# TAK1 Deficiency in Macrophages Increases Host Susceptibility to Leishmania Infection

Xiankai Cao, Kossiwa Kokou, Shi Yu, Mengdan Chen, Junling Niu, Hervé

Lecoeur, Eric Prina, Gerald Späth, Guangxun Meng

► To cite this version:

Xiankai Cao, Kossiwa Kokou, Shi Yu, Mengdan Chen, Junling Niu, et al. TAK1 Deficiency in Macrophages Increases Host Susceptibility to Leishmania Infection. Infectious Microbes and Diseases, 2023, 5 (3), pp.127-136. 10.1097/IM9.0000000000000127. pasteur-04608098

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# TAK1 Deficiency in Macrophages Increases Host Susceptibility to *Leishmania* Infection

Xiankai Cao¹#, Kossiwa C. Kokou¹#, Shi Yu¹, Mengdan Chen¹, Junling Niu¹, Hervé Lecoeur², Eric Prina², Gerald F. Späth², Guangxun Meng¹,³,⁴,⁵

**Abstract**

*Leishmania* parasites mainly infect macrophages and may cause severe immunopathologies in their host, which are called leishmaniases. In the current work, we infected human and mouse macrophages in vitro with *Leishmania major*, an etiological agent of cutaneous leishmaniasis, and found that inhibition or deletion of the transforming growth factor β-activated kinase 1 (TAK1) gene resulted in increased parasite loads. In vivo, following a challenge with *L. major*, mice with a macrophage-specific deletion of TAK1 showed increased clinical signs and higher parasite loads compared with wild-type controls. TAK1 deficiency in mouse macrophages led to biased Th2 cell responses during the acute stage of infection, characterized by a decrease in interferon-γ expression, and increased expression of IL-4, IL-5 and IL-10. Finally, we found that, in the late stage of *L. major* infection, excessive Th2-related cytokines led to high arginase 1 expression in mouse tissues and a significant reduction of NO production both locally and systemically, resulting in compromised control of *Leishmania*. These findings suggest that TAK1 plays a vital role in host resistance to *Leishmania* infection.

**Keywords:** TAK1; macrophage; *Leishmania major*; arginase 1; nitric oxide

Editor: Stijn van der Veen

Author affiliations: ¹The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology & Immunology, University of Chinese Academy of Sciences, Shanghai, China, ²Institute Pasteur, Paris City University, INSERM U1201, Molecular Parasitology and Signalling Unit, Paris, France, ³Pasteurien College, Soochow University, Suzhou, China, ⁴Nanjing Advanced Academy of Life and Health, Nanjing, China.

⁵Corresponding author: Guangxun Meng, The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology & Immunology, Chinese Academy of Sciences, 320 Yueyang Road, Life Science Research Building, Shanghai 200031, China. E-mail: gxmeng@jips.ac.cn

⁶XC and KCK contributed equally to this work.

Author contributions: GM conceived the project; XC and KCK conducted most of the experiments; SY, MC, JN, HL and EP helped with experiments; GM and GFS recruited grants; XC, KCK, GFS and GM analyzed the data and wrote the manuscript; GM supervised the study; all authors read and approved the final manuscript.

Ethics: All animal experiments were performed in compliance with the Regulations for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China, which enforces the ethical use of animals. The protocol was approved by the Institutional Animal Care and Use Committee at the Institute Pasteur of Shanghai, Chinese Academy of Sciences (permit number: P2021009; approved on April 29, 2021).

Data availability: Data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data sets can be obtained from the corresponding author on reasonable request.

Funding: This study was supported by grants from the Institute Pasteur International Direction, International Partnership Program (153831KYSB20190008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29030303), the National Key Basic Research Program (2018YFA0507300, 2022YFC2304700, 2022YFC2303200), the National Natural Science Foundation of China (81830049, 92269202), the Shanghai Municipal Science and Technology Major Project (#2019SHZDZX02), the Research Leader Program (#20XD1403900), and the Innovation Capacity Building Project of Jiangsu province (BM2020019). The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Conflicts of interest: The authors reported no conflicts of interest.

Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.
This is an open access article distributed under the Creative Commons Attribution
License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Infectious Microbes & Diseases (2023) 5:3

Received: 20 April 2023/Accepted: 28 June 2023/First published online: 25 July 2023

http://dx.doi.org/10.1097/IM9.0000000000000127

**Introduction**

Leishmaniases are immune pathologies caused by *Leishmania* parasites, which are transmitted between mammals by blood feeding of sandflies. There are different clinical forms of leishmaniases: cutaneous, mucocutaneous and visceral. Cutaneous leishmaniasis is the most common form of these pathologies, with an estimated one million new cases worldwide each year.¹

Host control of *Leishmania* infections involves reactive oxygen species production from monocytes, which can mediate respiratory oxygen bursts in both human and mouse cells to inhibit parasites.² In addition, cytokines such as IFN-γ and TNF-α can promote macrophage activation by inducing inducible nitric oxide synthase (iNOS) to eliminate *Leishmania*.³,⁴ In contrast, cytokines, including IL-4 and IL-13, produced by Th2 cells can activate alternative phagocytes, leading to pathogen amplification and chronic infections.⁵,⁶ Even in a Th1-dominated immune environment, IL-4-dependent tissue macrophages can facilitate *Leishmania major* expansion, leading to skin damage.⁷

From the pathogen side, *Leishmania* can inhibit NF-κB-mediated host inflammatory responses via targeting critical signaling molecules, including transforming growth factor β-activated kinase 1 (TAK1).⁸ TAK1 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) family that regulates cell homeostasis and inflammatory signaling.⁹ In IL-1β-, TNF-α- and Toll-like receptor (TLR)-mediated innate immune signaling, TAK1 binds to transforming growth factor β-activated kinase 1-binding protein 1 (TAB1), TAB2 and TAB3 proteins, then activates downstream NF-κB and MAPK signaling.¹⁰ The TAK1 function has been studied in different cells. In B cells, TAK1 is essential for TLR-mediated NF-κB and MAPK signaling and B-cell antigen receptor-induced c-Jun N-terminal kinase activation.¹¹ In T cells, the absence of TAK1 results in defects in T-cell maturation, leading to reduced T-cell numbers in peripheral tissues, and in lack of NF-κB and c-Jun N-terminal kinase activation in T-cell receptor-dependent stimulation of thymocytes.¹²,¹³ In addition, lack of
# TAK1 Deficiency in Macrophages Increases Host Susceptibility to *Leishmania* Infection

Xiankai Cao¹#, Kossiwa C. Kokou¹#, Shi Yu¹, Mengdan Chen¹, Junling Niu¹, Hervé Lecoeur², Eric Prina², Gerald F. Späth², Guangxun Meng¹,³,⁴,⁵

**Abstract**

*Leishmania* parasites mainly infect macrophages and may cause severe immunopathologies in their host, which are called leishmaniases. In the current work, we infected human and mouse macrophages in vitro with *Leishmania major*, an etiological agent of cutaneous leishmaniasis, and found that inhibition or deletion of the transforming growth factor β-activated kinase 1 (TAK1) gene resulted in increased parasite loads. In vivo, following a challenge with *L. major*, mice with a macrophage-specific deletion of TAK1 showed increased clinical signs and higher parasite loads compared with wild-type controls. TAK1 deficiency in mouse macrophages led to biased Th2 cell responses during the acute stage of infection, characterized by a decrease in interferon-γ expression, and increased expression of IL-4, IL-5 and IL-10. Finally, we found that, in the late stage of *L. major* infection, excessive Th2-related cytokines led to high arginase 1 expression in mouse tissues and a significant reduction of NO production both locally and systemically, resulting in compromised control of *Leishmania*. These findings suggest that TAK1 plays a vital role in host resistance to *Leishmania* infection.

**Keywords:** TAK1; macrophage; *Leishmania major*; arginase 1; nitric oxide

Editor: Stijn van der Veen

Author affiliations: ¹The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology & Immunology, University of Chinese Academy of Sciences, Shanghai, China, ²Institute Pasteur, Paris City University, INSERM U1201, Molecular Parasitology and Signalling Unit, Paris, France, ³Pasteurien College, Soochow University, Suzhou, China, ⁴Nanjing Advanced Academy of Life and Health, Nanjing, China.

⁵Corresponding author: Guangxun Meng, The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology & Immunology, Chinese Academy of Sciences, 320 Yueyang Road, Life Science Research Building, Shanghai 200031, China. E-mail: gxmeng@jips.ac.cn

⁶XC and KCK contributed equally to this work.

Author contributions: GM conceived the project; XC and KCK conducted most of the experiments; SY, MC, JN, HL and EP helped with experiments; GM and GFS recruited grants; XC, KCK, GFS and GM analyzed the data and wrote the manuscript; GM supervised the study; all authors read and approved the final manuscript.

Ethics: All animal experiments were performed in compliance with the Regulations for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China, which enforces the ethical use of animals. The protocol was approved by the Institutional Animal Care and Use Committee at the Institute Pasteur of Shanghai, Chinese Academy of Sciences (permit number: P2021009; approved on April 29, 2021).

Data availability: Data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data sets can be obtained from the corresponding author on reasonable request.

Funding: This study was supported by grants from the Institute Pasteur International Direction, International Partnership Program (153831KYSB20190008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29030303), the National Key Basic Research Program (2018YFA0507300, 2022YFC2304700, 2022YFC2303200), the National Natural Science Foundation of China (81830049, 92269202), the Shanghai Municipal Science and Technology Major Project (#2019SHZDZX02), the Research Leader Program (#20XD1403900), and the Innovation Capacity Building Project of Jiangsu province (BM2020019). The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Conflicts of interest: The authors reported no conflicts of interest.

Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Infectious Microbes & Diseases (2023) 5:3

Received: 20 April 2023/Accepted: 28 June 2023/First published online: 25 July 2023

http://dx.doi.org/10.1097/IM9.0000000000000127

**Introduction**

Leishmaniases are immune pathologies caused by *Leishmania* parasites, which are transmitted between mammals by blood feeding of sandflies. There are different clinical forms of leishmaniases: cutaneous, mucocutaneous and visceral. Cutaneous leishmaniasis is the most common form of these pathologies, with an estimated one million new cases worldwide each year.¹

Host control of *Leishmania* infections involves reactive oxygen species production from monocytes, which can mediate respiratory oxygen bursts in both human and mouse cells to inhibit parasites.² In addition, cytokines such as IFN-γ and TNF-α can promote macrophage activation by inducing inducible nitric oxide synthase (iNOS) to eliminate *Leishmania*.³,⁴ In contrast, cytokines, including IL-4 and IL-13, produced by Th2 cells can activate alternative phagocytes, leading to pathogen amplification and chronic infections.⁵,⁶ Even in a Th1-dominated immune environment, IL-4-dependent tissue macrophages can facilitate *Leishmania major* expansion, leading to skin damage.⁷

From the pathogen side, *Leishmania* can inhibit NF-κB-mediated host inflammatory responses via targeting critical signaling molecules, including transforming growth factor β-activated kinase 1 (TAK1).⁸ TAK1 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) family that regulates cell homeostasis and inflammatory signaling.⁹ In IL-1β-, TNF-α- and Toll-like receptor (TLR)-mediated innate immune signaling, TAK1 binds to transforming growth factor β-activated kinase 1-binding protein 1 (TAB1), TAB2 and TAB3 proteins, then activates downstream NF-κB and MAPK signaling.¹⁰ The TAK1 function has been studied in different cells. In B cells, TAK1 is essential for TLR-mediated NF-κB and MAPK signaling and B-cell antigen receptor-induced c-Jun N-terminal kinase activation.¹¹ In T cells, the absence of TAK1 results in defects in T-cell maturation, leading to reduced T-cell numbers in peripheral tissues, and in lack of NF-κB and c-Jun N-terminal kinase activation in T-cell receptor-dependent stimulation of thymocytes.¹²,¹³ In addition, lack of
TAK1 in keratinocytes and intestinal epithelial cells increases TNF-α-induced cell death.<sup>14,15</sup>

Of note, deficiency of TAK1 in macrophages causes activation of the nucleotide-binding and oligomerization domain–, leucine-rich repeat– and pyrin domain–containing protein 3 (NLRP3) inflammasome and cleavage of caspase-1 and gasdermin D, leading to cell death.<sup>16,17</sup> Moreover, a study has shown that inhibition of TAK1 in macrophages induces an increase in mitochondrial reactive oxygen species, which contributes to the abolishing of intracellular *Salmonella* colonization.<sup>18</sup> However, the potential function of TAK1 in *Leishmania* infection remains unclear thus far. In this study, we found that macrophage-specific deletion of TAK1 led to the inability of mice to mount an effective Th1 response, while the resultant high expression of IL-4 and arginase 1 (Arg1) led to a decrease in NO generation, thereby increasing the susceptibility of the host to *Leishmania* infection. Collectively, our results show that TAK1 is a key factor for host control of cutaneous leishmaniasis.

## Results

### TAK1 is essential to control *Leishmania* infection in macrophages

Macrophages are the main target cells for *Leishmania* infection. Therefore, we first investigated the role of TAK1 during *L. major* infection in cultured macrophages. To this end, cells of the human monocytic cell line THP-1 were differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) treatment and infected with *L. major* amastigotes for 48 hours in the absence or presence of 5Z-7-Oxozeaenol, a specific inhibitor of TAK1. After incubation, the small subunit ribosomal RNA (ssrRNA) of *L. major* was detected through both relative and absolute quantifications by real-time PCR method (Figures 1A and B). Results here showed that the parasite loads in cells were significantly increased after TAK1 inhibition. The cells infected for 48 hours were transferred to conditions suitable for promastigote differentiation and multiplication (rescue assay) in vitro. The results showed that THP-1 cells with TAK1 inhibition released significantly more parasites than the control cells (Figure 1C).

Next, we constructed a TAK1-specific knockout THP-1 cell line for *Leishmania* infection experiments (Figures S1A and B). Using the aforementioned methods, we found that the parasite loads in cells were significantly increased upon TAK1 deficiency (Figures 1D–F). Similar experiments were also conducted in mouse bone marrow–derived macrophages (BMDMs). Again, through both relative and absolute quantifications, we found that TAK1 inhibition resulted in elevated parasite loads in BMDMs (Figures 1G and H). As expected, when *L. major* promastigotes were cultured in vitro by transferring BMDMs infected for 48 hours to *Leishmania* culture conditions in the rescue assay, BMDMs with impaired TAK1 released more parasites than the control cells (Figure 1I). These results thus demonstrate that TAK1 is implicated in controlling *Leishmania* infection in both human and mouse macrophages.

### TAK1 in macrophages is essential for parasite clearance in vivo

To investigate the role of TAK1 in vivo, we inoculated $1 \times 10^5 L. major$ metacyclic promastigotes in footpads of macrophage-specific, conditional TAK1 (Map3k7 gene)-deficient mice (TAK1fl/fl; LysM-cre; Figure S1C). We confirmed that TAK1 deficiency was mainly in macrophages and there was no clear knockout of TAK1 in neutrophils (Figures S1C and D). Lesion development, quantifica-

tion of parasite loads and the presence of clinical signs were monitored up to 42 days after infection. Although a similar onset for the increase of footpad thickness was observed between TAK1fl/fl; LysM-cre and TAK1fl/fl mice, the TAK1-deficient mice showed faster lesion development and a larger lesion size (Figures 2A and B). In addition, the TAK1-deficient mice showed an earlier and more severe development of clinical signs, reaching a class 2 level by day 21, whereas no TAK1fl/fl mice exceeded a class 1 level (Figure 2B). Analysis of the lesion volume and parasite quantification carried out at day 42 clearly supported an increase in lesion size and parasite loads in TAK1-deficient mice (Figures 2C and D). Direct observation displayed enlarged lymph nodes in TAK1fl/fl; LysM-cre mice, and the feet of TAK1fl/fl; LysM-cre mice were swollen and reddish, and the mass change was palpable. This was confirmed by H&E staining of footpad sections that revealed infiltrates around the phalange ring, as well as in the connective tissue (Figure 2E). Therefore, these results suggest that TAK1 in macrophages is essential for the host clearance of *L. major* parasites.

### Macrophage-specific deletion of TAK1 impairs host resistance to *L. major* challenge due to compromised Th1 cell response

Th1 cell-mediated immune responses are important to control *L. major* infections.<sup>19</sup> We investigated Th1-related cytokine levels in TAK1fl/fl; LysM-cre and their littermate control mice at different time points after *L. major* infection. We found that there was no significant difference in IFN-γ and IL-1β transcript levels between the two groups of mice on day 14 after infection, with both cytokines expressed at a low basal level (Figure 3A). Notably, on 28 days after infection, the TAK1fl/fl; LysM-cre mice were unable to produce effective IFN-γ to control *Leishmania*, and the transcript levels of IL-12p40 were also lower compared with TAK1fl/fl mice, although transcript levels of other inflammatory cytokines, such as IL-6, IL-1β and TNF-α, did not show differences between these two groups (Figure 3B). When we evaluated the transcript levels of cytokines in mice infected for 42 days, we found that the levels of IFN-γ were still significantly lower in TAK1fl/fl; LysM-cre mice compared with control mice, and the same was true for IL-6. Furthermore, levels of IL-12, IL-1β and TNF-α were lower in TAK1fl/fl; LysM-cre mice (Figure 3C). These data indicated that T cells in TAK1fl/fl; LysM-cre mice showed a defect in differentiation to Th1 cells to resist *Leishmania* infection, thus leading to more pronounced clinical signs. In addition, the analysis of footpads also revealed that there was no significant difference in IFN-γ and IL-1β transcript levels on day 14 after infection, which were at extremely low levels (Figure S2A). Consistently, the levels of IFN-γ and IL-12 were lower in TAK1fl/fl; LysM-cre mice compared with their littermate control on day 28 after infection (Figure S2B). However, there was no significant difference in the levels of Th1-related proinflammatory factors such as IFN-γ, IL-6, IL-1β and TNF-α, between the two groups of mice on the 42nd day of infection (Figure S2C). This may be due to the fact that the disease began to resolve at day 42 (Figures 2A and B),<sup>20</sup> so the level of inflammation was reduced.

Next, we investigated the proportions of Th1 cells in lymph nodes between these two groups of mice. As expected, the percentages of IFN-γ<sup>+</sup> cells were lower in TAK1fl/fl; LysM-cre mice on days 28 and 42 after infection, whereas the percentages of CD4<sup>+</sup> cells were similar (Figures 3D–G). These data suggest that TAK1 deficiency in macrophages leads to the inability of mice to develop
TAK1 in keratinocytes and intestinal epithelial cells increases TNF-α-induced cell death.<sup>14,15</sup>

Of note, deficiency of TAK1 in macrophages causes activation of the nucleotide-binding and oligomerization domain–, leucine-rich repeat– and pyrin domain–containing protein 3 (NLRP3) inflammasome and cleavage of caspase-1 and gasdermin D, leading to cell death.<sup>16,17</sup> Moreover, a study has shown that inhibition of TAK1 in macrophages induces an increase in mitochondrial reactive oxygen species, which contributes to the abolishing of intracellular *Salmonella* colonization.<sup>18</sup> However, the potential function of TAK1 in *Leishmania* infection remains unclear thus far. In this study, we found that macrophage-specific deletion of TAK1 led to the inability of mice to mount an effective Th1 response, while the resultant high expression of IL-4 and arginase 1 (Arg1) led to a decrease in NO generation, thereby increasing the susceptibility of the host to *Leishmania* infection. Collectively, our results show that TAK1 is a key factor for host control of cutaneous leishmaniasis.

## Results

### TAK1 is essential to control *Leishmania* infection in macrophages

Macrophages are the main target cells for *Leishmania* infection. Therefore, we first investigated the role of TAK1 during *L. major* infection in cultured macrophages. To this end, cells of the human monocytic cell line THP-1 were differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) treatment and infected with *L. major* amastigotes for 48 hours in the absence or presence of 5Z-7-Oxozeaenol, a specific inhibitor of TAK1. After incubation, the small subunit ribosomal RNA (ssrRNA) of *L. major* was detected through both relative and absolute quantifications by real-time PCR method (Figures 1A and B). Results here showed that the parasite loads in cells were significantly increased after TAK1 inhibition. The cells infected for 48 hours were transferred to conditions suitable for promastigote differentiation and multiplication (rescue assay) in vitro. The results showed that THP-1 cells with TAK1 inhibition released significantly more parasites than the control cells (Figure 1C).

Next, we constructed a TAK1-specific knockout THP-1 cell line for *Leishmania* infection experiments (Figures S1A and B). Using the aforementioned methods, we found that the parasite loads in cells were significantly increased upon TAK1 deficiency (Figures 1D–F). Similar experiments were also conducted in mouse bone marrow–derived macrophages (BMDMs). Again, through both relative and absolute quantifications, we found that TAK1 inhibition resulted in elevated parasite loads in BMDMs (Figures 1G and H). As expected, when *L. major* promastigotes were cultured in vitro by transferring BMDMs infected for 48 hours to *Leishmania* culture conditions in the rescue assay, BMDMs with impaired TAK1 released more parasites than the control cells (Figure 1I). These results thus demonstrate that TAK1 is implicated in controlling *Leishmania* infection in both human and mouse macrophages.

