotherapy-induced toxicity
Given that previous studies have demonstrated that interferon-gamma (IFNγ) is a key upstream factor that induces IDO1 expression14,15, we further investigated its role in oxaliplatin-induced toxicity. First, ELISA revealed significantly elevated levels of IFNγ in both serum and fecal samples of WT mice following treatment with high-dose oxaliplatin (Fig. 3A). To further identify the cellular sources of IFNγ expression, we performed flow cytometric analysis on various immune cell populations and intestinal epithelial cells isolated from oxaliplatin-treated mice. Our results demonstrated a significant increase in IFNγ production specifically by CD8+ T cells (Fig. 3B). To elucidate the functional role of the IFNγ-IDO1 axis in chemotherapy toxicity, we administered a specific neutralizing antibody against IFNγ prior to high-dose oxaliplatin treatment (Fig. 3C). Compared with the control group without IFNγ neutralization, mice with IFNγ neutralization displayed significant improvements in systemic toxicity indicators (including weight loss, activity status) and intestinal toxicity parameters (including histopathological damage and colon length) (Fig. 3D–K and Fig. S2C). Critically, IFNγ neutralization significantly reduced IDO1 expression in intestinal tissues (Fig. S2D), directly confirming the IFNγ-dependent regulation of IDO1 in vivo. We also measured serum levels of L-kynurenine and found that IFNγ neutralization significantly reduced serum L-kynurenine levels compared with those in the control group (Fig. S2E). Collectively, these data indicate that the oxaliplatin triggers the production of IFNγ, which potently upregulates IDO1 expression, ultimately leading to severe chemotherapy-induced toxicity.

Alteration of IDO1 reshapes intestinal cellular components
To elucidate the role of IDO1 in intestinal tissues, we performed scRNA-seq on intestinal tissues from Ido1-/- and Ido1-/+ mice. Analysis of combined intestinal tissues from Ido1-/- and Ido1-/+ mice revealed 13 different cell clusters on the basis of the expression levels of the most variable genes (Fig. 4A). The cell clusters identified in intestinal tissues primarily consisted of myeloid cells (Ly6g+, Ly6a+ and Tgm3+), enterocytes (enterocyte 1: Slc13a1+, Slc10a2+ and Prap1+; enterocyte 2: Krt36+, Sele+ and Krt16+; enterocyte 3: Gsdmc4+, Krt84+ and Krtap6-5+), intestinal goblet cells (intestinal goblet cells 1: Muc5ac+, Muc2+ and Slc12a8+; intestinal goblet cells 2: Clca1+, Fcgbp+ and Tff3+), B lymphocytes (Cd74+, Cd79a+ and Cd79b+), T lymphocytes (Crtam+, Klrc1+, Trgc4+ and Pdcd1+), fibroblasts (Prrx1+, Clec3b+, Dcn+ and Ogn+), enteroendocrine cells (Chga+, Chgb+, Pyy+ and Sst+) and lymphatic endothelial cells (Prox1+, Kdr+ and Myct1+) (Fig. 4B). Specifically, the most prominent changes observed in Ido1-/- mice included a significant expansion of myeloid cells and a significant reduction in enterocyte 3 (Fig. 4C). Several genes associated with inflammatory and chemotactic processes were clearly downregulated in myeloid cells from Ido1-/- mice (Fig. 4D). Functional enrichment analysis revealed that alterations in these myeloid cells were associated with several inflammatory pathways, such as the PI3K-Akt signaling pathway, cytokine–cytokine receptor interactions, the chemokine signaling pathway, complement and coagulation cascades, and the IL-17 signaling and TNF signaling pathways (Fig. 4E). To further evaluate the cellular alteration upon changes in L-kynurenine metabolism, cell–cell interactions were subsequently constructed. Interestingly, myeloid cells played important roles in cellular interactions in the intestinal microenvironment (Fig. 4F). Strong interactions were observed between myeloid cells and enterocytes (Fig. 4F). Thus, we also characterized the alteration of enterocytes due to the role of L-kynurenine in intestinal toxicity. Consistent with the mouse phenotype, several genes associated with inflammatory processes, chemotactic processes and apoptosis were downregulated in Ido1-/- mice (Fig. 4G). Alteration of these three types enterocytes was associated with focal adhesion, cell adhesion molecules and ECM-receptor interaction (Fig. 4H). In addition, some inflammatory pathways such as Staphylococcus aureus infection, complement and coagulation cascades and intestinal immune network for IgA production were involved in the intestinal microenvironment (Fig. 4H). Collectively, these data emphasize the crucial role of myeloid cells and enterocytes in the L-kynurenine-associated chemotherapy toxicity.

L-kynurenine metabolism in myeloid cells is responsible for chemotherapy-induced toxicity
Although L-kynurenine-induced chemotherapy toxicity is associated with alterations in myeloid cells and enterocytes, the role of these cellular components in L-kynurenine biosynthesis during chemotherapy-induced toxicity remains unclear. To confirm the role of myeloid cell-associated IDO1 in chemotherapy toxicity, we constructed Ido1flox/flox Lyz2-Cre mice, which exhibited specific depletion of IDO1 in myeloid cells. A chemotherapy-induced toxicity model was established with Ido1flox/flox Lyz2-Cre and Ido1flox/flox mice, which were treated with oxaliplatin once a week (Fig. 5A). Compared with Ido1flox/flox mice, Ido1flox/flox Lyz2-Cre mice treated with oxaliplatin exhibited improvements in weight loss, clinical score and spleen index (Fig. 5B–D). Histopathological analysis also revealed a significant alleviation of intestinal toxicity in Ido1flox/flox Lyz2-Cre mice (Fig. 5E–H). To evaluate the role of myeloid cell-derived IDO1 in tumor growth, a subcutaneous tumor xenograft model was subsequently established. Similar alleviation of toxicity was observed in Ido1flox/flox Lyz2-Cre mice (Fig. S3A–J). We further measured serum levels of L-kynurenine in both Ido1flox/flox Lyz2-Cre and Ido1flox/flox mice, with or without OXA treatment. Consistent with the specific deletion of IDO1 in myeloid cells, serum L-kynurenine levels were significantly lower in Ido1flox/flox Lyz2-Cre mice than in control mice (Fig. 5I, J). To explore whether the expression of IDO1 in enterocytes modulates chemotherapy toxicity, we established a mouse model by using Ido1flox/flox Villin-Cre mice, which exhibited specific depletion of IDO1 in the epithelium (Fig. 5K). Similar trends in weight loss, clinical score and spleen index were observed in Ido1flox/flox Villin-Cre and Ido1flox/flox mice (Fig. 5L–N). These data confirmed that downregulation of IDO1 in myeloid cells but not in the epithelium improved chemotherapy-induced toxicity.

L-kynurenine metabolism modulates oxaliplatin-induced intestinal toxicity via the gut microbiota
Although alterations in L-kynurenine metabolism are associated with chemotherapy-induced toxicity, the underlying mechanism through which L-kynurenine metabolism modulates oxaliplatin-induced intestinal toxicity remains unclear. Previous studies have demonstrated that the gut microbiota plays a critical role in oxaliplatin-induced toxicity8. Thus, we established a chemotherapy-induced toxicity model with Ido1-/- mice and WT mice, both of which were treated with an antibiotic cocktail (Fig. 6A). The improvements in weight loss, clinical scores, spleen index and colon length in Ido1-/- mice disappeared after treatment with antibiotics, which was similar to the phenotypes in WT mice (Fig. 6B–E and Fig. S4A). Moreover, alleviation of intestinal toxicity induced by oxaliplatin was exclusively observed in Ido1-/- mice treated with the PBS control (Fig. 6F–I). Exacerbation of intestinal edema, decreased cellular proliferation, increased apoptosis of crypt cells and impairment of the intestinal barrier were detected in Ido1-/- mice treated with antibiotics and in WT mice (Fig. 6F–I). These data demonstrated that the influence of L-kynurenine metabolism on oxaliplatin-induced intestinal toxicity was associated with alterations of gut microbiota.

To further validate the role of the microbiota in oxaliplatin-induced toxicity, fecal microbiota transplantation (FMT) experiments were subsequently performed. Fecal samples collected from Ido1-/- mice or WT mice were transferred to recipient WT mice three times a week after 7 days of antibiotic treatment (Fig. 6J). The mice were challenged with a high dose of oxaliplatin once a week after FMT intervention (Fig. 6J). As expected, the recipient mice receiving microbiota from Ido1-/- mice exhibited significantly alleviated weight loss, clinical scores, spleen index and colon length (Fig. 6K–N and Fig. S4B). Moreover, a significant improvement in oxaliplatin-induced intestinal toxicity was also observed in recipient mice treated with microbiota from Ido1-/- mice (Fig. 6O–R). Therefore, these data indicated that the metabolism of L-kynurenine modulated oxaliplatin-induced intestinal toxicity via alteration of the gut microbiota.

Downregulation of L-kynurenine promotes the colonization of L. johnsonii to alleviate oxaliplatin toxicity
To clarify how L-kynurenine metabolism modulates the gut microbiota, fecal samples from donor and recipient mice were collected for 16S ribosomal RNA sequencing. Taxonomic analysis of the microbiome using principal coordinate analysis (PCoA) revealed that different genotypes of mice exhibited significant clustering and separation of the microbiome (Fig. 7A). The microbial structures of donor and recipient mice with the same genotype were similar (Fig. 7A, B). An increased proportion of Firmicutes was detected in the feces of Ido1-/- donor and recipient mice, whereas a greater proportion of Bacteroidetes was detected in WT mice (Fig. 7B). We subsequently detected marked alterations in microbes between different genotypes of donor mice and recipient mice. Specifically, significant accumulation of Lactobacillus was observed in Ido1-/- donor and recipient mice (Fig. 7C, D). Further identification of microbial units by the Basic Local Alignment Search Tool (BLAST) revealed that a stronger signal of Lactobacillus johnsonii (L. johnsonii) was detected in Ido1-/- donor and recipient mice (Fig. 7E). We subsequently evaluated the abundance of L. johnsonii in mouse feces using qPCR. As expected, oxaliplatin treatment significantly reduced the relative abundance of L. johnsonii in the feces of WT mice (Fig. 7F). In contrast, Ido1-/- and Ido1flox/flox Lyz2-Cre mice presented a higher baseline abundance of L. johnsonii, which was maintained after oxaliplatin treatment, resulting in consistently higher levels than those found in control group mice regardless of treatment (Fig. 7G, H). Moreover, in a clinical validation cohort of patients receiving oxaliplatin-based chemotherapy, we observed that serum L-kynurenine levels were significantly higher and that the abundance of fecal L. johnsonii was significantly lower in the high-toxicity group than those in the low-toxicity group (Fig. 7I). These results suggested that the downregulation of L-kynurenine metabolism enhanced the colonization of L. johnsonii.

To validate the role of L. johnsonii in oxaliplatin toxicity, we established a subcutaneous tumor xenograft model that was gavage with L. johnsonii three times a week followed by oxaliplatin treatment once a week (Fig. 7J). Notably, tumors size and tumor weight did not show significant differences between the groups, suggesting that supplementation with L. johnsonii did not affect the efficacy of oxaliplatin treatment (Fig. S5A–C). However, treatment with L. johnsonii significantly improved the oxaliplatin-induced toxicity, including the weight loss, clinical score, spleen index and colon length (Fig. 7K–N and Fig. S5D). Histopathological analysis also revealed a significant improvement of oxaliplatin-induced intestinal toxicity in mice treated with L. johnsonii (Fig. 7O–R). These data supported that downregulation of L-kynurenine metabolism promoted accumulation of L. johnsonii, therefore resulting in an alleviation of chemotherapy toxicity.