### TAK1 in macrophages is essential for parasite clearance in vivo

To investigate the role of TAK1 in vivo, we inoculated $1 \times 10^5 L. major$ metacyclic promastigotes in footpads of macrophage-specific, conditional TAK1 (Map3k7 gene)-deficient mice (TAK1fl/fl; LysM-cre; Figure S1C). We confirmed that TAK1 deficiency was mainly in macrophages and there was no clear knockout of TAK1 in neutrophils (Figures S1C and D). Lesion development, quantifica-

tion of parasite loads and the presence of clinical signs were monitored up to 42 days after infection. Although a similar onset for the increase of footpad thickness was observed between TAK1fl/fl; LysM-cre and TAK1fl/fl mice, the TAK1-deficient mice showed faster lesion development and a larger lesion size (Figures 2A and B). In addition, the TAK1-deficient mice showed an earlier and more severe development of clinical signs, reaching a class 2 level by day 21, whereas no TAK1fl/fl mice exceeded a class 1 level (Figure 2B). Analysis of the lesion volume and parasite quantification carried out at day 42 clearly supported an increase in lesion size and parasite loads in TAK1-deficient mice (Figures 2C and D). Direct observation displayed enlarged lymph nodes in TAK1fl/fl; LysM-cre mice, and the feet of TAK1fl/fl; LysM-cre mice were swollen and reddish, and the mass change was palpable. This was confirmed by H&E staining of footpad sections that revealed infiltrates around the phalange ring, as well as in the connective tissue (Figure 2E). Therefore, these results suggest that TAK1 in macrophages is essential for the host clearance of *L. major* parasites.

### Macrophage-specific deletion of TAK1 impairs host resistance to *L. major* challenge due to compromised Th1 cell response

Th1 cell-mediated immune responses are important to control *L. major* infections.<sup>19</sup> We investigated Th1-related cytokine levels in TAK1fl/fl; LysM-cre and their littermate control mice at different time points after *L. major* infection. We found that there was no significant difference in IFN-γ and IL-1β transcript levels between the two groups of mice on day 14 after infection, with both cytokines expressed at a low basal level (Figure 3A). Notably, on 28 days after infection, the TAK1fl/fl; LysM-cre mice were unable to produce effective IFN-γ to control *Leishmania*, and the transcript levels of IL-12p40 were also lower compared with TAK1fl/fl mice, although transcript levels of other inflammatory cytokines, such as IL-6, IL-1β and TNF-α, did not show differences between these two groups (Figure 3B). When we evaluated the transcript levels of cytokines in mice infected for 42 days, we found that the levels of IFN-γ were still significantly lower in TAK1fl/fl; LysM-cre mice compared with control mice, and the same was true for IL-6. Furthermore, levels of IL-12, IL-1β and TNF-α were lower in TAK1fl/fl; LysM-cre mice (Figure 3C). These data indicated that T cells in TAK1fl/fl; LysM-cre mice showed a defect in differentiation to Th1 cells to resist *Leishmania* infection, thus leading to more pronounced clinical signs. In addition, the analysis of footpads also revealed that there was no significant difference in IFN-γ and IL-1β transcript levels on day 14 after infection, which were at extremely low levels (Figure S2A). Consistently, the levels of IFN-γ and IL-12 were lower in TAK1fl/fl; LysM-cre mice compared with their littermate control on day 28 after infection (Figure S2B). However, there was no significant difference in the levels of Th1-related proinflammatory factors such as IFN-γ, IL-6, IL-1β and TNF-α, between the two groups of mice on the 42nd day of infection (Figure S2C). This may be due to the fact that the disease began to resolve at day 42 (Figures 2A and B),<sup>20</sup> so the level of inflammation was reduced.

Next, we investigated the proportions of Th1 cells in lymph nodes between these two groups of mice. As expected, the percentages of IFN-γ<sup>+</sup> cells were lower in TAK1fl/fl; LysM-cre mice on days 28 and 42 after infection, whereas the percentages of CD4<sup>+</sup> cells were similar (Figures 3D–G). These data suggest that TAK1 deficiency in macrophages leads to the inability of mice to develop
<figure><img src="image_1.png" /><figcaption>**Figure 1.** Inhibition or deficiency of TAK1 enhances parasite loads in cultured cells. A–C: THP-1 cells were treated with phorbol 12-myristate 13-acetate (100 ng/mL) to differentiate into macrophages. THP-1-derived macrophages were then infected with LMa for 48 hours at MOI = 3. TAK1 inhibitor (TAK1, 5Z-7:5Z-7-Oxozeaenol, 1 µM) was applied as indicated. A: Parasite-specific ssrRNA was detected through relative real-time quantitative PCR to determine the parasite loads. B: Ten nanograms of genomic DNA was processed, and the parasite-specific ssrRNA was detected by absolute real-time quantitative PCR to determine the parasite loads. C: Cells were lysed with 0.1% Triton X-100 and cultured in M199 complete medium for 4 days to count *Leishmania major* promastigotes. D–F: TAK1 knockout and sgControl THP-1 cells were infected with LMa for 48 hours at MOI = 3 and analyzed as in A–C. G–I: BMDMs from wild-type mice were infected with LMa for 48 hours at MOI = 3, and TAK1 inhibitor 5Z-7:5Z-7-Oxozeaenol was applied at 100 nM, a low dose of 5Z-7-Oxozeaenol to impair TAK1 in BMDM without inducing clear cell death. Relative expression is normalized to GAPDH. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. P values were determined by Student's *t* test. BMDM, bone marrow-derived macrophage; LMa, *Leishmania major* amastigotes; N.D., not detectable; ssrRNA, small subunit ribosomal RNA; TAK1, transforming growth factor β-activated kinase 1; UN, untreated.</figcaption></figure>

effective Th1 immune responses and inflammation, resulting in uncontrolled leishmaniasis.

**Macrophage-specific deletion of TAK1 results in high expression of Arg1 and impaired NO production leading to enhanced infections in mice**

The reduced Th1 response in TAK1-deficient mice may have been the result from an increased Th2 response.²¹ We thus focused our analysis on molecules representative of a Th2 response, that is, the cytokines IL-4, IL-5, IL-13 and IL-10, and the enzyme Arg1. Our results revealed that IL-4 expression in TAK1<sup>fl/fl</sup>; LysM-cre

mice was significantly lower than that in control mice on day 14 after infection (Figure 4A). On the 28th day after infection, the trend was reversed: IL-4 levels in TAK1<sup>fl/fl</sup>; LysM-cre mice became slightly higher than that in TAK1<sup>fl/fl</sup> mice, even though there was no significant difference between these two groups of animals (Figure 4B). Of note, 42 days after infection, the levels of IL-4, IL-5 and IL-10 in TAK1<sup>fl/fl</sup>; LysM-cre mice were all significantly higher than those in the control mice (Figure 4C). We analyzed Arg1 transcriptional changes on days 14, 28 and 42, respectively. Surprisingly, Arg1 levels increased from day 14 to day 42, which was consistent with the IL-4 increase (Figures 4A–D). In addition, Western blot results also proved that the expression
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Inhibition or deficiency of TAK1 enhances parasite loads in cultured cells. A–C: THP-1 cells were treated with phorbol 12-myristate 13-acetate (100 ng/mL) to differentiate into macrophages. THP-1-derived macrophages were then infected with LMa for 48 hours at MOI = 3. TAK1 inhibitor (TAK1, 5Z-7:5Z-7-Oxozeaenol, 1 µM) was applied as indicated. A: Parasite-specific ssrRNA was detected through relative real-time quantitative PCR to determine the parasite loads. B: Ten nanograms of genomic DNA was processed, and the parasite-specific ssrRNA was detected by absolute real-time quantitative PCR to determine the parasite loads. C: Cells were lysed with 0.1% Triton X-100 and cultured in M199 complete medium for 4 days to count *Leishmania major* promastigotes. D–F: TAK1 knockout and sgControl THP-1 cells were infected with LMa for 48 hours at MOI = 3 and analyzed as in A–C. G–I: BMDMs from wild-type mice were infected with LMa for 48 hours at MOI = 3, and TAK1 inhibitor 5Z-7:5Z-7-Oxozeaenol was applied at 100 nM, a low dose of 5Z-7-Oxozeaenol to impair TAK1 in BMDM without inducing clear cell death. Relative expression is normalized to GAPDH. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. P values were determined by Student's t test. BMDM, bone marrow-derived macrophage; LMa, *Leishmania major* amastigotes; N.D., not detectable; ssrRNA, small subunit ribosomal RNA; TAK1, transforming growth factor β-activated kinase 1; UN, untreated.</figcaption></figure>

effective Th1 immune responses and inflammation, resulting in uncontrolled leishmaniasis.

**Macrophage-specific deletion of TAK1 results in high expression of Arg1 and impaired NO production leading to enhanced infections in mice**

The reduced Th1 response in TAK1-deficient mice may have been the result from an increased Th2 response.²¹ We thus focused our analysis on molecules representative of a Th2 response, that is, the cytokines IL-4, IL-5, IL-13 and IL-10, and the enzyme Arg1. Our results revealed that IL-4 expression in TAK1<sup>fl/fl</sup>; LysM-cre

mice was significantly lower than that in control mice on day 14 after infection (Figure 4A). On the 28th day after infection, the trend was reversed: IL-4 levels in TAK1<sup>fl/fl</sup>; LysM-cre mice became slightly higher than that in TAK1<sup>fl/fl</sup> mice, even though there was no significant difference between these two groups of animals (Figure 4B). Of note, 42 days after infection, the levels of IL-4, IL-5 and IL-10 in TAK1<sup>fl/fl</sup>; LysM-cre mice were all significantly higher than those in the control mice (Figure 4C). We analyzed Arg1 transcriptional changes on days 14, 28 and 42, respectively. Surprisingly, Arg1 levels increased from day 14 to day 42, which was consistent with the IL-4 increase (Figures 4A–D). In addition, Western blot results also proved that the expression
<figure><img src="image_2.png" /><figcaption>**Figure 2.** Macrophage-specific deletion of TAK1 aggravates clinical signs of *Leishmania* infection in mice. Mice (n = 6 per group) were injected with 1 × 10⁵ *Leishmania major* metacyclic promastigotes in footpads. A and B: On the indicated days, the thickness of the mouse footpad was recorded by Vernier caliper, and disease scores were calculated. C: Volume of mouse footpads at day 42 is shown. D: Mice were sacrificed on day 42, and parasite loads were determined based on absolute parasite quantification of *Leishmania* ssrRNA by real-time quantitative PCR. E: Mice were sacrificed on day 42, images of footpads and H&E staining are shown. Scale bar is 50 µm, m = muscle, c = phalanx cavity, * = infiltrated cells. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.0001. P values were determined by two-way ANOVA (A, B) or Student's t test (C, D). ssrRNA, small subunit ribosomal RNA; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

of Arg1 in TAK1<sup>fl/fl</sup>; LysM-cre mice was higher than for control mice on day 42 after infection, but not on days 14 and 28 (Figures 4E–G).