To investigate the effect of L-kynurenine on L. johnsonii, we first cultured the bacterium in vitro using medium supplemented with L-kynurenine. The results demonstrated that L-kynurenine significantly inhibited the growth of L. johnsonii (Fig. S5E). We subsequently performed transcriptome sequencing on L-kynurenine-treated and untreated L. johnsonii. Principal component analysis (PCA) of the sequencing data revealed distinct separations among the control group, the low-concentration L-kynurenine group (LKL), and the high-concentration L-kynurenine group (LKH) (Fig. S5F). In total, 1477 genes were annotated, and 32 genes were significantly downregulated in the LKL group compared with the control group (Fig. S5G). In the LKH group, 175 genes were significantly downregulated, whereas 82 genes were significantly upregulated compared with those in the control group (Fig. S5H). The functional annotation of the differentially expressed genes (DEGs) revealed that the DEGs were enriched primarily in processes related to adhesion, biofilm formation, exotoxin production, immune modulation, invasion mechanisms, motility, nutrient and metabolic factors, and stress response (Fig. S5I). After treatment with L-kynurenine, we observed a significant downregulation of several genes essential for the survival and proliferation of L. johnsonii. Specifically, the expression of AcpP16,17 (fatty acid synthesis), LepB18,19 and LspA20 (signal peptidases), DltB21 (cell wall modification), and RpoD22 (RNA polymerase) were decreased (Fig. S5J). These findings suggest that L-kynurenine may inhibit the growth of L. johnsonii, compromising its ability to colonize the gut.

L-kynurenine exacerbates apoptosis via TNFα/JNK pathway activation and L. johnsonii intervention attenuates this effect
Studies have demonstrated that radiotherapy-induced intestinal damage is mediated by activation of the TNFα (tumor necrosis factor-α)/JNK (c-Jun N-terminal kinase) signaling pathway23. To elucidate the downstream mechanism through which L-kynurenine exacerbates chemotherapy-induced intestinal injury, we focused on the TNFα/JNK cascade–a key pathway regulating epithelial apoptosis. TNFα levels were markedly elevated in the intestinal tissue of L-kynurenine-treated mice (Fig. S6A). Importantly, administration of L. johnsonii reversed this effect, resulting in a marked downregulation of TNFα expression (Fig. S6A). To further clarify the underlying mechanism, we performed in vitro experiments using epithelial cells treated with L-kynurenine in combination with OXA. This combined treatment led to a significant increase in phosphorylated JNK (p-JNK) levels (Fig. S6B). Probiotic treatment with L. johnsonii effectively attenuated activation of p-JNK (Fig. S6B). Meanwhile, L-kynurenine/OXA exposure markedly increased epithelial apoptosis (Fig. S6C), and L. johnsonii significantly reduced epithelial apoptosis (Fig. S6D). Together, these findings indicate that L. johnsonii alleviates chemotherapy-induced intestinal damage by suppression of TNFα/JNK signaling, thus inhibiting epithelial apoptosis.

Pharmacological and microbial therapeutic strategies attenuate IDO1/L-kynurenine-mediated chemotoxicity
On the basis of the protective effects observed in the genetic model, we further sought to confirm the therapeutic potential of pharmacologically inhibiting IDO1 using the specific inhibitor Epacadostat. WT mice were gavaged with PBS or Epacadostat twice a day and high-dose oxaliplatin was administered on day 0 and 7 (Fig. 8A). Consistent with the phenotype observed in Ido1-/- mice, Epacadostat treatment exhibited a significant protection against oxaliplatin-induced toxicity. Compared with control mice, Epacadostat-treated mice exhibited markedly reduced severity of toxicity symptoms (Fig. 8B–E and Fig. S7A). Histopathological assessment of intestinal tissues revealed substantial improvement in the Epacadostat group compared with the vehicle group (Fig. 8F–I). Collectively, these results demonstrate that pharmacological inhibition of IDO1 with Epacadostat could attenuate chemotherapy-induced toxicity.

While suppressing L-kynurenine biosynthesis in host cells is effective, we hypothesized that directly enhancing its metabolism in the gut lumen could offer a complementary strategy. We established engineered bacteria by using Escherichia coli strain BL21, which overexpresses the downstream enzyme kynurenine aminotransferase (KAT) (BL21-KAT) (Fig. 8J). A significantly lower level of L-kynurenine was found in the supernatant from BL21-KAT (Fig. 8K). To evaluate the therapeutic potential of BL21-KAT in oxaliplatin-induced toxicity, we established a subcutaneous tumor xenograft model in which mice were gavaged with BL21-KAT three times a week followed by oxaliplatin treatment once a week (Fig. 8L). Similar tumor size and tumor weight were observed in mice treated with BL21-KAT or BL21 after oxaliplatin intervention, indicating that BL21-KAT intervention did not influence the antitumor efficacy of oxaliplatin (Fig. S7B, C). Moreover, compared with mice treated with BL21 and oxaliplatin, mice treated with BL21-KAT and oxaliplatin exhibited significant improvements in weight loss, clinical score, spleen index and colon length (Fig. 8M–P and Fig. S7D). Histopathological analysis also revealed a significant alleviation of oxaliplatin-induced intestinal toxicity in mice treated with BL21-KAT and oxaliplatin (Fig. 8Q–T). More importantly, the concentration of L-kynurenine in the plasma of mice treated with BL21-KAT and oxaliplatin was significantly lower than that in the plasma of mice treated with BL21 and oxaliplatin, whereas similar concentrations of L-kynurenine were observed between mice treated with BL21-KAT alone and those treated with BL21-KAT and oxaliplatin (Fig. 8U). Therapeutic targeting of the IDO1–L-kynurenine axis, either through pharmacological inhibition (Epacadostat) or engineered L-kynurenine-metabolizing probiotics, significantly reduces chemotherapy-induced toxicity, offering clinically actionable strategies to improve patient tolerance.

Methods
Humansamples
A cohort of colorectal cancer (CRC) patients treated with oxaliplatin-based chemotherapy was recruited from The Sixth Affiliated Hospital of Sun Yat-sen University. Sixty four patients were recruited and stratified into higher toxicity group (n=30) and lower toxicity group (n=34) based on the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI-CTCAE v5.0). Demographic and clinical characteristics of the cohort are summarized in Table S1. While an independent validation cohort of 28 patients was additionally collected to verify the findings. The study was approved by the Human Medical Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from all participants prior to their inclusion in the study. The approve number of this study was 2020ZSLYEC-266. Serum samples were collected after chemotherapy and stored at −80°C.

Mice
Six-to eight-week-old male mice on the C57BL/6J (WT) background were used. C57BL/6J WT mice, Ido1-/- mice, Ido1flox/flox mice, Lyz2-Cre mice and Villin-Cre mice were obtained from GemPharmatech company. Myeloid-specific (Ido1fl/fl Lyz2-Cre) and intestinal epithelial-specific (Ido1fl/fl Villin-Cre) Ido1 knockout mice were generated through strategic cross-breeding of the respective Cre and floxed lines. All animals were maintained under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle and ad libitum access to food and water at the Sixth Affiliated Hospital of Sun Yat-sen University and Guangzhou Ruige Biological Technology Co., Ltd. Experimental procedures were strictly conducted following protocols approved by the Institutional Animal Care and Use Committee (IACUC). The approve number of this study were IACUC-2023060701 and 20220801-001.

Cell culture
The cell lines were cultured in Gibco Dulbecco’s Modified Eagle Medium (DMEM) or RPMI-1640 medium with 10% fetal bovine serum (GIBCO) and 1% penicillin–streptomycin (GIBCO) at 37°C in 5% CO2 and 95% humidity.

Bacteria culture
Media for cultivation of organisms were listed in the Method Details were pre-reduced in the aerobic chamber (90% N2, 5% CO2, 5% H2) for 48h before inoculation. Bacterial cultures intended for in vitro and in vivo assays were grown overnight at 37°C in the aerobic or anaerobic chamber (CAW1100, Guangzhou Huanghe Instrument Technology Co., Ltd.). Bacterial isolation and identification were achieved by colony picker (RapidPick SP, GuangZhou Jinke Chian Technology Co., Ltd.), detection system (CMI-1600, Guangzhou Chengyi Imp & Exp Co., Ltd.) and dilution system (Easy spiral dilute, Zhongke Scientific & Technical Co., Ltd.).

Oxaliplatin toxicity model
A toxic dose of oxaliplatin (20 mg/kg body weight) or PBS control was administered to mice via peritoneal injection once a week. The mice were then housed in sterile autoclaved cages and provided standard chow and water ad libitum, unless otherwise noted. Body weight, clinical scores (determined using a cumulative scoring system, based on weight loss, temperature changes, physical appearance, posture, and mobility), and survival were monitored. To evaluate the role of L-kynurenine, mice were gavaged with L-kynurenine (20 mg/kg body weight) or PBS control three times a week. To evaluate the role of IDO1, Ido1 -/- mice, Ido1flox/flox Lyz2-Cre mice and Ido1flox/flox Villin-Cre mice were established for oxaliplatin toxicity model as described above. To evaluate the role of microbiota, mice were treated with broad-spectrum antibiotic cocktail (ampicillin 0.2 g/L, metronidazole 0.2 g/L, neomycin 0.2 g/L, vancomycin 0.1 g/L) for one week before oxaliplatin treatment and continued until the end of the experiment. In accordance with the protocols approved by IACUC, the maximal allowable body weight loss was 30% relative to the initial weight.

Subcutaneous tumor xenograft experiment
To establish a subcutaneous tumor xenograft model, CRC cell line MC38 (1×106/100ul) were injected into the loose subcutaneous tissues of the mice right back. After 7 days, C57BL/6J WT mice were treated with oxaliplatin (20 mg/kg body weight) intraperitoneally once a week. To evaluate the role of L. johnsonii, mice were gavaged with L. johnsonii (1x109 CFU/dose) three times a week for one week before tumor injection and continued until the end of the experiment. To evaluate the role of IDO1, Ido1flox/flox Lyz2-Cre mice was established for subcutaneous tumor xenograft model as described above. To evaluate the role of engineered bacteria, mice were gavaged with BL21-KAT or BL21 (1x109 CFU/dose) three times a week for one week before tumor injection and continued until the end of the experiment. In accordance with the protocols approved by the IACUC, the maximal permitted tumor volume was 2000 mm3.

Fecal microbiota transplantation
Feces from WT C57BL/6J and Ido1-/- donor mice were collected in 20% autoclaved glycerol and quickly suspended and homogenized. After centrifugation at 100×g at 4°C for 30s, the supernatants were collected. Recipient mice were treated with a broad-spectrum antibiotic cocktail for one week and subsequently were gavaged with 0.2 mL fecal suspension three times a week. Subsequently, mice were challenged with oxaliplatin once a week at 1 week post-transplantation.

IDO1 inhibitor animal experiment
Mice were administered Epacadostat (50 mg/kg) or PBS (control) via oral gavage twice daily until the end of the experiment. Following the initiation of Epacadostat or PBS treatment, the mice received oxaliplatin or PBS control therapy. A toxic dose of oxaliplatin (20 mg/kg body weight) or PBS was administered once per week via intraperitoneal injection.

In vivo IFNγ neutralization
To assess the role of IFNγ, mice were injected intraperitoneally on days −1, 2, 5, and 8, with 300 µg anti-IFNγ (clone R4–6A2; BioXCell) or isotype rat IgG1 controls (clone HRPN; BioXCell). Following the initiation of anti-IFNγ treatment, the mice received oxaliplatin therapy. A toxic dose of oxaliplatin (20 mg/kg body weight) or PBS was administered once per week via intraperitoneal injection.

Isolation and culture of Lactobacillus johnsonii
L. johnsonii was isolated from healthy individual and verified using 16S rRNA sequencing (V4 sequences). The isolation of L. johnsonii using an automated colony picker (RapidPick SP, GuangZhou Jinke Chian Technology Co., Ltd.), detection system (CMI-1600, Guangzhou Chengyi Imp & Exp Co., Ltd.) and dilution system (Easy spiral dilute, Zhongke Scientific & Technical Co., Ltd.). L. johnsonii was cultured in MRS medium at 37℃ in an anaerobic chamber using gas mix consisting of 5% hydrogen, 10% carbon dioxide and 85% nitrogen for 24h.

Histopathological analysis of spleen and colon
Spleens and Swiss-rolled like colon tissues were freshly collected and immersed in 10% neutral buffered formalin. The tissue was then embedded in paraffin and sliced into 5mm sections, which were sequentially performed following standard procedures. The pathological changes in spleens and colon were quantified by hematoxylin and eosin (H&E) staining.

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IFN-γ in serum and fecal samples were quantified using a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit (Telenbiotech, TL-E1382), according to the manufacturer’s instructions. Briefly, serum samples were diluted and fecal samples were homogenized in PBS followed by centrifugation to obtain supernatants. Standards and samples were incubated in antibody-precoated plates. After a series of incubations with a biotinylated detection antibody and streptavidin-HRP conjugate, the reaction was developed with tetramethylbenzidine (TMB) substrate and stopped with acid. The absorbance was measured at 450nm. The cytokine concentration was determined by interpolating values from the standard curve. For fecal samples, results were normalized to the initial wet weight of the sample.

Immunohistochemistry (IHC)
Paraffin-embedded tissue sections (4μm) were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to heat-induced antigen retrieval in sodium citrate buffer (10 mM, pH=6.0) at 95°C for 10 min. Endogenous peroxidase activity was quenched with 3% H2O2, followed by blocking with 5% BSA in PBS for 30 min. Sections were incubated overnight at 4°C with anti-Ki67 antibody (#GB121141, Servicebio, 1:250 dilution). Next, washed with PBST, and treated with HRP-conjugated secondary antibody (#G1214, servicebio) for 30 min at room temperature in the dark. Signal was visualized using DAB chromogen, followed by counterstaining with hematoxylin for 1 min, and mounted with resin. The slides were scanned by slide scanning system (Shengqiang, China). The Ki-67 positive area was evaluated by ImageJ.

Immunofluorescence staining
The tissues sections were freshly isolated and fixed with 10% formalin before embedding in paraffin wax. The whole staining was performed on paraffin-embedded sections (4mm). After deparaffination with dimethylbenzene and rehydration with ethanol, the slides were immersed in boiling sodium citrate buffer (10 mM, pH=6.0) for 10 min to retrieve antigen. Antigen retrieval was performed by boiling slides in 10 mM sodium citrate buffer (pH 6.0) for 10 min, followed by cooling to room temperature. Sections were permeabilized and blocked with 0.6% Triton X-100 in PBS for 15 min and washed three times with PBST (PBS containing 0.1% Tween-20, 5 min per wash). The tissues samples were immune-stained with primary antibody (#GB120002, servicebio, 1:250 dilution) by incubating overnight at 4C. After three PBST washes, sections were incubated with secondary antibody (#ab150077, Abcam, Alexa Fluor 488) for 30 min at room temperature in the dark. Nuclei were counterstained with DAPI, and slides were cover slipped and sealed. Then examined slides under a laser scanning confocal microscope (Zeiss, Germany).

TdT-mediated dUTP nick-end labeling (TUNEL) assay
Tissue apoptosis was detected with an in-situ cell death detection kit (#101-131, Goonie, China) following the manufacture’s instruction. Briefly, paraffin-embedded intestinal sections were dewaxed in xylene and rehydrated through gradient alcohols. The slides were then incubated with 20ug/mL proteinase K for 5 min followed by PBS washing. Next, the samples were incubated with the TUNEL reaction mixture in a humidified chamber at room temperature for 60 min avoid light. This was followed by PBS washes and incubation with 1mg/mL DAPI. After staining, the sections were scanned under a laser scanning confocal microscope (Zeiss, Germany). Quantification was performed with ImageJ.

RNA and DNA extraction and quantitative real-time PCR
Quantitative real-time PCR RNA extraction was performed using the FastPure Cell/Tissue Total RNA Isolation Kit according to the manufacturer instructions, and the HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper was used to reverse transcribe the RNA into cDNA. Fecal or bacterial DNA was obtained using an AmPure Microbial DNA Kit (D7111, Megan). qPCR was performed using the SYBR Green Supermix Kit on the QuantStudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific). Primer sequences used in this study was shown in Table S2.

Heterologous expression of KAT in E. coli
The kynurenine aminotransferase (KAT) expression system was established as previously described30. Briefly, the KAT open reading frame (ORF) (Table S4) was codon-optimized for E. coli expression using the OPTIMIZER online tool, OptimumGene™ (Genscript), and manual adjustments to eliminate rare codons, optimize GC content, and remove negative CIS elements or repeats. The optimized sequence was cloned into the pET15b vector. For protein overexpression, the pET15b-KAT plasmid and an empty pET15b control were transformed into chemically competent E. coli BL21 (DE3) via standard heat-shock to generate E. coli BL21-KAT and E. coli BL21-WT, respectively. Transformants were selected on ampicillin plates (100 mg/mL) at 37°C. A single colony was expanded overnight and inoculated into 50 mL of LB medium. Once the culture reached an OD600 of 0.6–0.8, KAT expression was induced with 1 mM IPTG at 37°C under agitation (220 rpm).

Cell isolation of spleen mononuclear cells
Spleens were completely isolated from mice and crushed with forceps, and single cells were isolated in PBS using a 70-μm cell strainer. The cells were washed with 1x PBS and centrifuged (100×g for 5 min), and then red blood cell lysis containing splenocytes was pipetted up. The culture medium was then added to the cells and centrifuged at 100×g at 4°C for 5 min. Single-cell suspensions were diluted in Roswell Park Memorial Institute (RPMI) medium.

Cell culture and cellular stimulation
The murine epithelial cell line NCM460 was purchased from the American Type Culture Collection and cultured at 37°C in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a 5% CO2 atmosphere. 5×105 cells were grown in a 6-well plate and co-cultured with bacteria, L-kynurenine, or oxaliplatin for 24h. Subsequently, protein was extracted for Western blot analysis or cells were collected for apoptosis analysis by flow cytometry.

Flow cytometry analysis
The effectors IFN-g (BD Biosciences, 562303) were detected by adding Activation Cocktails (BioLegend, 423303) to 1ml of cell suspension and incubating for 6h at 37°C. Then, 1×106 cells were stained with Fixable Viability Stain 700 (BD Biosciences, 564997) in serum-free buffer for 30 min. Then, cells were incubated with anti-CD16/32 antibody (BioLegend, 101320) for 10 min to block non-specific binding of immunoglobulin to the Fc receptors. Afterwards, the appropriate amount of pre-diluted fluorescent labeled antibody (BD Biosciences, 563024, 553051, 552877, 557659, 564143; STARTER, S0B5093) was added to each tube as recommended by the manufacturer. Cells were incubated in the 4°C refrigerator for 30 min and fixed with Fixation Buffer (BD Biosciences, 562574) for 30 min at room temperature. After washing, the membranes were ruptured (BD Biosciences, 562574) and intracellular fluorescent antibodies were added to each tube and incubated for 30 min at room temperature. Prepared cells were analyzed by flow cytometry (Beckman Coulter). The gating strategy is illustrated in the Fig. S8.

Cell apoptosis assay
2×105 of cells were seeded into a 6-well plate, and then treated with bacteria, L-kynurenine, or oxaliplatin for 24h. Cell apoptosis was detected by Apoptosis Detection Kit (Multisciences, AP107). Prepared cells were analyzed by flow cytometry (Beckman Coulter).

Western blot
Total protein was extracted using RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors (Servicebio, Wuhan, China) followed by sonication and centrifugation at 4000 rpm for 15 min. Then, Protein samples (20μg) were separated by Bis/Tris-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Shanghai, China). Membranes were blocked in 5% skimmed milk and incubated with primary antibodies and secondary antibodies. The protein bands were visualized using Enhanced Chemiluminescence (ECL) Reagent (Millipore, 2236001). Protein signals were captured and imaged using the Azure 280 Imaging System (Azure Biosystems, USA).

Targeted metabolomics for tryptophan metabolism
Chemicals and reagents. Serum tryptophan and its metabolites were quantified by Maiwei Metabolic Biotechnology Co., Ltd. (Wuhan, China). HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany), while acetic acid and all analytical standards were sourced from Sigma-Aldrich or OlChemim (Olomouc, Czech Republic). Ultrapure water was prepared using a Milli-Q system (Millipore, USA). Individual standard stock solutions (1 mg/mL in MeOH) were stored at −20°C and further diluted with MeOH to prepare working solutions.

Sample preparation. Briefly, 50 mg of thawed serum was extracted with 500 μL of MeOH, supplemented with 20 μL of an internal standard (IS) mixture (250 ng/mL). The mixture was vortexed for 3 min, incubated at −20°C for 30 min, and centrifuged at 3000×g for 10 min at 4°C. A 250 μL aliquot of the supernatant was collected and subjected to a second centrifugation (3000×g for 5 min at 4°C). Finally, 150 μL of the resulting supernatant was recovered for LC-MS/MS analysis.

LC-MS/MS conditions. Metabolomic profiling was performed using an ExionLC AD UPLC system coupled with a QTRAP 6500+ mass spectrometer (Sciex). Chromatographic separation was achieved on a Waters ACQUITY UPLC HSS T3 C18 column (100 mm × 2.1 mm i.d., 1.8 μm) maintained at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was as follows: 0–1 min, 10% B; 1–8 min, 10–95% B; 8–9.5 min, 95% B; and 9.6–12 min, 10% B. The flow rate was 0.35 mL/min with a 5 μL injection volume.

The mass spectrometer, equipped with an ESI Turbo Ion-Spray interface, operated in both positive and negative ionization modes. Key source parameters included: source temperature, 550°C; ion spray voltage (IS) 5500 V (Positive), −4500 V (Negative); curtain gas (CUR) was set at 35 psi. Metabolites were quantified using scheduled multiple reaction monitoring (MRM). Data acquisition and quantification were conducted using Analyst 1.6.3 and MultiQuant 3.0.3 software (Sciex), respectively.

Metabolomics analysis. The Wilcoxon rank-sum test was used to identify concentrations that differed in abundance between higher and lower toxicity group. The linear mixed effects modeling with the R package MaAsLin2 to perform the association of each clinical parameter and specific metabolites. The p values and beta coefficients generated from this model indicate the significance and effect estimates of examining whether the concentration of a specific metabolite changes across the change of clinical parameter in a dose-response manner.