We also analyzed the changes in Th2-related cytokine levels in the lymph nodes of experimental mice on days 14, 28 and 42 after *Leishmania* infection. On the 14th day after infection, the levels of IL-4, IL-5 and IL-13 did not show a significant difference between the two groups of mice, but the levels of IL-10 were higher in TAK1<sup>fl/fl</sup>; LysM-cre mice (Figure S3A). On the 28th day after infection, the levels of IL-4, IL-5 and IL-13 were significantly higher in TAK1<sup>fl/fl</sup>; LysM-cre mice compared with control mice (Figure S3B). On the 42nd day after infection, high levels of Th2-associated cytokines persisted in TAK1<sup>fl/fl</sup>; LysM-cre mice (Figure S3C). The analysis of lymph nodes also showed that Arg1 was highly expressed in TAK1<sup>fl/fl</sup>; LysM-cre mice (Figures S3D–F).

It has been reported that iNOS catalyzes arginine to NO,²² whereas Arg1 competes with iNOS to reduce NO production and to favor the production of polyamines.²³,²⁴ We further examined NO concentrations in the footpads of mice. To this end, no significant difference in the NO concentration between control mice and TAK1<sup>fl/fl</sup>; LysM-cre mice was found on days 14 and 28 after infection (Figures 4H and I). On the 42nd day after *Leishmania* challenge, the NO concentration in the footpads of TAK1<sup>fl/fl</sup>; LysM-cre mice was significantly reduced (Figure 4J). Analysis of the concentration of NO in serum also revealed that the concentration of NO in TAK1<sup>fl/fl</sup>; LysM-cre mice was significantly lower than that in control mice (Figures S3G and H). These data suggest that elevation of Arg1 potentially increases the level of polyamines, which facilitates parasite growth and may be responsible for the

decrease in NO levels in local tissues, and attenuates the ability of hosts to control *Leishmania* infections.

## Discussion

The mechanisms of host resistance to *Leishmania* infections are largely elusive. Our current work found that TAK1 plays an important role in antileishmanial responses. When control C57BL/6 mice were infected with *L. major*, Th1 cytokines were induced and arginine was catalyzed by iNOS to NO, and effective control of parasites was achieved. When *TAK1* was deleted from macrophages, *Leishmania* infection led to enhanced production of IL-4 and IL-5, which induced high expression of Arg1 and decreased generation of NO. Such a Th2-biased response failed to the control parasite burden and caused more severe disease (Figure 5).

In cutaneous leishmaniasis, the host resists *Leishmania* infection largely through IFN-γ.²⁵ In the C57BL/6 mouse strain, *L. major* induces a Th1 cell-mediated resistance response. Although CD4⁺ T cells do not evenly distribute and contact infected macrophages, the IFN-γ produced by CD4⁺ T cells can stimulate other cells to produce NO.²⁶ Especially, *Leishmania*-infected macrophages require IFN-γ to effectively suppress parasite growth.²⁷ Our data in the present study demonstrate that the absence of TAK1 from macrophages leads to the inability of the host to produce sufficient IFN-γ to control *Leishmania*, albeit the mechanism accounting for compromised IFN-γ generation awaits further investigation.

Contrasting to C57BL/6 mice, BALB/c mice infected with *L. major* develop a nonhealing Th2 response, and treatment with anti-IL-4 antibody shifts the mice from a Th2 to a Th1 response.²⁸
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Macrophage-specific deletion of TAK1 aggravates clinical signs of *Leishmania* infection in mice. Mice (n = 6 per group) were injected with $1 \times 10^5$ *Leishmania major* metacyclic promastigotes in footpads. A and B: On the indicated days, the thickness of the mouse footpad was recorded by Vernier caliper, and disease scores were calculated. C: Volume of mouse footpads at day 42 is shown. D: Mice were sacrificed on day 42, and parasite loads were determined based on absolute parasite quantification of *Leishmania* ssrRNA by real-time quantitative PCR. E: Mice were sacrificed on day 42, images of footpads and H&E staining are shown. Scale bar is 50 µm, m = muscle, c = phalanx cavity, * = infiltrated cells. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.0001. P values were determined by two-way ANOVA (A, B) or Student's t test (C, D). ssrRNA, small subunit ribosomal RNA; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

of Arg1 in TAK1$^{fl/fl}$; LysM-cre mice was higher than for control mice on day 42 after infection, but not on days 14 and 28 (Figures 4E–G).

We also analyzed the changes in Th2-related cytokine levels in the lymph nodes of experimental mice on days 14, 28 and 42 after *Leishmania* infection. On the 14th day after infection, the levels of IL-4, IL-5 and IL-13 did not show a significant difference between the two groups of mice, but the levels of IL-10 were higher in TAK1$^{fl/fl}$; LysM-cre mice (Figure S3A). On the 28th day after infection, the levels of IL-4, IL-5 and IL-13 were significantly higher in TAK1$^{fl/fl}$; LysM-cre mice compared with control mice (Figure S3B). On the 42nd day after infection, high levels of Th2-associated cytokines persisted in TAK1$^{fl/fl}$; LysM-cre mice (Figure S3C). The analysis of lymph nodes also showed that Arg1 was highly expressed in TAK1$^{fl/fl}$; LysM-cre mice (Figures S3D–F).

It has been reported that iNOS catalyzes arginine to NO,²² whereas Arg1 competes with iNOS to reduce NO production and to favor the production of polyamines.²³,²⁴ We further examined NO concentrations in the footpads of mice. To this end, no significant difference in the NO concentration between control mice and TAK1$^{fl/fl}$; LysM-cre mice was found on days 14 and 28 after infection (Figures 4H and I). On the 42nd day after *Leishmania* challenge, the NO concentration in the footpads of TAK1$^{fl/fl}$; LysM-cre mice was significantly reduced (Figure 4J). Analysis of the concentration of NO in serum also revealed that the concentration of NO in TAK1$^{fl/fl}$; LysM-cre mice was significantly lower than that in control mice (Figures S3G and H). These data suggest that elevation of Arg1 potentially increases the level of polyamines, which facilitates parasite growth and may be responsible for the

decrease in NO levels in local tissues, and attenuates the ability of hosts to control *Leishmania* infections.

## Discussion

The mechanisms of host resistance to *Leishmania* infections are largely elusive. Our current work found that TAK1 plays an important role in antileishmanial responses. When control C57BL/6 mice were infected with *L. major*, Th1 cytokines were induced and arginine was catalyzed by iNOS to NO, and effective control of parasites was achieved. When TAK1 was deleted from macrophages, *Leishmania* infection led to enhanced production of IL-4 and IL-5, which induced high expression of Arg1 and decreased generation of NO. Such a Th2-biased response failed to the control parasite burden and caused more severe disease (Figure 5).

In cutaneous leishmaniasis, the host resists *Leishmania* infection largely through IFN-γ.²⁵ In the C57BL/6 mouse strain, *L. major* induces a Th1 cell-mediated resistance response. Although CD4⁺ T cells do not evenly distribute and contact infected macrophages, the IFN-γ produced by CD4⁺ T cells can stimulate other cells to produce NO.²⁶ Especially, *Leishmania*-infected macrophages require IFN-γ to effectively suppress parasite growth.²⁷ Our data in the present study demonstrate that the absence of TAK1 from macrophages leads to the inability of the host to produce sufficient IFN-γ to control *Leishmania*, albeit the mechanism accounting for compromised IFN-γ generation awaits further investigation.

Contrasting to C57BL/6 mice, BALB/c mice infected with *L. major* develop a nonhealing Th2 response, and treatment with anti-IL-4 antibody shifts the mice from a Th2 to a Th1 response.²⁸
<figure><img src="image_3.png" /><figcaption>**Figure 3.** Macrophage-specific deletion of TAK1 alleviates effective Th1 responses in mice. A–C: Mice were injected with $1 \times 10^5$ *Leishmania major* metacyclic promastigotes in footpads and sacrificed at different time points after infection, namely, on day 14 (A), day 28 (B) and day 42 (C). Transcriptional analysis of lymph nodes was performed for various immune response-related targets via real-time quantitative PCR. Relative expression was normalized to GAPDH. D–G: Flow cytometry analysis of popliteal lymph node cells from TAK1$^{fl/fl}$ and TAK1$^{fl/fl}$; LysM$^{cre}$ mice after indicated infection days by *L. major* upon staining with antibodies against the indicated targets. The percentages of CD4$^+$ T-cell populations and intracellular IFN-γ levels from CD4$^+$ T cells were analyzed. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001, \*\*\*\*P < 0.0001; NS, not significant. *P* values were determined by Student's *t* test. TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

Our results demonstrate that IL-4 levels are elevated in mice from the early to late stages of *Leishmania* infection, and the absence of TAK1 from macrophages exacerbates this process. Of note, Arg1

increases susceptibility and parasite loads in mice,²¹ and IL-4 induces alternative macrophage activation and high expression of Arg1.²⁹ In susceptible mice, high levels of IL-4 and Arg1 were
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Macrophage-specific deletion of TAK1 alleviates effective Th1 responses in mice. A–C: Mice were injected with $1 \times 10^5$ *Leishmania major* metacyclic promastigotes in footpads and sacrificed at different time points after infection, namely, on day 14 (A), day 28 (B) and day 42 (C). Transcriptional analysis of lymph nodes was performed for various immune response-related targets via real-time quantitative PCR. Relative expression was normalized to GAPDH. D–G: Flow cytometry analysis of popliteal lymph node cells from TAK1$^{fl/fl}$ and TAK1$^{fl/fl}$; LysM$^{cre}$ mice after indicated infection days by *L. major* upon staining with antibodies against the indicated targets. The percentages of CD4$^+$ T-cell populations and intracellular IFN-γ levels from CD4$^+$ T cells were analyzed. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001, \*\*\*\*P < 0.0001; NS, not significant. *P* values were determined by Student's *t* test. TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

Our results demonstrate that IL-4 levels are elevated in mice from the early to late stages of *Leishmania* infection, and the absence of TAK1 from macrophages exacerbates this process. Of note, Arg1

increases susceptibility and parasite loads in mice,²¹ and IL-4 induces alternative macrophage activation and high expression of Arg1.²⁹ In susceptible mice, high levels of IL-4 and Arg1 were
<figure><img src="image_4.png" /><figcaption>**Figure 4.** Deletion of TAK1 in macrophages induces Arg1 elevation and attenuates NO production. A–J: Mice were injected with 1 × 10⁵ Leishmania major metacyclic promastigotes in footpads. Host factors related to NO metabolism were analyzed at different time points on footpad extracts by real-time quantitative PCR (A–D), Western blotting (E–G) and NO detection assays (H–J). Relative expression is normalized to GAPDH. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01; NS, not significant. P values were determined by Student's *t* test. Arg1, arginase 1; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Deletion of TAK1 in macrophages induces Arg1 elevation and attenuates NO production. A–J: Mice were injected with 1 × 10⁵ Leishmania major metacyclic promastigotes in footpads. Host factors related to NO metabolism were analyzed at different time points on footpad extracts by real-time quantitative PCR (A–D), Western blotting (E–G) and NO detection assays (H–J). Relative expression is normalized to GAPDH. Results presented are representative of two independent experiments. \*P < 0.05, \*\*P < 0.01; NS, not significant. P values were determined by Student's *t* test. Arg1, arginase 1; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 5. Working model showing that enhanced Th2 response due to TAK1 deficiency in macrophages leads to more severe disease upon *Leishmania* infection in mice. When TAK1<sup>f/f</sup> mice are infected with *Leishmania*, they produce IFN-γ and IL-12 cytokines, which causes a Th1 cell-biased inflammatory response; in this case, arginine is catalyzed by iNOS to generate NO, thereby effectively controlling parasite infection. When the TAK1<sup>f/f</sup>; LysM-cre mice are infected with *Leishmania*, they produce more IL-4 and IL-5, which induce high expression of Arg1, resulting in a decrease in NO production; such Th2 cell-biased response renders the host being unable to control the parasite effectively, leading to increased infection and disease. Arg1, arginase 1; iNOS, inducible nitric oxide synthase; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

detected after *Leishmania* infection.²¹ In vivo, knockout of *Arg1* in hematopoietic and endothelial cells reduced footpad swelling and parasite loads.³⁰ Our results indicate that Th2-related cytokines increase Arg1 expression, especially on day 42 after parasite infection in the TAK1f/f; LysM-cre mice.