Liquid chromatograph mass spectrometer (LC-MS) analysis
For the serum samples 100 μL samples were mixed with 900 μL mass spectrometry grade pre-chilled methanol, then vortexed for 5 min. The mixture was then centrifuged at 3000xg and 4°C for 10 minutes, and the supernatant was collected. An Agilent 1290 Infinity II liquid chromatography system coupled to an Agilent 6495A triple quadrupole LC-MS system was used to quantitate L-Kyn. Data were collected by Mass Hunter workstation. For data processing, the peak area was integrated by use of Agilent Mass Hunter Vista Flux software, and Agilent Metabolite ID software.

16S ribosomal RNA gene sequencing and analysis
DNA from frozen stool samples was extracted using a FastDNA Spin Kit for Soil. For 16S rRNA gene sequencing, the V3-V4 variable region was amplified using 2-step PCR. In the first step, 10 ng genomic DNA was used as a template for the first PCR with a total volume of 20 μl using the 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) primers appended with Illumina adaptor sequences. The amplicons were purified, checked on a fragment analyzer, quantified, followed by equimolar multiplexing, and sequenced on an Illumina MiSeq PE300 platform.

Single cell RNA sequencing
Tissues dissociation and preparation of mice intestinal tissues. Intestinal tissues were harvested from Ido1-/- mice and their corresponding littermate controls. To minimize environmental bias, all mice were co-housed from birth and received no prior experimental treatment. Single-cell RNA sequencing (scRNA-seq) was performed by LC-Bio Technologies Co., Ltd. (Hangzhou, China).

Pre-processing. Fresh tissues were placed in sterile, RNase-free dishes containing ice-cold, calcium/magnesium-free 1 × PBS. Tissues were minced into 0.5 mm2 fragments, washed with PBS, and meticulously cleared of blood stains and mesenteric fat.

Enzymatic digestion. Tissue fragments were dissociated into single cells using a digestion cocktail (0.35% collagenase IV 5, 2 mg/ml papain, 120 Units/ml DNase I) in a 37°C water bath with shaking for 20 min at 100×g.

Cell recovery. The reaction was neutralized with 1 × PBS supplemented with 10% (v/v) fetal bovine serum (FBS). After mechanical trituration using a Pasteur pipette, the suspension was filtered through a 70 μm cell strainer and centrifuged at 300g for 5 min at 4°C.

Purification. The cell pellet was resuspended in 100 ul 1 × PBS (0.04% BSA). Residual red blood cells were removed using 1 × RBC Lysis Buffer (MACS 130-094-183), followed by dead cell removal using the Miltenyi Dead Cell Removal Kit (MACS 130-090-101).

Quality control. The final pellet was washed twice and resuspended in 50 μl of 1 × PBS (0.04% BSA). Cell viability was assessed via trypan blue exclusion (threshold >85%). Cells were counted using a Countess II Automated Cell Counter and adjusted to a final concentration of 700–1200 cells/μl.

Chromium 10x Genomics library and sequencing. Single-cell Gel Bead-in-Emulsions (GEMs) were generated by loading cellular suspensions onto a 10x Genomics GemCode instrument. Libraries were constructed using the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1. Full-length barcoded cDNAs were amplified via PCR. R1 sequences were introduced during GEM incubation, while P5, P7, sample indices, and R2 sequences were added during library construction via end repair, A-tailing, and adapter ligation. The final Illumina-ready libraries contained 16bp 10x Barcodes and 10bp UMIs in Read 1, with cDNA fragments sequenced in Read 2.

Data quality control. Raw data were processed using Cell Ranger (v3.1.0) for FASTQ conversion, genome alignment, and UMI counting. Low-quality barcodes were filtered, and valid cell droplets were identified using the EmptyDrops method. Cell-by-gene matrices were generated via UMI counting and cell barcode calling. Downstream analyses were performed by Seurat v5.043. Cells with mitochondrial gene ≥25%, Hemoglobin gene ≥3%, n.features <200 genes, or n.count >6000 genes were filtered out. Doublets were identified and removed using DoubletFinder (v2.0.4). Through the quality control steps above, low-quality cells, droplets with ambient RNA, and suspicious doublets were removed. Samples were integrated and batch effects were minimized with canonical correlation analysis (CCA).

Unsupervised clustering and markers identification. Data were log-normalized and scaled. After PCA-based linear dimensional reduction on highly variable genes, cells were clustered and visualized using UMAP. Cluster-specific markers were identified using the FindMarkers function (Wilcoxon rank-sum test). Differentially expressed genes (DEGs) were defined by >10% cells in one of the two conditions, with |log2FC| > 0.5, and adjusted p value <0.05 (Bonferroni correction) are defined as differentially expressed.

Cell-cell interaction analysis. Potential cell-cell interactions between different immune cells were identified using CellChat v1.1.344. Communication probabilities were calculated by integrating gene expression with the CellChatDB database of ligand-receptor pairs. The aggregated signaling network was visualized using the netVisual_bubble function to highlight significant interaction strengths between immune cell subsets.

Bacterial strand-specific RNA-seq
Total RNA was extracted from the tissue using CTAB method and genomic DNA was removed. Only high-quality RNA sample was used to construct sequencing library.

Library construction and sequencing. Ribosomal RNA (rRNA) depletion instead of poly(A) purification is performed by RiboCop rRNA Depletion Kit for Mixed Bacterial Samples (lexogen, USA) and then all mRNAs were broken into short (200 nt) fragments by adding fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized with random hexamer primers (Illumina). When the second strand cDNA was synthesized, dUTP was incorporated in place of dTTP. Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. RNA-seq transcriptome library was prepared following Illumina® Stranded mRNA Prep, Ligation (San Diego, CA) using of total RNA. paired-end RNA-seq library was sequenced with the Illumina Novaseq X plus (or other new sequenator) (Illumina Inc., San Diego, CA, USA). The processing of original images to sequences, base-calling, and quality value calculations. The clean reads by removing low-quality sequences, reads with more than 10% of N bases (unknown bases) and reads containing adaptor sequences.

Expression analysis. Quantify gene and transcript abundances from single-end or paired-end RNA-Seq data using RSEM, RSEM computes maximum likelihood abundance estimates using the Expectation Maximization (EM) algorithm for its statistical model, including the modeling of paired-end (PE) and variable-length reads, fragment length distributions, and quality scores, to determine which transcripts are isoforms of the same gene. The FPKM and TPM are able to eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression. Therefore, the calculated gene expression can be directly used for comparing the difference of gene expression among samples.

Differential expression analysis
For each dataset, and for each alignment and quantification protocol, we identified differentially expressed genes by using the edgeR, DESeq2, or DESeq packages.