We found that NO was significantly reduced in mouse serum and lesions at the late stage of *Leishmania* infection. However, there was no significant difference in the protein levels of iNOS in the tissues of mice. Only on the 28th day after infection, iNOS was slightly lower in the lesions of TAK1f/f; LysM-cre mice. This suggested that the decrease of NO in TAK1f/f; LysM-cre mice was likely caused by the high levels of Arg1, which competed with iNOS for the common substrate L-arginine, resulting in a decrease in NO and thus compromised control of parasites.³¹

TAK1 is an important protein for signal transduction initiated by IL-1β, TNF-α and TLR ligands. The absence of TAK1 from macrophages may attenuate host inflammatory responses to invading pathogens. A lack of such inflammatory responses may lead to inadequate Th1 responses, which are subsequently necessary to mount important adaptive immune responses. Moreover, *Leishmania* infection of BMDMs inhibits lipopolysaccharide-stimulated IL-12 production³² and NF-κB/NLRP3-mediated inflammatory responses.⁸

Thus, inhibition of cell activation after infection with *Leishmania* may be exacerbated in macrophages deficient for TAK1.

In summary, our current study demonstrates that TAK1 in macrophages is essential for host control of *Leishmania* infections, thus providing a new perspective to consider future drugs enhancing TAK1 control of leishmaniasis.

## Materials and methods

### L. major parasite culture

The *Leishmania* strain *L. major* Friedlin V1 has been reported.³³ These parasites were propagated in Nude mice (NU/NU, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). *L. major* amastigotes were isolated 2 months after infection from mouse footpad lesions. Briefly, after euthanasia, lesions were excised and homogenized in cold PBS (abs962; Absin) containing 1% antibiotics penicillin-streptomycin (15140122; Gibco) with a Dounce tissue grinder. The debris was removed by centrifugation at 30g for 10 minutes at 4°C. The supernatant was further centrifuged at 1730g for 5 minutes at 4°C. After removal of red blood cells, the pelleted amastigotes were washed with PBS and then used to infect cells or resuspended in M199 culture medium for differentiation into promastigotes.

### Isolation of L. major metacyclic promastigotes

After differentiation of the initial amastigotes into promastigotes, *L. major* parasites were cultured at 26°C in M199 medium (12350-039; Gibco) supplemented with 10% FBS (04-001-1A; Biological Industries), 20 mM HEPES (15630080; Gibco), 100 µM Adenin (A2786-5G; Sigma-Aldrich), 50 µg/mL hemin (H9039-1G; Sigma-Aldrich), 10 µg/mL Biotin (B4639; Sigma), 8 µM Biopterin (B2517; Sigma), 4.1 mM NaHCO₃ (SB0482; Bio Basic Inc.), and 1% penicillin-streptomycin. Metacyclic promastigotes were isolated from day 7 stationary phase cultures using a discontinuous density gradient as described previously.³⁴

### Mouse infection with L. major

All animal studies were conducted according to the guidelines from the national care and use of laboratory animals and were approved by the institutional ethics committee of the Institute Pasteur of Shanghai, Chinese Academy of Sciences. TAK1f/f mice (Jax#: 011039)³⁵ and LysM-cre (Jax#: 4781) mice were crossed to obtain TAK1f/f; LysM-cre mice. TAK1f/f mice and TAK1f/f; LysM-cre in a C57BL/6 background were kept and bred in specific pathogen-free conditions. Eight-week-old mice were infected with 1 × 10⁵ *L. major* metacyclic promastigotes in a volume of 50 µL per footpad. The development of lesions in *L. major*-infected mice was monitored weekly using a Vernier caliper. Calculations for the lesions' volumes were based on the triaxial ellipsoid formula. According the length *a*, width *b* and height *c*, the volume was given by $V = \frac{4}{3}\pi abc$. Clinical scores were assessed using an arbitrary scale from 0 to 5 (0, absence of lesion; 1, low induration; 2, high induration; 3, small ulcer; 4, large ulcer; 5, perforated tissue). According to the experimental design, lymph nodes, sera and footpads were collected on days 14, 28 and 42 for subsequent experiments.

### BMDM culture and infection with L. major

Bone marrow cells from the tibia and femurs of 8-week-old C57BL/6 mice were harvested in DMEM. Cells were cultured in
<figure><img src="image_6.png" /><figcaption>Figure 5. Working model showing that enhanced Th2 response due to TAK1 deficiency in macrophages leads to more severe disease upon *Leishmania* infection in mice. When TAK1<sup>f/f</sup> mice are infected with *Leishmania*, they produce IFN-γ and IL-12 cytokines, which causes a Th1 cell-biased inflammatory response; in this case, arginine is catalyzed by iNOS to generate NO, thereby effectively controlling parasite infection. When the TAK1<sup>f/f</sup>; LysM-cre mice are infected with *Leishmania*, they produce more IL-4 and IL-5, which induce high expression of Arg1, resulting in a decrease in NO production; such Th2 cell-biased response renders the host being unable to control the parasite effectively, leading to increased infection and disease. Arg1, arginase 1; iNOS, inducible nitric oxide synthase; TAK1, transforming growth factor β-activated kinase 1.</figcaption></figure>

detected after *Leishmania* infection.²¹ In vivo, knockout of *Arg1* in hematopoietic and endothelial cells reduced footpad swelling and parasite loads.³⁰ Our results indicate that Th2-related cytokines increase Arg1 expression, especially on day 42 after parasite infection in the TAK1f/f; LysM-cre mice.

We found that NO was significantly reduced in mouse serum and lesions at the late stage of *Leishmania* infection. However, there was no significant difference in the protein levels of iNOS in the tissues of mice. Only on the 28th day after infection, iNOS was slightly lower in the lesions of TAK1f/f; LysM-cre mice. This suggested that the decrease of NO in TAK1f/f; LysM-cre mice was likely caused by the high levels of Arg1, which competed with iNOS for the common substrate L-arginine, resulting in a decrease in NO and thus compromised control of parasites.³¹

TAK1 is an important protein for signal transduction initiated by IL-1β, TNF-α and TLR ligands. The absence of TAK1 from macrophages may attenuate host inflammatory responses to invading pathogens. A lack of such inflammatory responses may lead to inadequate Th1 responses, which are subsequently necessary to mount important adaptive immune responses. Moreover, *Leishmania* infection of BMDMs inhibits lipopolysaccharide-stimulated IL-12 production³² and NF-κB/NLRP3-mediated inflammatory responses.⁸

Thus, inhibition of cell activation after infection with *Leishmania* may be exacerbated in macrophages deficient for TAK1.

In summary, our current study demonstrates that TAK1 in macrophages is essential for host control of *Leishmania* infections, thus providing a new perspective to consider future drugs enhancing TAK1 control of leishmaniasis.

## Materials and methods

### L. major parasite culture

The *Leishmania* strain *L. major* Friedlin V1 has been reported.³³ These parasites were propagated in Nude mice (NU/NU, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). *L. major* amastigotes were isolated 2 months after infection from mouse footpad lesions. Briefly, after euthanasia, lesions were excised and homogenized in cold PBS (abs962; Absin) containing 1% antibiotics penicillin-streptomycin (15140122; Gibco) with a Dounce tissue grinder. The debris was removed by centrifugation at 30g for 10 minutes at 4°C. The supernatant was further centrifuged at 1730g for 5 minutes at 4°C. After removal of red blood cells, the pelleted amastigotes were washed with PBS and then used to infect cells or resuspended in M199 culture medium for differentiation into promastigotes.

### Isolation of L. major metacyclic promastigotes

After differentiation of the initial amastigotes into promastigotes, *L. major* parasites were cultured at 26°C in M199 medium (12350-039; Gibco) supplemented with 10% FBS (04-001-1A; Biological Industries), 20 mM HEPES (15630080; Gibco), 100 µM Adenin (A2786-5G; Sigma-Aldrich), 50 µg/mL hemin (H9039-1G; Sigma-Aldrich), 10 µg/mL Biotin (B4639; Sigma), 8 µM Biopterin (B2517; Sigma), 4.1 mM NaHCO₃ (SB0482; Bio Basic Inc.), and 1% penicillin-streptomycin. Metacyclic promastigotes were isolated from day 7 stationary phase cultures using a discontinuous density gradient as described previously.³⁴

### Mouse infection with L. major

All animal studies were conducted according to the guidelines from the national care and use of laboratory animals and were approved by the institutional ethics committee of the Institute Pasteur of Shanghai, Chinese Academy of Sciences. TAK1f/f mice (Jax#: 011039)³⁵ and LysM-cre (Jax#: 4781) mice were crossed to obtain TAK1f/f; LysM-cre mice. TAK1f/f mice and TAK1f/f; LysM-cre in a C57BL/6 background were kept and bred in specific pathogen-free conditions. Eight-week-old mice were infected with 1 × 10⁵ *L. major* metacyclic promastigotes in a volume of 50 µL per footpad. The development of lesions in *L. major*-infected mice was monitored weekly using a Vernier caliper. Calculations for the lesions' volumes were based on the triaxial ellipsoid formula. According the length *a*, width *b* and height *c*, the volume was given by $V = \frac{4}{3}\pi abc$. Clinical scores were assessed using an arbitrary scale from 0 to 5 (0, absence of lesion; 1, low induration; 2, high induration; 3, small ulcer; 4, large ulcer; 5, perforated tissue). According to the experimental design, lymph nodes, sera and footpads were collected on days 14, 28 and 42 for subsequent experiments.