Quantification and statistical analysis
Statistical evaluations were conducted using R (version 4.3) or GraphPad Prism 9 (GraphPad Software, San Diego, CA). Comparative analysis between two groups was performed using the Student’s t-test for parametric data or the Mann–Whitney U test for non-parametric data. For multi-group comparisons (≥3 groups), one-way ANOVA or the Kruskal–Wallis test was employed, depending on the data distribution. Results were considered statistically significant at an adjusted p <0.05. Significance levels are denoted in figures as follows: *, p <0.05; **, p <0.01; ***, p <0.001 and ****, p <0.0001; “ns” represents non-significance. Detailed information regarding sample sizes (n), specific statistical tests, and biological replicates for each experiment is provided in the corresponding figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Chemotherapy induced gastrointestinal toxicity is a major limiting factor in cancer treatment and understanding the mechanisms associated with this adverse side-effect has merit. This manuscript describes the development of oxaliplatin-induced gastrointestinal toxicity, specifically the breakdown of the gut epithelial barrier due to alteration in tryptophan metabolism. The authors show that patients treated with oxaliplatin develop GI toxicity that can be stratified as high or low toxicity that correlates with lower levels of white blood cells and neutrophils. The authors also note that there is significant upregulation of L-kynurenine in patients demonstrating higher toxicity. They then test the hypothesis that enhanced L-kynurenine is associated with GI toxicity by oxaliplatin in mice models. The gut toxicity is evaluated by body weight, clinical score, histology, spleen index, and expression of MUC2 and apoptosis across the various treatments.\nThe studies in mice have the following findings: 1) Oxaliplatin causes significant GI toxicity correlating with increase in kynurenine in serum, 2) deletion of the metabolizing enzyme (IDO) reduces oxaliplatin-induced GI toxicity, 3) depletion of gut microbiome improves GI toxicity due to oxaliplatin in WT, but worsens in IDO knock out mice (with respect to spleen index, colon length, separation from muscularis mucosae and cell apoptosis; but not in clinical score: see below my comments on this). This suggests that the protective effect of IDO1 KO is dependent on the gut microbiome. The authors have further conducted fecal transplant to show that WT mice receiving microbiome from IDO1 KO donors protect against oxaliplatin induce GI toxicity and it is L. johnsonii that protects against GI toxicity. They conclude that lack of kynurenine metabolism allows for the increase in L. johnsonii. In a final series of experiments, the authors show that myeloid cells are the main site for L-kynurenine biosynthesis and that providing bacteria that degrade L-kynurenine protects against oxaliplatin – induced GI toxicity.\nThere are several major issues with this manuscript in its current form.\n1) The experimental methodology is not provided in detail and thus it is difficult to evaluate the specific experimental paradigms.\nThere is a discrepancy in the time frame of OXA treatment and kynurenine measurement in mice. In Fig 1, the model is 3 OXA treatments over two weeks, while the rest of the studies are conducted with 2 OXA injections over one week. Thus, it remains unclear if KYN measurements reflect changes over the 2 weeks. What is meant by clinical score? There is no description of the scoring system or any reference to previous studies. What specific physical appearance and mobility parameters were determined? What is the spleen index? The histology images in figures S1 – S5 of the spleen are unclear. What is being shown with regard to the differences in spleen index.\nThere is no indication of how many males/females were used and no stratification based on sex. Some studies e.g. Figure 3 have only 4 mice (males or females?) and it is unclear how statistical analysis were carried out with this small numbers.\nOne of the cardinal features of chemotherapy-induced gastrointestinal toxicity is the development of severe diarrhea. This has not been measured and neither are studies to show changes in intestinal permeability.\nThroughout the manuscript it is stated that kynurenine was gavaged 3 time/seek. I assume every alternate day?\n2) There are some important controls missing from the overall study. IDO1 Knock out mice show improved GI toxicity, however the levels of tryptophan or kynurenine are not determined.\nHow effective is one week of ABX treatment in depleting gut bacteria. There is no difference in the clinical score between IDO1 K.O. and IDO KO + ABX while other parameters of GI toxicity are significantly altered. This needs to be addressed.\nThe studies on the microbiome analysis are confusing. Are both donor and recipient mice treated with OXA or just the recipient mice? If both groups are treated than a control of untreated microbiome is missing. This needs to be validated. Also Figure 4E and F are not clear. In 4 what is the difference between WT and control (4E). If mice are treated with OXA in 4F, please explain why expression is higher in WT than control of 4E. Figure 4G needs better labeling. It is unclear which class is illustrated based on the color scheme.\nIf OXA treatment is only given to recipient mice, then there appears to be no effect of OXA on the microbiome and the only differences are in the genotype.\n3) The conclusion of this study is unclear. It would help for the authors to provide a schematic illustration on the mechanism of how OXA alters GI toxicity. An important control missing is whether L. johnsonii shifts with OXA treatment in WT mice, resulting in increased expression of kynurenine and if this is prevented in IDOfl/fl lyzCre mice.\nMinor issues\nThe manuscript is not well written and some conclusions are difficult to follow.\nThe color scheme of the figures need to be better presented so that significant differences can be observed.\nFig 2K: Kynurenine structure is incorrect\nFig 3R: Should conduct an outlier test for the FMT-Ido-/- MUC2 expression\nFig 5H: Unclear of the IDO -/- vs IDO -/+", "reviewer_2": "Xie et al. focus on mechanisms underlying chemotherapy-induced intestinal toxicity. They found changes in tryptophan metabolites and traced the toxicity (in their models) to IDO1-mediated production on kynurenine pathway (KP) products. KP products then altered the microbiota, driving intestinal damage. Although the manuscript reports some important observations, it has several problems of logic and lack of appreciation of alternative possibilities.\nSpecific comments:\nFigure 1. In the first part of the figure, changes (relative) to metabolites are shown but from the plasma. KP pathway metabolites are generally detected in any scenario where a strong IFN response has happened or is ongoing – a good example is SARS-CoV-2 infection – as IFNs drive IDO1 expression. Thus, in this figure, the issues center on (i) the source of IFN and especially IFN-gamma in the patients and mouse model, (ii) the cells expressing IDO1, and most importantly, (iii) the concentration of KP pathway metabolites in the gut relative to the blood.\nFigure 2. Following from Figure 1, the authors tie IDO1 to the toxicity (e.g., panel M-Q) using a whole animal IDO1 KO. However, these effects are partial (panels N, O). We still don’t know who is making IDO1 at this stage. An orthogonal approach would to start OXA treatment and then treat with epacadostat and/or lindrostat in both WT and IDO1 KO models. This would give two important pieces of information: (i) any “remnant” effects could plausibly be attributed to TDO2, and (ii) adaptation of the IDO1 KO to the loss of IDO1 from development would be countered.\nFigure 3. I find the authors’ interpretation of the data shown in B and C difficult to follow. The authors conclusions are reported in lines 157-159 but are vague. Looking carefully at the data, the IDO1 KO and WT mice seem fundamentally different. Yet the authors focus on the “reversal” of the IDO1 “protective” effect (the red vs. orange symbols) but the WT ABX treated mice have the reverse phenotype. Thus. ABX treatment triggers a different outcome in the WT vs. KO mice, which is not properly explained. Bone marrow chimeras would probably help here as one set of variables can be control relative to the others.\nFigure 4. This figure contains descriptive microbiota-related data. As this reviewer understands it, the absence of IDO1 creates a permissive environment for an altered microbiota that is protective (and does not happen in the WT, see supra). If true, then administration of IDO1 inhibitors should do the same in the WT (but not the KO). The authors don’t explain how lower KP pathway metabolites control these effects or any molecular targets in host or microbiota. Overall, the section is confusing and incomplete. Other things can happen here – for example, KP pathway metabolites have been reported to control ferroptosis and stress signaling – this could occur in the intestinal epithelia (but less of in the IDO1 KO) and drive changes in microbiota.\nFigure 5. Loss of enterocytes 2, 3: this could be via an effect such as loss of NRF2/AHR signaling (see, key papers from Opitz, Murray, etc.). We still have no information on the IDO1+ cells in this figure or the source of IFNs. It is not clear in the scRNAseq how the experiment was done – is this steady-state or with OXA, ABX, etc. The conclusion drawn (lines 231-232) is vague.\nFigure 6. This line of investigation lacks a basis in logic given the issues raised above. Use of E. coli BL21 may be irrelevant. LysM-cre also deletes in neurophils and can cause mosaic deletion in many hematopoietic lineages. In my view, the use of the Cre delete here ( even through it has drawbacks) should have been used earlier. Lines 253-254 – “better therapeutic strategy” for what? Overall, this part of the paper was weak.", "reviewer_3": "Recent advances in cancer chemotherapy include the development of molecularly targeted agents and immune checkpoint inhibitors, which have raised expectations for reducing treatment-associated side effects. Nevertheless, traditional cytotoxic agents that non-selectively target proliferating cells continue to be used. These drugs often cause severe side effects, sometimes forcing treatment discontinuation, which remains a significant clinical issue. In this study, Xie and colleagues report that patients treated with oxaliplatin—a platinum-based chemotherapeutic agent—who experienced severe side effects exhibited elevated levels of L-kynurenine, a tryptophan metabolite. Their mouse model further demonstrated that oxaliplatin-induced intestinal toxicity was associated with increased L-kynurenine levels. Importantly, genetic ablation of IDO-1, an enzyme essential for L-kynurenine synthesis, mitigated this toxicity in mice, highlighting the key role of L-kynurenine in this process. Intriguingly, the attenuation of toxicity observed in Ido1-knockout mice was abolished by antibiotic treatment, suggesting a critical involvement of microbiota in mediating L-kynurenine's effects. The authors further identified Lactobacillus johnsonii as a key bacterial species whose reduction due to elevated L-kynurenine levels exacerbates oxaliplatin-induced intestinal toxicity in mice. These findings provide novel insights into the regulation of chemotherapy-associated side effects and represent a significant contribution to the field.\nHowever, there are several important unresolved issues.\nIt is particularly disappointing that the authors did not investigate why elevated L-kynurenine levels lead to the reduction of L. johnsonii, and by what mechanism L. johnsonii confers protection against oxaliplatin-induced intestinal toxicity.\nSpecific concerns:\n1. Previous research has already suggested that tryptophan metabolism is involved in responses to chemotherapy and that the gut microbiota contributes to gastrointestinal toxicity. Therefore, the finding that tryptophan metabolism modulates chemotherapy-induced intestinal toxicity via the microbiota is somewhat expected. Nonetheless, this study's identification of Lactobacillus johnsonii as a central microbial player is highly novel and noteworthy. Still, the most critical question—why elevated L-kynurenine reduces L. johnsonii, and how this bacterium mechanistically protects against toxicity—remains essentially unaddressed. The authors should explore these aspects in more depth.\n2. It would also be interesting to know whether Lactobacillus johnsonii is reduced in patients treated with oxaliplatin who have higher levels of L-kynurenine.\n3. Since L-kynurenine can also be synthesized via TDO (tryptophan 2,3-dioxygenase), its concentration in Ido1-knockout mice is unlikely to be zero.The authors should clarify the actual concentration of L-kynurenine in Ido1-KO mice. This would enable the identification of a threshold for sufficient L-kynurenine reduction to mitigate oxaliplatin-induced intestinal toxicity.\n4. In lines 151–153, the authors state“The improvement of the weight loss, clinical scores, colon length and spleen index in Ido1-knockout mice were disappeared in Ido1-knockout mice after treatment with antibiotics.”\nHowever, in Figure 3C, the clinical scores appear similar between Ido1-KO mice treated with or without antibiotics. Why?\nMoreover, antibiotic treatment in WT mice improves clinical scores (Figure 3C), suggesting the existence of specific bacteria that exacerbate toxicity. This interpretation is further supported by Figure 3B, where antibiotic-treated WT mice appear to recover body weight.\nThe authors should provide a clear explanation of these results.\n5. In Figure 3P, fecal transplantation from Ido1-KO mice leads to lower Ki67 expression than transplantation from WT mice. The authors should clarify why this phenomenon occurs."}, "author_response": "Dear Editor,\nThank you for the thorough and constructive feedback provided by the reviewers on our manuscript.\nWe greatly appreciate the reviewers' insights and in response we have undertaken considerable\nadditional work, which we believe has strengthened the manuscript. We enclose a point-by-point\nresponse to the reviewers' comments and have highlighted all changes to the manuscript in blue.\nReviewer #1\nChemotherapy induced gastrointestinal toxicity is a major limiting factor in cancer treatment and\nunderstanding the mechanisms associated with this adverse side-effect has merit. This manuscript\ndescribes the development of oxaliplatin-induced gastrointestinal toxicity, specifically the\nbreakdown of the gut epithelial barrier due to alteration in tryptophan metabolism. The authors show\nthat patients treated with oxaliplatin develop GI toxicity that can be stratified as high or low\ntoxicity that correlates with lower levels of white blood cells and neutrophils. The authors also note that\nthere is significant upregulation of L-kynurenine in patients demonstrating higher toxicity. They\nthen test the hypothesis that enhanced L-kynurenine is associated with GI toxicity by oxaliplatin in\nmice models. The gut toxicity is evaluated by body weight, clinical score, histology, spleen index,\nand expression of MUC2 and apoptosis across the various treatments.\nThe studies in mice have the following findings: 1) Oxaliplatin causes significant GI toxicity\ncorrelating with increase in kynurenine in serum, 2) deletion of the metabolizing enzyme (IDO)\nreduces oxaliplatin-induced GI toxicity, 3) depletion of gut microbiome improves GI toxicity due\nto oxaliplatin in WT, but worsens in IDO knock out mice (with respect to spleen index, colon length,\nseparation from muscularis mucosae and cell apoptosis; but not in clinical score: see below my\ncomments on this). This suggests that the protective effect of IDO1 KO is dependent on the gut\nmicrobiome. The authors have further conducted fecal transplant to show that WT mice receiving\nmicrobiome from IDO1 KO donors protect against oxaliplatin induce GI toxicity and it is L.\njohnsonii that protects against GI toxicity. They conclude that lack of kynurenine metabolism allows\nfor the increase in L. johnsonii. In a final series of experiments, the authors show that myeloid cells\nare the main site for L-kynurenine biosynthesis and that providing bacteria that degrade L-\nkynurenine protects against oxaliplatin – induced GI toxicity. There are several major issues with\nthis manuscript in its current form.\nResponse: We sincerely thank the reviewer for the insightful comments and constructive feedback\non our manuscript. We appreciate the time and effort dedicated to reviewing our work. In the\nfollowing comments, we have clarified any changes made to our manuscript to address specific\nreviewer questions.\n1) The experimental methodology is not provided in detail and thus it is difficult to evaluate the\nspecific experimental paradigms.\nThere is a discrepancy in the time frame of OXA treatment and kynurenine measurement in mice.\nIn Fig 1, the model is 3 OXA treatments over two weeks, while the rest of the studies are conducted\nwith 2 OXA injections over one week. Thus, it remains unclear if KYN measurements reflect\nchanges over the 2 weeks.\nResponse: The initial experiment employed a regimen of three intraperitoneal injections of\noxaliplatin (OXA; 20 mg/kg) administered once per week over two weeks. However, for subsequent\nexperiments, the dosing regimen was standardized to two injections of OXA (20 mg/kg)\nadministered once per week. This modification was implemented because administration of the\nthird dose significantly increased mortality rates in our model, likely due to the cumulative\nexacerbation of chemotherapy-induced toxicity. In follow-up experiments, we longitudinally\nquantified serum and fecal L-kynurenine levels in the two-dose model. These results (Figure R1)\ndemonstrate that L-kynurenine concentrations remain persistently elevated following the second\nOXA dose, thereby capturing the sustained biochemical changes relevant to the model's\npathophysiology.\nFigure R1. Serum levels of L-kynurenine of WT mice before and after oxaliplatin treatment.\nWhat is meant by clinical score? There is no description of the scoring system or any reference to\nprevious studies. What specific physical appearance and mobility parameters were determined?\nWhat is the spleen index? The histology images in figures S1 – S5 of the spleen are unclear. What\nis being shown with regard to the differences in spleen index.\nResponse: The clinical score was assessed using a validated composite grading system\n(supplementary Table 2), adapted from established models of chemotherapy-induced toxicity. This\nscoring system has been previously employed by our group in a similar manner to evaluate the\nseverity of chemotherapy-related adverse effects (PMID: 38400752). The spleen index was\ncalculated as: Spleen weight (mg) / Body weight (g). This metric quantitatively reflects splenic\nhypertrophy or atrophy, where increased values indicate immune activation and decreased values\nsuggest immunosuppression. Histological differences in spleen sections (Figs S1-S5) demonstrate:\nwhite pulp atrophy in severe toxicity (PMID: 33122357). High-resolution images have been re-\nuploaded.\nThere is no indication of how many males/females were used and no stratification based on sex.\nSome studies e.g. Figure 3 have only 4 mice (males or females?) and it is unclear how statistical\nanalysis were carried out with this small numbers.\nResponse: We thank the reviewer for highlighting these critical methodological issues. All studies\nused male C57BL/6 mice to control for sex-specific variables. Regarding sample sizes, we have\nnow expanded all cohorts to ensure robust conclusions. Revised figures present in Figure 6A-I.\nOne of the cardinal features of chemotherapy-induced gastrointestinal toxicity is the development\nof severe diarrhea. This has not been measured and neither are studies to show changes in intestinal\npermeability.\nResponse: We appreciate this insightful comment. While diarrhea and changes in intestinal\npermeability are recognized features of chemotherapy-induced gastrointestinal toxicity, our study\nfocused on a comprehensive evaluation of colonic length, intestinal edema and various intestinal\npathological changes, such as Ki-67 (proliferation), MUC2 (goblet cell function), and TUNEL\n(apoptosis). These established metrics provide robust evidence of intestinal damage and dysfunction,\noffering a detailed understanding of the structural and functional integrity of the gut barrier in\nresponse to chemotherapy. In future investigations, we will consider incorporating experimental\nassays of intestinal permeability (e.g., FITC-dextran assay) to more comprehensively evaluate\nchemotherapy-induced impairment of intestinal barrier function.\n2) There are some important controls missing from the overall study. IDO1 Knock out mice show\nimproved GI toxicity, however the levels of tryptophan or kynurenine are not determined.\nResponse: We thank the reviewer for this insightful suggestion. Our new data show that L-\nkynurenine levels in Ido1-/- mice are significantly lower than in controls, both before and after\noxaliplatin treatment (Figure S2B).\nHow effective is one week of ABX treatment in depleting gut bacteria. There is no difference in the\nclinical score between IDO1 K.O. and IDO KO + ABX while other parameters of GI toxicity are\nsignificantly altered. This needs to be addressed.\nResponse: We sincerely appreciate the reviewer's meticulous scrutiny. In response to the reviewer's\nquestion regarding the efficacy of our one-week antibiotic (ABX) regimen, we confirmed its\neffectiveness through bacterial culture of fecal samples collected from the mice after one week of\nreceiving antibiotic-supplemented water. The results demonstrated a marked reduction in viable\nbacterial colonies, confirming that the treatment was effective in depleting the gut microbiota\n(Figure R2). They are correct in identifying an error in the original Figure 3C where the group\nlabels for ' Ido1-/- ' and 'WT+ABX' were inadvertently swapped. This occurred during figure\nassembly due to misaligned data identifiers in our analysis spreadsheet. Repeated experiments with\nnew cohorts, and replaced all affected figures (Figure 6A-I).\nFigure R2. Representative images of fecal bacterial cultures.\nThe studies on the microbiome analysis are confusing. Are both donor and recipient mice treated\nwith OXA or just the recipient mice? If both groups are treated than a control of untreated\nmicrobiome is missing. This needs to be validated. Also Figure 4E and F are not clear. In 4 what is\nthe difference between WT and control (4E). If mice are treated with OXA in 4F, please explain\nwhy expression is higher in WT than control of 4E. Figure 4G needs better labeling. It is unclear\nwhich class is illustrated based on the color scheme.\nResponse: We apologize for any confusion in the microbiome study description. Key clarifications:\nDonor mice received NO OXA treatment, FMT donors were healthy untreated mice. Recipient mice\nunderwent OXA challenge post-FMT - as per Figure 6A workflow. Revised Figures 7E with\nexplicit labeling and unified color scheme.\nThank you for your insightful comment regarding the observed differences in Lactobacillus\nabundance between our two independent animal experiments. Figure 4E compares the fecal\nabundance of Lactobacillus in wild-type mice following treatment with or without oxaliplatin.\nFigure 4F compares the abundance of Lactobacillus in fecal samples from wild-type and Ido1-/- mice\nfollowing oxaliplatin treatment. We agree this point warrants careful consideration. The primary\nreason for such discrepancies often stems from batch effects during sample processing and analysis.\nSpecifically, variations can arise from the fecal DNA extraction process, where subtle differences\nin lysis efficiency, DNA yield, and purity may occur across different extraction batches. Similarly,\nqPCR detection can be subject to batch effects due to slight variations in reagent lots, instrument\ncalibration between different assay runs. These cumulative factors can significantly influence the\nfinal quantification of specific bacterial taxa. To minimize such batch effects and enhance the\ncomparability of the data, we have subsequently switched from an absolute quantification method\nbased on standard curves to a relative quantification approach using a reference gene for\nnormalization (Figure 7F-7H).\nIf OXA treatment is only given to recipient mice, then there appears to be no effect of OXA on the\nmicrobiome and the only differences are in the genotype.\nResponse: That's an interesting point. While genotype certainly plays a significant role in shaping\nthe gut microbiome, we've found that OXA treatment also has a demonstrable impact on the fecal\nmicrobiota in mice. Our previously published work provides direct evidence of OXA's influence on\nthe murine gut microbiome, even when considering genotypic variations. This indicates that OXA's\neffects on the microbiome are independent of, or additive to, any genotypic differences among the\nmice. Therefore, it's crucial to consider both genotype and OXA treatment as contributing factors to\nchanges observed in the gut microbiome of recipient mice.\n3) The conclusion of this study is unclear. It would help for the authors to provide a schematic\nillustration on the mechanism of how OXA alters GI toxicity.\nResponse: We agree that a clear illustration of the mechanism would be beneficial. As per your\nsuggestion, we've now added a schematic illustration detailing how oxaliplatin (OXA) is proposed\nto alter gastrointestinal (GI) toxicity in the revised manuscript (Figure 9). We believe this visual aid\nwill significantly enhance the understanding of our proposed mechanism.\nAn important control missing is whether L. johnsonii shifts with OXA treatment in WT mice,\nresulting in increased expression of kynurenine and if this is prevented in IDOfl/fl lyzCre mice.\nResponse: We have investigated the effects of OXA treatment on L. johnsonii of WT mice. Our data\nindicate that OXA treatment leads to a decrease in the abundance of L. johnsonii in the feces of WT\nmice (Figure 7F). We performed the suggested experiments in IDOfl/fl lyzCre mice treated with OXA.\nOur analysis of these mice showed that while serum kynurenine levels did not significantly increase\ncompared to control groups after OXA treatment (Figure 5I and 5J), the abundance of L. johnsonii\nin their feces significantly increased (Figure 7H).\nMinor issues\nThe manuscript is not well written and some conclusions are difficult to follow.\nThe color scheme of the figures need to be better presented so that significant differences can be\nobserved.\nResponse: We sincerely apologize if the manuscript's writing made some conclusions difficult to\nfollow. We understand the importance of clear and concise communication. We have thoroughly\nreviewed the entire manuscript and made revisions to enhance its readability and logical flow. We\nalso acknowledge your valuable comment on the color schemes of our figures and their impact on\ndistinguishing significant differences. We agree that visual clarity is paramount for data\ninterpretation. We have therefore reviewed our figures and adjusted the color palettes in areas where\nvisual differentiation was less clear, aiming to improve contrast and ensure that key differences are\nmore readily observable. We've focused on enhancing the distinctiveness of particularly critical data\npoints and groups. The manuscript has been restructured to ensure a more logical and rational flow.\nFig 2K: Kynurenine structure is incorrect\nResponse: Thank you for catching the error in the kynurenine structure. We apologize for this\noversight. We have corrected the kynurenine structure in the revised manuscript to ensure accuracy.\nFig 3R: Should conduct an outlier test for the FMT-Ido-/- MUC2 expression\nResponse: We appreciate your attention to detail in our statistical analysis. You are correct that our\ndata for MUC2 expression in the FMT-Ido1-/- group contains an outlier. We have conducted an\noutlier test, use Grubb's test to identify and remove the outliers from the data. To ensure the\nrobustness of our conclusions, we have now performed further calculations both with and without\nthis outlier. Importantly, our analysis consistently shows significant differences between the groups\nin both scenarios, whether the outlier is included or excluded. This demonstrates that our findings\nregarding MUC2 expression are robust and not driven by a single data point.