### BMDM culture and infection with L. major

Bone marrow cells from the tibia and femurs of 8-week-old C57BL/6 mice were harvested in DMEM. Cells were cultured in
L929 cell-conditioned DMEM (C11995500BT; Gibco) supplemented with 10% FBS (FBSSA500-S; AusGeneX), 1% nonessential amino acids (11140-050; Gibco), 1% penicillin-streptomycin and 0.05 mM β-mercaptoethanol (M3148; Sigma-Aldrich), and incubated at 37°C with 5% CO₂ for 6 days. On day 6, differentiated BMDMs were collected and seeded at $1 \times 10^6$ cells/mL in 24-well plates.³⁶ Specific inhibition of TAK1 in BMDMs was carried out with 100 nM 5Z-7-Oxoeaenol (HY-12686; MedChemExpress). The BMDMs were infected with *L. major* at a MOI of three amastigotes per macrophage for 2 days. TAK1 deficiency in macrophages was examined in BMDMs by PCR detection of the *Map3k7* gene (forward primer: CTCGCTGGTAGTGGTGTTAG; reverse primer: CTTGCCTCTTCAGACTATGC).

## THP-1 cell culture and infection with *L. major*

Human monocytic leukemia THP-1 (ATCC#TIB-202) cells were grown in suspension in RPMI 1640 medium (C11875500BT; Gibco) supplemented with 10% FBS (SA500-S; AusGeneX), 1% penicillin-streptomycin and 0.05 mM β-mercaptoethanol at 37°C with 5% CO₂. TAK1-deficient cells were generated by the CRISPR/Cas9 approach. Briefly, HEK293T cells were transfected with packaging plasmids PXA2, VSVG and lenti-crispr V2 lentivirus vector carrying the *sgTAK1* gene sequence GATCGACTACAAGGAGATCG to generate viruses. THP-1 cells were infected with these viruses, and monoclonal cells were obtained by puromycin screening. TAK1 deficiency was examined in the THP-1 cell line by PCR detection of the *MAP3K7* gene (forward primer AGCGGAGCCAGAGCCGGACA; reverse primer AGGCAGCTACTCCCGAGCAGT). SgControl and sgTAK1 THP1 monocytes were treated with PMA (100 ng/mL, P1585; Sigma-Aldrich) for 12 hours, washed and resuspended in medium for 24 hours to differentiate into macrophages.³⁷ Prior to infection, the macrophages were treated with 5Z-7-Oxoeaenol or a vehicle control. The PMA-differentiated THP1 cells were infected with *L. major* at a MOI of 3 for 2 days.

## NO and cytokine detection

Tissues were lysed and homogenized in lysis buffer (0.5% NP40, 145 mM NaCl, 25 mM Tris, pH 7.3, 5% glycerine) and centrifuged at 13,000g at 4°C for 10 minutes to remove debris. NO production was determined in the collected supernatants and in serum by using the Griess Reagent System kit (G2930; Promega).³⁸ Briefly, gradient standard solutions of 100 µM were prepared. A volume of 50 µL of sample was added to each well of a 96-well plate, and then 50 µL sulfanilamide and 50 µL N-1-naphthylethylene diamine were added. Each reagent addition was followed by a 10-minute incubation period in the dark at room temperature. Finally, the absorbance at 550 nm was measured using a Multiskan microplate reader, and the concentrations were estimated from the NO standard curve.

## Western blotting

Tissue was lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0) containing protease inhibitors (04693132001; Roche) followed by homogenization. The homogenized samples were centrifuged at 13,000g at 4°C for 10 minutes. The supernatants were boiled in loading buffer at 95°C for 10 minutes. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes.³⁹ Membranes were blocked with PBS supplemented with 0.1% Tween20 and 2.5% bovine serum

albumin and then probed overnight at 4°C with the following primary antibodies: TAK1 (4505; CST), iNOS (18985-1-AP; Peprotech), Arg1 (16001-1-AP; Peprotech) and β-actin (12620S; CST). Following incubation with secondary antirabbit horseradish peroxidase antibodies (Jackson Immunoresearch; 111035003), protein bands were visualized by PicoLight substrate-enhanced chemiluminescence solution (EpizymeBiotech; SQ202L). Immunoblot images were captured digitally using a 5200 chemiluminescent imaging system (Tanon 5200 Multi, Tanon).

## DNA extraction and absolute quantification of *L. major* loads

Genomic DNA was extracted with a DNeasy Qiagen Kit according to the manufacturer's instructions or with the phenol/chloroform method using Phenol/Chloroform/isoamyl alcohol. Briefly, tissues or cells were resuspended in lysis buffer (200 mM NaCl, 0.2% SDS, 5 mM EDTA, 100 nM Tris-HCl, pH 8.5) with 100 µg/mL Proteinase K (Milliprepro, 539480) overnight at 55° C. DNA was extracted in phenol-CHCl₃-isoamyl alcohol (25:24:1) (AP1012-100 ml). Samples were ethanol precipitated and resuspended in water. The measurement of parasite loads by quantitative PCR was performed by absolute quantitation, based on a standard curve produced from DNA samples extracted from *L. major* promastigotes. Serial dilutions of 1:10 were made to produce five points of the standard curves, ranging from $10^7$ to $10^3$ parasites. Primers were designed targeting the ssRNA of *Leishmania*: ssRNA forward CCATGTCGGATTTGGT; ssRNA reverse CGAAACGGTAGCCTAGAG.⁴⁰ The 12 µL reaction mixture contained TOROGreen quantitative PCR Master Mix, each primer at a concentration of 1 µM, and 2 µL of template DNA. The reactions were performed on a ABI QuantStudio 6 flex, run at 95°C for 1 minute followed by 40 cycles of 95°C for 15 seconds, 54°C for 25 seconds, and 72°C for 30 seconds, and a final melting curve. Total parasites per tissue or harvested cell was obtained by multiplying the relative parasite numbers by the factor volume.

## Transcriptional analysis via real-time quantitative PCR

Total RNA was extracted using TRIzol reagent (15596018, Ambion) according to the manufacturer's instructions. RNA was reverse-transcribed using the GoScriptTM Reverse Transcription System (A5001; Promega). Relative transcript abundance of ssRNA, IFN-γ, IL-1β, IL-12p40, TNF-α, IL-4, IL-5, IL-6, IL-10 and Arg1 was normalized to that of GAPDH levels using the $2^{-\Delta Cq}$ method (see Supplementary Information for primer sequences). mRNA levels were quantified on an ABI QuantStudio 6 flex using the TOROGreen quantitative PCR Master Mix kit (QST-100; TOROIVD). Cycling conditions were as follows: 1 cycle at 95°C for 1 minute followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A melting curve analysis was performed on all reactions.

## *L. major* promastigote rescue transformation assay

The assay was performed according to the promastigote rescue transformation assay described before.⁴¹ Two days after infection, cells were washed and lysed with 0.1% Triton X-100 to liberate *Leishmania* from the parasite vacuoles in infected macrophages. *Leishmania* M199 medium was added, and the plate was incubated at 26°C. After 4 days of incubation, the amastigotes differentiate into promastigotes. The parasite loads were determined by counting the promastigotes in each well.
L929 cell-conditioned DMEM (C11995500BT; Gibco) supplemented with 10% FBS (FBSSA500-S; AusGeneX), 1% nonessential amino acids (11140-050; Gibco), 1% penicillin-streptomycin and 0.05 mM β-mercaptoethanol (M3148; Sigma-Aldrich), and incubated at 37°C with 5% CO₂ for 6 days. On day 6, differentiated BMDMs were collected and seeded at $1 \times 10^6$ cells/mL in 24-well plates.³⁶ Specific inhibition of TAK1 in BMDMs was carried out with 100 nM 5Z-7-Oxoeaenol (HY-12686; MedChemExpress). The BMDMs were infected with *L. major* at a MOI of three amastigotes per macrophage for 2 days. TAK1 deficiency in macrophages was examined in BMDMs by PCR detection of the *Map3k7* gene (forward primer: CTCGCTGGTAGTGGTGTTAG; reverse primer: CTTGCCTCTTCAGACTATGC).

## THP-1 cell culture and infection with *L. major*

Human monocytic leukemia THP-1 (ATCC#TIB-202) cells were grown in suspension in RPMI 1640 medium (C11875500BT; Gibco) supplemented with 10% FBS (SA500-S; AusGeneX), 1% penicillin-streptomycin and 0.05 mM β-mercaptoethanol at 37°C with 5% CO₂. TAK1-deficient cells were generated by the CRISPR/Cas9 approach. Briefly, HEK293T cells were transfected with packaging plasmids PXA2, VSVG and lenti-crispr V2 lentivirus vector carrying the *sgTAK1* gene sequence GATCGACTACAAGGAGATCG to generate viruses. THP-1 cells were infected with these viruses, and monoclonal cells were obtained by puromycin screening. TAK1 deficiency was examined in the THP-1 cell line by PCR detection of the *MAP3K7* gene (forward primer AGCGGAGCCAGAGCCGGACA; reverse primer AGGCAGCTACTCCCGAGCAGT). SgControl and sgTAK1 THP1 monocytes were treated with PMA (100 ng/mL, P1585; Sigma-Aldrich) for 12 hours, washed and resuspended in medium for 24 hours to differentiate into macrophages.³⁷ Prior to infection, the macrophages were treated with 5Z-7-Oxoeaenol or a vehicle control. The PMA-differentiated THP1 cells were infected with *L. major* at a MOI of 3 for 2 days.

## NO and cytokine detection

Tissues were lysed and homogenized in lysis buffer (0.5% NP40, 145 mM NaCl, 25 mM Tris, pH 7.3, 5% glycerine) and centrifuged at 13,000g at 4°C for 10 minutes to remove debris. NO production was determined in the collected supernatants and in serum by using the Griess Reagent System kit (G2930; Promega).³⁸ Briefly, gradient standard solutions of 100 µM were prepared. A volume of 50 µL of sample was added to each well of a 96-well plate, and then 50 µL sulfanilamide and 50 µL N-1-naphthylethylene diamine were added. Each reagent addition was followed by a 10-minute incubation period in the dark at room temperature. Finally, the absorbance at 550 nm was measured using a Multiskan microplate reader, and the concentrations were estimated from the NO standard curve.

## Western blotting

Tissue was lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0) containing protease inhibitors (04693132001; Roche) followed by homogenization. The homogenized samples were centrifuged at 13,000g at 4°C for 10 minutes. The supernatants were boiled in loading buffer at 95°C for 10 minutes. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes.³⁹ Membranes were blocked with PBS supplemented with 0.1% Tween20 and 2.5% bovine serum

albumin and then probed overnight at 4°C with the following primary antibodies: TAK1 (4505; CST), iNOS (18985-1-AP; Peprotech), Arg1 (16001-1-AP; Peprotech) and β-actin (12620S; CST). Following incubation with secondary antirabbit horseradish peroxidase antibodies (Jackson Immunoresearch; 111035003), protein bands were visualized by PicoLight substrate-enhanced chemiluminescence solution (EpizymeBiotech; SQ202L). Immunoblot images were captured digitally using a 5200 chemiluminescent imaging system (Tanon 5200 Multi, Tanon).