\nFig 5H: Unclear of the IDO -/- vs IDO -/+\nResponse: In single-cell sequencing, we opted to use littermate heterozygous (+/−) mice as the\ncontrol group for the homozygous knockout mice. Our primary rationale for this approach was to\nminimize potential confounding variables arising from differences in genetic background, maternal\neffects, and early-life environmental exposures. By utilizing littermates from the same dam and\nhoused under identical conditions, we aimed to control for a multitude of external factors that can\nsignificantly influence complex biological phenotypes, including the microbiome. Crucially, we\nalso validated the protein expression of IDO1 in both heterozygous and homozygous knockout mice.\nOur Western blot results clearly show that heterozygous mice express IDO1 protein, whereas\nhomozygous mice show no IDO1 expression (Figure R3).\nFigure R3. Western blot analysis of IDO1 and β-Tubulin in samples from Mouse IDO -/- and Mouse IDO\n-/+.\nReviewer #2 (Remarks to the Author):\nXie et al. focus on mechanisms underlying chemotherapy-induced intestinal toxicity. They found\nchanges in tryptophan metabolites and traced the toxicity (in their models) to IDO1-mediated\nproduction on kynurenine pathway (KP) products. KP products then altered the microbiota, driving\nintestinal damage. Although the manuscript reports some important observations, it has several\nproblems of logic and lack of appreciation of alternative possibilities.\nResponse: Thank you very much for the careful review and valuable feedback on our manuscript.\nWe greatly appreciate you raising important points regarding the study's logic and the lack of\nappreciation for alternative possibilities. We agree that a deeper exploration of the mechanisms and\nlogical chain connecting our findings is essential. As such, in the detailed responses and revisions\nthat follow.\nSpecific comments:\nFigure 1. In the first part of the figure, changes (relative) to metabolites are shown but from the\nplasma. KP pathway metabolites are generally detected in any scenario where a strong IFN response\nhas happened or is ongoing – a good example is SARS-CoV-2 infection – as IFNs drive IDO1\nexpression. Thus, in this figure, the issues center on (i) the source of IFN and especially IFN-gamma\nin the patients and mouse model, (ii) the cells expressing IDO1, and most importantlxy, (iii) the\nconcentration of KP pathway metabolites in the gut relative to the blood.\nResponse: Thank you for your insightful comments regarding Figure 1 and the critical role of the\nL-Kynurenine Pathway (KP) in our study. You've raised important points concerning the source of\nIFN-γ, the cells expressing IDO1, and the compartmentalization of KP metabolites. We appreciate\nyou highlighting the relevance of IFN-γ in driving IDO1 expression, as seen in various inflammatory\ncontexts. To address your specific concerns: we have indeed investigated the IFN-γ response in our\nmouse model. Our data show that OXA treatment leads to an increase in IFN-γ concentration in\nboth the serum and fecal samples of mice (Figure 3A). This indicates a systemic immune activation.\nWe've performed flow cytometric analysis to identify the cellular sources of IFN-γ in our mouse\nmodel. Our results (Figure 3B) demonstrate that oxaliplatin treatment specifically increased IFN-γ\nproduction by CD8⁺ T cells, but not by other immune cells or intestinal epithelial cells. This helps\nto pinpoint the cellular contributors to the observed IFN-γ elevation. To further substantiate the\ncritical role of IFN-γ, we conducted in vivo neutralization experiments. Administration of anti-IFN-\nγantibody prior to oxaliplatin treatment exhibited a significant alleviation of both systemic side\neffects and intestinal damage (Figure 3C-K and Figure S2C). Concurrently, this depletion of IFN-\nγ led to a marked reduction in IDO1 expression (Figure S2D), directly confirming the IFNγ-\ndependent regulation of IDO1 in vivo. While a direct comparison of absolute L-kynurenine\nconcentrations between gut contents and blood is challenging due to differing biological matrices\nand units, our finding is that the relative changes in fecal L-kynurenine levels parallel those in the\nblood (Figure R4).\nFigure R4. L-kynurenine levels in feces from mice treated with oxaliplatin.\nFigure 2. Following from Figure 1, the authors tie IDO1 to the toxicity (e.g., panel M-Q) using a\nwhole animal IDO1 KO. However, these effects are partial (panels N, O). We still don’t know who\nis making IDO1 at this stage. An orthogonal approach would to start OXA treatment and then treat\nwith epacadostat and/or lindrostat in both WT and IDO1 KO models. This would give two important\npieces of information: (i) any “remnant” effects could plausibly be attributed to TDO2, and (ii)\nadaptation of the IDO1 KO to the loss of IDO1 from development would be countered.\nResponse: Thank you for your highly insightful comments regarding Figure 2, particularly\nconcerning the observed effects with whole-animal IDO1 KO and the crucial question of the cellular\nsource of IDO1. We also appreciate your valuable suggestion for an orthogonal approach using\ninhibitors. Regarding Partial Effects: We acknowledge that while IDO1 deletion significantly\nameliorates OXA-induced toxicity as shown, the effects are not a complete abrogation. This\nsuggests that while IDO1 plays a crucial role, other pathways or compensatory mechanisms may\nalso contribute to the overall toxicity. Your point about pinpointing the specific cellular source of\nIDO1 is critical. To address this, our study also utilizes the IDO1fl/fl lyzCre mouse model to\ninvestigate the role of IDO1 specifically in myeloid cells. As discussed in the manuscript and\nsupported by data in Figure 5A-I, these experiments allow us to dissect the contribution of myeloid-\nderived IDO1 to the observed toxicity. Our findings from this conditional knockout model indicate\nthat myeloid cells are indeed a significant source of IDO1 contributing to OXA-induced effects in\nour context.\nWe appreciate your suggestion to utilize inhibitors for a more comprehensive understanding. We\nagree that a pharmacological approach offers valuable mechanistic insights, and to further\nstrengthen our findings, we have now conducted experiments using an IDO1 inhibitor. The results\nfrom these experiments, presented in Figure 8A-I, these results demonstrate that pharmacological\ninhibition of IDO1 with Epacadostat replicates the protective effect seen with genetic ablation of\nIDO1. Regarding TDO2, our current data, particularly the observed undetectable expression of\nTDO2 in intestinal tissue (Figure R5), suggest that TDO2 may not play a primary role in the\nintestinal side effects we are investigating. We acknowledge that inhibitor studies could offer\nadditional insights into the developmental adaptation of constitutive IDO1 KOs, and we consider\nthis an excellent direction for future investigations.\nFigure R5. Western blot analysis of IDO1 and TDO2 in intestinal samples from wildtype mice.\nFigure 3. I find the authors’ interpretation of the data shown in B and C difficult to follow. The\nauthors conclusions are reported in lines 157-159 but are vague. Looking carefully at the data, the\nIDO1 KO and WT mice seem fundamentally different. Yet the authors focus on the “reversal” of\nthe IDO1 “protective” effect (the red vs. orange symbols) but the WT ABX treated mice have the\nreverse phenotype. Thus. ABX treatment triggers a different outcome in the WT vs. KO mice, which\nis not properly explained. Bone marrow chimeras would probably help here as one set of variables\ncan be control relative to the others.\nResponse: We sincerely appreciate the reviewer's meticulous scrutiny. They are correct in\nidentifying an error in the original Figure 3C where the group labels for ' Ido1-/- ' and 'WT+ABX'\nwere inadvertently swapped. This occurred during figure assembly due to misaligned data identifiers\nin our analysis spreadsheet. Repeated experiments with new cohorts, and replaced all affected\nfigures (Figure 6A-I). We believe this correction resolves the discrepancies you noted, including\nthe seemingly \"reverse phenotype\" in WT ABX-treated mice.\nFigure 4. This figure contains descriptive microbiota-related data. As this reviewer understands it,\nthe absence of IDO1 creates a permissive environment for an altered microbiota that is protective\n(and does not happen in the WT, see supra). If true, then administration of IDO1 inhibitors should\ndo the same in the WT (but not the KO). The authors don’t explain how lower KP pathway\nmetabolites control these effects or any molecular targets in host or microbiota. Overall, the section\nis confusing and incomplete. Other things can happen here – for example, KP pathway metabolites\nhave been reported to control ferroptosis and stress signaling – this could occur in the intestinal\nepithelia (but less of in the IDO1 KO) and drive changes in microbiota.\nResponse: Thank you for your comprehensive feedback on Figure 4. Regarding your insightful point\nabout IDO1 inhibitors, we agree. As detailed in our response to your comments on Figure 8A-I, we\nhave indeed conducted experiments using an IDO1 inhibitor in WT mice. As mentioned in the reply\nabove.\nYou highlighted the need for a clearer explanation of how lower KP pathway metabolites control\nthese effects and the underlying molecular targets. This is a critical mechanistic question. To address\nthis, we have now included data exploring the direct impact of L-Kynurenine on L. johnsonii growth.\nSpecifically, our in vitro experiments demonstrate that kynurenine can directly inhibit the growth of\nL. johnsonii (Figure S5E). Following transcriptome sequencing analysis, treatment with L-\nkynurenine led to a significant downregulation of several genes essential for the survival and\nproliferation of L. johnsonii. Specifically, the expression of AcpP (fatty acid synthesis), LepB and\nLspA (signal peptidases), DltB (cell wall modification), and RpoD (RNA polymerase) was\ndecreased (Figure S5J). These findings suggest that L-kynurenine may inhibit the growth of L.\njohnsonii, compromising its ability to colonize the gut.\nIn addition to KP pathway metabolites controlling ferroptosis and stress signaling in intestinal\nepithelia, their potential role in regulating epithelial cell apoptosis may also indirectly contribute to\nmicrobiota alterations. We agree that these are intriguing possibilities. Furthermore, we have now\ninvestigated the downstream mechanisms by which L-kynurenine-induced microbiota dysbiosis\nenhances intestinal side effects. Our findings suggest that this may occur through the activation of\nthe TNF-α/JNK pathway, which promotes epithelial cell apoptosis. Moreover, we found that\nsupplementation with L. johnsonii can inhibit this pathway-induced apoptosis and ameliorate\nchemotherapy-induced intestinal toxicity (Figure S6 A-D). This provides a more detailed molecular\nlink between the microbiota alterations caused by kynurenine and the host's inflammatory response,\ncontributing to the observed gastrointestinal toxicity.\nFigure 5. Loss of enterocytes 2, 3: this could be via an effect such as loss of NRF2/AHR signaling\n(see, key papers from Opitz, Murray, etc.). We still have no information on the IDO1+ cells in this\nfigure or the source of IFNs. It is not clear in the scRNAseq how the experiment was done – is this\nsteady-state or with OXA, ABX, etc. The conclusion drawn (lines 231-232) is vague.\nResponse: We understand your concern about the lack of specific information on IDO1+ cells and\nIFN sources within the context of Figure 5. To clarify these aspects: IDO1+ Cells: Our investigations\ninto the cellular source of IDO1, as discussed in our response to your comments on Figure 2,\nindicate that myeloid cells are a significant source of IDO1 in our model.\nSource of IFNs: As detailed in our response to your comments on Figure 1, we have assessed IFN-\nγ levels. Our data demonstrate an increase in IFN-γ in both serum and fecal samples following OXA\ntreatment (Figure 3A). This indicates a systemic immune activation. We've performed flow\ncytometric analysis to identify the cellular sources of IFN-γ in our mouse model. Our results (Figure\n3B) demonstrate that oxaliplatin treatment specifically increased IFN-γ production by CD8⁺ T cells,\nbut not by other immune cells or intestinal epithelial cells. This helps to pinpoint the cellular\ncontributors to the observed IFN-γ elevation.\nYou correctly point out that the scRNAseq experimental setup and the conclusions drawn were\nvague. We apologize for this lack of clarity. To address this: scRNAseq experimental design: we\nhave revised the methods section to explicitly state that the scRNAseq experiment was performed\non intestinal samples from untreated littermate Ido1-/- mice and Ido1+/- mice. This ensures it's clear\nthat these analyses represent a steady-state comparison between the genotypes without additional\ninterventions. We've also added more detail to the figure legend to precisely define the groups\nanalyzed in the scRNAseq.\nFigure 6. This line of investigation lacks a basis in logic given the issues raised above. Use of E.\ncoli BL21 may be irrelevant. LysM-cre also deletes in neurophils and can cause mosaic deletion in\nmany hematopoietic lineages. In my view, the use of the Cre delete here (even through it has\ndrawbacks) should have been used earlier. Lines 253-254 – “better therapeutic strategy” for what?\nOverall, this part of the paper was weak.\nResponse: We acknowledge the concerns about the logical flow of this section as it was previously\npresented. We agree that a clearer progression of our findings would enhance the paper's overall\ncoherence. To address this, we have significantly restructured the results section, adjusting the order\nin which data from different models (including the conditional knockout) are presented. We believe\nthis new logical sequence better builds the case for the role of IDO1 and the subsequent therapeutic\nimplications.\nYou raised a valid point regarding the relevance of BL21-KAT in our study. We would like to clarify\nthat our use of this engineered strain was carefully considered: it serves as a model to demonstrate\nhow a gut microbe can be designed to degrade L-kynurenine. This approach directly aligns with our\nproposed mechanism, in which elevated L-kynurenine contributes to gut toxicity (Figure 8J-8U).