## DNA extraction and absolute quantification of *L. major* loads

Genomic DNA was extracted with a DNeasy Qiagen Kit according to the manufacturer's instructions or with the phenol/chloroform method using Phenol/Chloroform/isoamyl alcohol. Briefly, tissues or cells were resuspended in lysis buffer (200 mM NaCl, 0.2% SDS, 5 mM EDTA, 100 nM Tris-HCl, pH 8.5) with 100 µg/mL Proteinase K (Milliprepro, 539480) overnight at 55° C. DNA was extracted in phenol-CHCl₃-isoamyl alcohol (25:24:1) (AP1012-100 ml). Samples were ethanol precipitated and resuspended in water. The measurement of parasite loads by quantitative PCR was performed by absolute quantitation, based on a standard curve produced from DNA samples extracted from *L. major* promastigotes. Serial dilutions of 1:10 were made to produce five points of the standard curves, ranging from $10^7$ to $10^3$ parasites. Primers were designed targeting the ssRNA of *Leishmania*: ssRNA forward CCATGTCGGATTTGGT; ssRNA reverse CGAAACGGTAGCCTAGAG.⁴⁰ The 12 µL reaction mixture contained TOROGreen quantitative PCR Master Mix, each primer at a concentration of 1 µM, and 2 µL of template DNA. The reactions were performed on a ABI QuantStudio 6 flex, run at 95°C for 1 minute followed by 40 cycles of 95°C for 15 seconds, 54°C for 25 seconds, and 72°C for 30 seconds, and a final melting curve. Total parasites per tissue or harvested cell was obtained by multiplying the relative parasite numbers by the factor volume.

## Transcriptional analysis via real-time quantitative PCR

Total RNA was extracted using TRIzol reagent (15596018, Ambion) according to the manufacturer's instructions. RNA was reverse-transcribed using the GoScriptTM Reverse Transcription System (A5001; Promega). Relative transcript abundance of ssRNA, IFN-γ, IL-1β, IL-12p40, TNF-α, IL-4, IL-5, IL-6, IL-10 and Arg1 was normalized to that of GAPDH levels using the $2^{-\Delta Cq}$ method (see Supplementary Information for primer sequences). mRNA levels were quantified on an ABI QuantStudio 6 flex using the TOROGreen quantitative PCR Master Mix kit (QST-100; TOROIVD). Cycling conditions were as follows: 1 cycle at 95°C for 1 minute followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. A melting curve analysis was performed on all reactions.

## *L. major* promastigote rescue transformation assay

The assay was performed according to the promastigote rescue transformation assay described before.⁴¹ Two days after infection, cells were washed and lysed with 0.1% Triton X-100 to liberate *Leishmania* from the parasite vacuoles in infected macrophages. *Leishmania* M199 medium was added, and the plate was incubated at 26°C. After 4 days of incubation, the amastigotes differentiate into promastigotes. The parasite loads were determined by counting the promastigotes in each well.
## H&E staining

The tissues were fixed in 4% paraformaldehyde at 4°C for 24 to 48 hours. Following fixation, the mouse footpads were decalcified using formic acid aluminum chloride decalcifying fluid for up to 2 weeks. Tissues were immersed in reagents at least 20 times, and tissue volume and decalcification reagents were replaced twice every 3–4 days. After the bones melted, tissues were washed to flush out the decalcifier. Footpads were then dehydrated in graded ethanol, cleared in xylenes and infiltrated with paraffin mix. They were embedded in paraffin, and 5- to 7-µm sections were prepared and stained with hematoxylin and eosin. Briefly, sections were deparaffinized with xylene and gradually rehydrated through graded alcohols for staining. The sections were submerged in hematoxylin, differentiated in a solution of 1% hydrochloric acid in 70% alcohol, blued using water and stained with eosin.

## Flow cytometry

Isolated popliteal lymph node cells were washed with FACS buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA), nonspecific binding was blocked with an FcR blocking antibody (anti-Mouse CD16/32; eBioscience) before staining with labeled monoclonal antibodies against CD45.2 (BD Biosciences), CD3 (BD Biosciences) and CD4 (BD Biosciences). Intracellular staining of INF-γ (BD Biosciences) was carried out after permeabilization with Permfix (BD Biosciences). The fluorescence was examined on a BD LSR Fortessa.⁴² Data were analyzed using FlowJo software.

## Neutrophil isolation

Mouse bone marrow cells were counted and resuspended in 1 mL of ice-cold sterile PBS. Histopaque 1119 (Sigma; RNBF0417) was added in a 15-mL conical tube. Then 3 mL of Histopaque 1077 (Sigma; RNBF2365) was added as overlay on 3 mL of Histopaque 1119, and the bone marrow cell suspension was added as overlay on top of the Histopaque 1077. Tubes were centrifuged for 30 minutes at 2000 rpm at 25°C. Neutrophils were collected at the interface of the Histopaque 1119 and Histopaque 1077 layers. Finally, the collected neutrophils were washed twice with RPMI 1640 1× supplemented with 10% FBS and 1% penicillin-streptomycin and centrifuged at 1400 rpm for 7 minutes at 4°C. TAK1 deficiency was examined in neutrophils by PCR detection of the Map3k7 gene.

## Statistical analysis

The data were presented as the mean and SEM. GraphPad Prism v7.04 software (La Jolla, California) was used for data analysis. Statistically significant differences were determined by Student's two-tailed *t* test for two groups or one-way ANOVA with Bonferroni post-hoc test for three or more groups. *P* < 0.05 was considered statistically significant.

## Supplemental digital content

Supplemental materials are available at http://links.lww.com/IMD/A48. http://links.lww.com/IMD/A49, http://links.lww.com/IMD/A50, http://links.lww.com/IMD/A51.

## References

[1] Burza S, Croft SL, Boelaert M. Leishmaniasis. *Lancet* 2018;392(10151):951–970. https://doi.org/10.1016/s0140-6736(18)31204-2

[2] Serafim TD, Coutinho-Abreu IV, Dey R, et al. Leishmaniasis: the act of transmission. *Trends Parasitol* 2021;37(11):976–987. https://doi.org/10.1016/j.pt.2021.07.003

[3] Gurjar D, Kumar Patra S, Bodhale N, Lenka N, Saha B. *Leishmania* intercepts IFN-γR signaling at multiple levels in macrophages. *Cytokine* 2022;157:155956. https://doi.org/10.1016/j.cyto.2022.155956

[4] Rodrigues LS, Barreto AS, Bomfim LGS, et al. Multifunctional, TNF-α and IFN-γ-secreting CD4 and CD8 T cells and CD8(high) T cells are associated with the cure of human visceral leishmaniasis. *Front Immunol* 2021;12:773983. https://doi.org/10.3389/fimmu.2021.773983

[5] Zaatar MT, Simaan Y, Karam MC. Exogenous IL-13 exacerbates *Leishmania major* infection and abrogates acquired immunity to re-infection. *Parasitol Res* 2022;121(7):2009–2017. https://doi.org/10.1007/s00436-022-07539-y

[6] Varikuti S, Verma C, Natarajan G, Oghumu S, Satoskar AR. MicroRNA155 plays a critical role in the pathogenesis of cutaneous *Leishmania major* infection by promoting a Th2 response and attenuating dendritic cell activity. *Am J Pathol* 2021;191(5):809–816. https://doi.org/10.1016/j.ajpath.2021.01.012

[7] Lee SH, Charmoy M, Romano A, et al. Mannose receptor high, M2 dermal macrophages mediate nonhealing *Leishmania major* infection in a Th1 immune environment. *J Exp Med* 2018;215(1):357–375. https://doi.org/10.1084/jem.20171389

[8] Lecoeur H, Prina E, Rosazza T, et al. Targeting macrophage histone H3 modification as a *Leishmania* strategy to dampen the NF-κB/NLRP3-mediated inflammatory response. *Cell Rep* 2020;30(6):1870–1882.e4. https://doi.org/10.1016/j.celrep.2020.01.030

[9] Xu YR, Lei CQ. TAK1-TABs complex: a central signalosome in inflammatory responses. *Front Immunol* 2020;11:608976. https://doi.org/10.3389/fimmu.2020.608976

[10] Ajibade AA, Wang HY, Wang RF. Cell type-specific function of TAK1 in innate immune signaling. *Trends Immunol* 2013;34(7):307–316. https://doi.org/10.1016/j.it.2013.03.007

[11] Sato S, Sanjo H, Takeda K, et al. Essential function for the kinase TAK1 in innate and adaptive immune responses. *Nat Immunol* 2005;6(11):1087–1095. https://doi.org/10.1038/ni1255

[12] Liu HH, Xie M, Schneider MD, Chen ZJ. Essential role of TAK1 in thymocyte development and activation. *Proc Natl Acad Sci U S A* 2006; 103(31):11677–11682. https://doi.org/10.1073/pnas.0603089103

[13] Wang XD, Zhao CS, Wang QL, et al. The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1. *EMBO Rep* 2020;21(7):e48035. https://doi.org/10.15252/embr.201948035

[14] Omori E, Matsumoto K, Sanjo H, et al. TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis. *J Biol Chem* 2006;281(28):19610–19617. https://doi.org/10.1074/jbc.M603384200

[15] Kajino-Sakamoto R, Inagaki M, Lippert E, et al. Enterocyte-derived TAK1 signaling prevents epithelium apoptosis and the development of ileitis and colitis. *J Immunol* 2008;181(2):1143–1152. https://doi.org/10.4049/jimmunol.181.2.1143

[16] Malireddi RKS, Gurung P, Mavuluri J, et al. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation. *J Exp Med* 2018;215(4):1023–1034. https://doi.org/10.1084/jem.20171922

[17] Malireddi RKS, Gurung P, Kesavardhana S, et al. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. *J Exp Med* 2020;217(3):e20191644. https://doi.org/10.1084/jem.20191644

[18] López-Pérez W, Sai K, Sakamachi Y, Parsons C, Kathariou S, Ninomiya-Tsuji J. TAK1 inhibition elicits mitochondrial ROS to block intracellular bacterial colonization. *Proc Natl Acad Sci U S A* 2021;118(25):e2023647118. https://doi.org/10.1073/pnas.2023647118

[19] Carvalho AM, Guimarães LH, Costa R, et al. Impaired Th1 response is associated with therapeutic failure in patients with cutaneous leishmaniasis caused by *Leishmania braziliensis*. *J Infect Dis* 2021;223(3):527–535. https://doi.org/10.1093/infdis/jiaa374

[20] Lecoeur H, Buffet PA, Milon G, Lang T. Early curative applications of the aminoglycoside WR279396 on an experimental *Leishmania major*-loaded cutaneous site do not impair the acquisition of immunity. *Antimicrob Agents Chemother* 2010;54(3):984–990. https://doi.org/10.1128/AAC.01310-09

[21] Pessenda G, da Silva JS. Arginase and its mechanisms in *Leishmania* persistence. *Parasite Immuno* 2020;42(7):e12722. https://doi.org/10.1111/pim.12722

[22] Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. *Cell* 2022;185(16):2853–2878. https://doi.org/10.1016/j.cell.2022.06.010

[23] Carter NS, Kawasaki Y, Nahata SS, et al. Polyamine metabolism in *Leishmania* parasites: a promising therapeutic target. *Med Sci (Basel)* 2022;10(2):24. https://doi.org/10.3390/medsci10020024

[24] Phillips MA. Polyamines in protozoan pathogens. *J Biol Chem* 2018; 293(48):18746–18756. https://doi.org/10.1074/jbc.TM118.003342
## H&E staining

The tissues were fixed in 4% paraformaldehyde at 4°C for 24 to 48 hours. Following fixation, the mouse footpads were decalcified using formic acid aluminum chloride decalcifying fluid for up to 2 weeks. Tissues were immersed in reagents at least 20 times, and tissue volume and decalcification reagents were replaced twice every 3–4 days. After the bones melted, tissues were washed to flush out the decalcifier. Footpads were then dehydrated in graded ethanol, cleared in xylenes and infiltrated with paraffin mix. They were embedded in paraffin, and 5- to 7-µm sections were prepared and stained with hematoxylin and eosin. Briefly, sections were deparaffinized with xylene and gradually rehydrated through graded alcohols for staining. The sections were submerged in hematoxylin, differentiated in a solution of 1% hydrochloric acid in 70% alcohol, blued using water and stained with eosin.