\nBy showing that a selectively modified bacterium can reduce L-kynurenine levels, we hope to\nprovide preliminary yet insightful evidence that the gut microbiota can be therapeutically targeted.\nWe believe these findings help build a preclinical foundation for potential future interventions—\nsuch as engineered probiotics—that may alleviate chemotherapy-induced side effects through\nmodulation of the kynurenine pathway. Furthermore, we have also conducted related animal\nexperiments using the IDO1 inhibitor Epacadostat. Collectively, these results demonstrate that\npharmacological inhibition of IDO1 replicates the protective effect observed with genetic ablation\nof IDO1 (Figure 8A-8I), underscoring the translational potential of targeting this pathway for\nclinical applications.\nWe appreciate your comment regarding the Lyz-Cre model. As Lyz-Cre drives recombination in\nmyeloid cell lineages, which inherently include neutrophils and other hematopoietic cells, its use\nallows us to specifically target IDO1 in these key immune populations. We believe that by\ncombining data from this myeloid-specific conditional knockout with our whole-animal IDO1\nknockout data and pharmacological inhibition studies, we provide a robust and comprehensive\npicture of IDO1's role in the context of relevant immune cells.\nReviewer #3 (Remarks to the Author):\nRecent advances in cancer chemotherapy include the development of molecularly targeted agents\nand immune checkpoint inhibitors, which have raised expectations for reducing treatment-\nassociated side effects. Nevertheless, traditional cytotoxic agents that non-selectively target\nproliferating cells continue to be used. These drugs often cause severe side effects, sometimes\nforcing treatment discontinuation, which remains a significant clinical issue. In this study, Xie and\ncolleagues report that patients treated with oxaliplatin—a platinum-based chemotherapeutic\nagent—who experienced severe side effects exhibited elevated levels of L-kynurenine, a tryptophan\nmetabolite. Their mouse model further demonstrated that oxaliplatin-induced intestinal toxicity was\nassociated with increased L-kynurenine levels. Importantly, genetic ablation of IDO-1, an enzyme\nessential for L-kynurenine synthesis, mitigated this toxicity in mice, highlighting the key role of L-\nkynurenine in this process. Intriguingly, the attenuation of toxicity observed in Ido1-knockout mice\nwas abolished by antibiotic treatment, suggesting a critical involvement of microbiota in mediating\nL-kynurenine's effects. The authors further identified Lactobacillus johnsonii as a key bacterial\nspecies whose reduction due to elevated L-kynurenine levels exacerbates oxaliplatin-induced\nintestinal toxicity in mice. These findings provide novel insights into the regulation of\nchemotherapy-associated side effects and represent a significant contribution to the field.\nHowever, there are several important unresolved issues.\nIt is particularly disappointing that the authors did not investigate why elevated L-kynurenine levels\nlead to the reduction of L. johnsonii, and by what mechanism L. johnsonii confers protection against\noxaliplatin-induced intestinal toxicity.\nResponse: We thank the reviewer for their insightful comments and constructive feedback. We\nacknowledge that the raised questions are critical for a deeper understanding of our findings. In the\nfollowing sections, we will address each of these points in detail.\nSpecific concerns:\n1. Previous research has already suggested that tryptophan metabolism is involved in responses to\nchemotherapy and that the gut microbiota contributes to gastrointestinal toxicity. Therefore, the\nfinding that tryptophan metabolism modulates chemotherapy-induced intestinal toxicity via the\nmicrobiota is somewhat expected. Nonetheless, this study's identification of Lactobacillus johnsonii\nas a central microbial player is highly novel and noteworthy. Still, the most critical question—why\nelevated L-kynurenine reduces L. johnsonii, and how this bacterium mechanistically protects\nagainst toxicity—remains essentially unaddressed. The authors should explore these aspects in more\ndepth.\nResponse: Thank you for your constructive feedback and for acknowledging the novelty of\nidentifying Lactobacillus johnsonii as a central microbial player in our study. We agree that while\nthe general involvement of tryptophan metabolism and the gut microbiota in chemotherapy-induced\ngastrointestinal toxicity is recognized, elucidating the specific mechanistic links is crucial for\nadvancing the field. To this end, we have conducted new experiments and significantly expanded\nour mechanistic investigation in the revised manuscript. Specifically, our in vitro experiments\ndemonstrate that kynurenine can directly inhibit the growth of L. johnsonii (Figure S5E). Following\ntranscriptome sequencing analysis, treatment with L-kynurenine led to a significant downregulation\nof several genes essential for the survival and proliferation of L. johnsonii. Specifically, the\nexpression of AcpP (fatty acid synthesis), LepB and LspA (signal peptidases), DltB (cell wall\nmodification), and RpoD (RNA polymerase) was decreased (Figure S5J). This provides a direct\nmechanistic explanation, suggesting that the elevated L-kynurenine levels, resulting from increased\nhost IDO1 activity, create an unfavorable gut environment that suppresses L. johnsonii proliferation.\nMechanism of L. johnsonii's protective action: We have further investigated the downstream\nconsequences of kynurenine-induced dysbiosis, particularly focusing on the reduction of L.\njohnsonii. Our findings suggest that this may occur through the activation of the TNF-α/JNK\npathway, which promotes epithelial cell apoptosis. Moreover, we found that supplementation with\nL. johnsonii can inhibit this pathway-induced apoptosis and ameliorate chemotherapy-induced\nintestinal toxicity (Figure S6 A-D). This suggests that L. johnsonii (or the balanced microbiota it\ncontributes to) plays a role in counteracting this specific apoptosis signaling pathway, thereby\nconferring protection against intestinal toxicity.\n2. It would also be interesting to know whether Lactobacillus johnsonii is reduced in patients treated\nwith oxaliplatin who have higher levels of L-kynurenine.\nResponse: Thank you for your excellent question regarding whether elevated L-kynurenine levels\ncorrelate with reduced L. johnsonii in oxaliplatin-treated patients. This is indeed a crucial point for\nclinical translation. To address your query, we have now supplemented our study with data from a\nnew clinical cohort, comprising 10 patients with low toxicity and 18 with high toxicity. We observed\nthat serum L-kynurenine levels were significantly higher, and fecal L. johnsonii abundance was\nsignificantly lower, in the high-toxicity group compared to the low-toxicity group (Figure 7I). This\nclinical finding strongly corroborates the mechanistic insights observed in our mouse models,\nfurther supporting the inverse relationship between L-kynurenine and L. johnsonii. It also highlights\nthe potential clinical relevance of this mechanism in chemotherapy-associated gastrointestinal\ntoxicity.\n3. Since L-kynurenine can also be synthesized via TDO (tryptophan 2,3-dioxygenase), its\nconcentration in Ido1-knockout mice is unlikely to be zero. The authors should clarify the actual\nconcentration of L-kynurenine in Ido1-KO mice. This would enable the identification of a threshold\nfor sufficient L-kynurenine reduction to mitigate oxaliplatin-induced intestinal toxicity.\nResponse: To address this, we have quantified the L-kynurenine levels in the serum samples of our\nIdo1-/- mice (Figure S2B). Our data show that while L-kynurenine levels are indeed significantly\nreduced in Ido1-/- mice compared to wild-type controls, they are not completely absent, confirming\nyour point about TDO.\n4. In lines 151–153, the authors state“The improvement of the weight loss, clinical scores, colon\nlength and spleen index in Ido1-knockout mice were disappeared in Ido1-knockout mice after\ntreatment with antibiotics.”\nHowever, in Figure 3C, the clinical scores appear similar between Ido1-KO mice treated with or\nwithout antibiotics. Why?\nMoreover, antibiotic treatment in WT mice improves clinical scores (Figure 3C), suggesting the\nexistence of specific bacteria that exacerbate toxicity. This interpretation is further supported by\nFigure 3B, where antibiotic-treated WT mice appear to recover body weight.\nThe authors should provide a clear explanation of these results.\nResponse: We sincerely appreciate the reviewer's meticulous scrutiny. They are correct in\nidentifying an error in the original Figure 3C where the group labels for ' Ido1-/- ' and 'WT+ABX'\nwere inadvertently swapped. This occurred during figure assembly due to misaligned data identifiers\nin our analysis spreadsheet. Repeated experiments with new cohorts, and replaced all affected\nfigures (Figure 6A-I). We are confident that the updated figures will now provide a clear and\nconsistent representation of our results, resolving the apparent contradictions and allowing for a\nstraightforward interpretation of how antibiotic treatment impacts Ido1-/- and WT mice in the context\nof oxaliplatin-induced toxicity. We believe this correction is crucial for the clarity and validity of\nour conclusions.\n5. In Figure 3P, fecal transplantation from Ido1-KO mice leads to lower Ki67 expression than\ntransplantation from WT mice. The authors should clarify why this phenomenon occurs.\nResponse: We thank the reviewer for identifying the error in the original Figure 3P (now Figure 6P\nin the revised manuscript) regarding Ki67 expression. The group labels were inadvertently swapped,\ncreating a misleading impression. We have corrected this in the revised manuscript and apologize\nfor the oversight."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded to most of my concerns. I still feel that the manuscript needs further improvement in the writing as there are several grammatical errors.", "reviewer_2": "The authors have done quite a bit of work for this manuscript and a number of improvements were made. Amazingly, despite being told the chemical structure of Kyn was wrong, the authors have how shown (Fig. 2K) that Kyn is shown as Top and the upper \"Top\" structure - which should be Kyn, is ALSO wrong (the amine group of the aryl ring is in the wrong place). Structures of Tryptophan and Kynurenine are available on the internet.", "reviewer_3": "The authors have provided extensive new data, corrected previous errors, and articulated a coherent mechanistic framework linking IDO1-dependent kynurenine production, microbiota dysbiosis, and intestinal toxicity. In my view, all major concerns that I raised in the initial review have been satisfactorily addressed, and the revised manuscript now represents a solid and well-substantiated contribution to the field. I therefore consider this manuscript suitable for publication in Nature Communications."}, "author_response": "POINT-BY-POINT REPLIES TO THE REVIEWER COMMENTS\nDear Editor,\nThank you very much for your email and for the good news regarding our manuscript. We are\ndelighted to learn that the journal is happy, in principle, to publish our work under an open access\nlicense. We are pleased to accept this offer.\nThank you also for the thorough and constructive feedback provided by the reviewers. We enclose\na point-by-point response to the reviewers' comments and have highlighted all changes to the\nmanuscript in blue. We hope that these revisions satisfactorily address all concerns raised.\nReviewer #1\nThe authors have responded to most of my concerns. I still feel that the manuscript needs further\nimprovement in the writing as there are several grammatical errors.\nResponse: We thank the reviewer for this final important comment regarding the writing quality\nand remaining grammatical errors. We sincerely apologize for this oversight in the previous revision.\nWe agree that linguistic clarity is paramount for the manuscript's readability.\nTo address this concern comprehensively, we have commissioned a premium professional English\nlanguage editing service to thoroughly review and polish the entire manuscript. This service was\nprovided by an editing company affiliated with Nature Research, specializing in scientific and\ntechnical documentation.\nReviewer #2:\nThe authors have done quite a bit of work for this manuscript and a number of improvements were\nmade. Amazingly, despite being told the chemical structure of Kyn was wrong, the authors have\nhow shown (Fig. 2K) that Kyn is shown as Top and the upper \"Top\" structure - which should be\nKyn, is ALSO wrong (the amine group of the aryl ring is in the wrong place). Structures of\nTryptophan and Kynurenine are available on the internet.\nResponse: We sincerely thank the reviewer for their continued vigilance regarding the chemical\nstructures. We are deeply sorry that the structural error for Kynurenine (Kyn) persisted in Figure 2K\ndespite previous feedback, specifically concerning the incorrect placement of the amine group (-\nNH ) on the aryl ring. We agree this is a critical error. To prevent any recurrence of such errors, all\n2\nchemical structures throughout the figures have been redrawn using the Nature Chemistry template\nsettings. This adherence to a professional, high-precision standard ensures that all compounds,\nincluding Kynurenine and Tryptophan, are chemically accurate and correctly represented. We thank\nthe reviewer once again for identifying this persistent error and helping us maintain the chemical\nintegrity of the manuscript.\nReviewer #3:\nThe authors have provided extensive new data, corrected previous errors, and articulated a coherent\nmechanistic framework linking IDO1-dependent kynurenine production, microbiota dysbiosis, and\nintestinal toxicity. In my view, all major concerns that I raised in the initial review have been\nsatisfactorily addressed, and the revised manuscript now represents a solid and well-substantiated\ncontribution to the field. I therefore consider this manuscript suitable for publication in Nature\nCommunications.\nResponse: We are exceptionally grateful for this final, highly positive assessment of our revised\nmanuscript. We deeply appreciate the time and effort the reviewer dedicated to providing such\nthorough and constructive feedback throughout the entire review process."}]

Decision letter