## Flow cytometry

Isolated popliteal lymph node cells were washed with FACS buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA), nonspecific binding was blocked with an FcR blocking antibody (anti-Mouse CD16/32; eBioscience) before staining with labeled monoclonal antibodies against CD45.2 (BD Biosciences), CD3 (BD Biosciences) and CD4 (BD Biosciences). Intracellular staining of INF-γ (BD Biosciences) was carried out after permeabilization with Permfix (BD Biosciences). The fluorescence was examined on a BD LSR Fortessa.⁴² Data were analyzed using FlowJo software.

## Neutrophil isolation

Mouse bone marrow cells were counted and resuspended in 1 mL of ice-cold sterile PBS. Histopaque 1119 (Sigma; RNBF0417) was added in a 15-mL conical tube. Then 3 mL of Histopaque 1077 (Sigma; RNBF2365) was added as overlay on 3 mL of Histopaque 1119, and the bone marrow cell suspension was added as overlay on top of the Histopaque 1077. Tubes were centrifuged for 30 minutes at 2000 rpm at 25°C. Neutrophils were collected at the interface of the Histopaque 1119 and Histopaque 1077 layers. Finally, the collected neutrophils were washed twice with RPMI 1640 1× supplemented with 10% FBS and 1% penicillin-streptomycin and centrifuged at 1400 rpm for 7 minutes at 4°C. TAK1 deficiency was examined in neutrophils by PCR detection of the Map3k7 gene.

## Statistical analysis

The data were presented as the mean and SEM. GraphPad Prism v7.04 software (La Jolla, California) was used for data analysis. Statistically significant differences were determined by Student's two-tailed *t* test for two groups or one-way ANOVA with Bonferroni post-hoc test for three or more groups. *P* < 0.05 was considered statistically significant.

## Supplemental digital content

Supplemental materials are available at http://links.lww.com/IMD/A48. http://links.lww.com/IMD/A49, http://links.lww.com/IMD/A50, http://links.lww.com/IMD/A51.

## References

[1] Burza S, Croft SL, Boelaert M. Leishmaniasis. *Lancet* 2018;392(10151):951–970. https://doi.org/10.1016/s0140-6736(18)31204-2

[2] Serafim TD, Coutinho-Abreu IV, Dey R, et al. Leishmaniasis: the act of transmission. *Trends Parasitol* 2021;37(11):976–987. https://doi.org/10.1016/j.pt.2021.07.003

[3] Gurjar D, Kumar Patra S, Bodhale N, Lenka N, Saha B. *Leishmania* intercepts IFN-γR signaling at multiple levels in macrophages. *Cytokine* 2022;157:155956. https://doi.org/10.1016/j.cyto.2022.155956

[4] Rodrigues LS, Barreto AS, Bomfim LGS, et al. Multifunctional, TNF-α and IFN-γ-secreting CD4 and CD8 T cells and CD8(high) T cells are associated with the cure of human visceral leishmaniasis. *Front Immunol* 2021;12:773983. https://doi.org/10.3389/fimmu.2021.773983

[5] Zaatar MT, Simaan Y, Karam MC. Exogenous IL-13 exacerbates *Leishmania major* infection and abrogates acquired immunity to re-infection. *Parasitol Res* 2022;121(7):2009–2017. https://doi.org/10.1007/s00436-022-07539-y

[6] Varikuti S, Verma C, Natarajan G, Oghumu S, Satoskar AR. MicroRNA155 plays a critical role in the pathogenesis of cutaneous *Leishmania major* infection by promoting a Th2 response and attenuating dendritic cell activity. *Am J Pathol* 2021;191(5):809–816. https://doi.org/10.1016/j.ajpath.2021.01.012

[7] Lee SH, Charmoy M, Romano A, et al. Mannose receptor high, M2 dermal macrophages mediate nonhealing *Leishmania major* infection in a Th1 immune environment. *J Exp Med* 2018;215(1):357–375. https://doi.org/10.1084/jem.20171389

[8] Lecoeur H, Prina E, Rosazza T, et al. Targeting macrophage histone H3 modification as a *Leishmania* strategy to dampen the NF-κB/NLRP3-mediated inflammatory response. *Cell Rep* 2020;30(6):1870–1882.e4. https://doi.org/10.1016/j.celrep.2020.01.030

[9] Xu YR, Lei CQ. TAK1-TABs complex: a central signalosome in inflammatory responses. *Front Immunol* 2020;11:608976. https://doi.org/10.3389/fimmu.2020.608976

[10] Ajibade AA, Wang HY, Wang RF. Cell type-specific function of TAK1 in innate immune signaling. *Trends Immunol* 2013;34(7):307–316. https://doi.org/10.1016/j.it.2013.03.007

[11] Sato S, Sanjo H, Takeda K, et al. Essential function for the kinase TAK1 in innate and adaptive immune responses. *Nat Immunol* 2005;6(11):1087–1095. https://doi.org/10.1038/ni1255

[12] Liu HH, Xie M, Schneider MD, Chen ZJ. Essential role of TAK1 in thymocyte development and activation. *Proc Natl Acad Sci U S A* 2006; 103(31):11677–11682. https://doi.org/10.1073/pnas.0603089103

[13] Wang XD, Zhao CS, Wang QL, et al. The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1. *EMBO Rep* 2020;21(7):e48035. https://doi.org/10.15252/embr.201948035

[14] Omori E, Matsumoto K, Sanjo H, et al. TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis. *J Biol Chem* 2006;281(28):19610–19617. https://doi.org/10.1074/jbc.M603384200

[15] Kajino-Sakamoto R, Inagaki M, Lippert E, et al. Enterocyte-derived TAK1 signaling prevents epithelium apoptosis and the development of ileitis and colitis. *J Immunol* 2008;181(2):1143–1152. https://doi.org/10.4049/jimmunol.181.2.1143

[16] Malireddi RKS, Gurung P, Mavuluri J, et al. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation. *J Exp Med* 2018;215(4):1023–1034. https://doi.org/10.1084/jem.20171922

[17] Malireddi RKS, Gurung P, Kesavardhana S, et al. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. *J Exp Med* 2020;217(3): e20191644. https://doi.org/10.1084/jem.20191644

[18] López-Pérez W, Sai K, Sakamachi Y, Parsons C, Kathariou S, Ninomiya-Tsuji J. TAK1 inhibition elicits mitochondrial ROS to block intracellular bacterial colonization. *Proc Natl Acad Sci U S A* 2021;118(25):e2023647118. https://doi.org/10.1073/pnas.2023647118

[19] Carvalho AM, Guimarães LH, Costa R, et al. Impaired Th1 response is associated with therapeutic failure in patients with cutaneous leishmaniasis caused by *Leishmania braziliensis*. *J Infect Dis* 2021;223(3):527–535. https://doi.org/10.1093/infdis/jiaa374

[20] Lecoeur H, Buffet PA, Milon G, Lang T. Early curative applications of the aminoglycoside WR279396 on an experimental *Leishmania major*-loaded cutaneous site do not impair the acquisition of immunity. *Antimicrob Agents Chemother* 2010;54(3):984–990. https://doi.org/10.1128/AAC.01310-09

[21] Pessenda G, da Silva JS. Arginase and its mechanisms in *Leishmania* persistence. *Parasite Immuno* 2020;42(7):e12722. https://doi.org/10.1111/pim.12722

[22] Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. *Cell* 2022;185(16):2853–2878. https://doi.org/10.1016/j.cell.2022.06.010

[23] Carter NS, Kawasaki Y, Nahata SS, et al. Polyamine metabolism in *Leishmania* parasites: a promising therapeutic target. *Med Sci (Basel)* 2022;10(2):24. https://doi.org/10.3390/medsci10020024

[24] Phillips MA. Polyamines in protozoan pathogens. *J Biol Chem* 2018; 293(48):18746–18756. https://doi.org/10.1074/jbc.TM118.003342
[25] Toepp AJ, Petersen CA. The balancing act: immunology of leishmaniosis. *Res Vet Sci* 2020;130:19–25. https://doi.org/10.1016/j.rvsc.2020.02.004

[26] Müller AJ, Filipe-Santos O, Eberl G, Aebischer T, Späth GF, Bousso P. CD4+ T cells rely on a cytokine gradient to control intracellular pathogens beyond sites of antigen presentation. *Immunity* 2012;37(1):147–157. https://doi.org/10.1016/j.immuni.2012.05.015

[27] Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. *Nat Rev Immunol* 2016;16(9):581–592. https://doi.org/10.1038/nri.2016.72

[28] Chatelain R, Varkila K, Coffman RL. IL-4 induces a Th2 response in *Leishmania major*-infected mice. *J Immunol* 1992;148(4):1182–1187. https://doi.org/10.4049/jimmunol.148.4.1182

[29] Shapouri-Moghaddam A, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization, and function in health and disease. *J Cell Physiol* 2018;233(9):6425–6440. https://doi.org/10.1002/jcp.26429

[30] Schleicher U, Paduch K, Debus A, et al. TNF-mediated restriction of arginase 1 expression in myeloid cells triggers type 2 NO synthase activity at the site of infection. *Cell Rep* 2016;15(5):1062–1075. https://doi.org/10.1016/j.celrep.2016.04.001

[31] Caldwell RW, Rodriguez PC, Toque HA, Narayanan SP, Caldwell RB. Arginase: a multifaceted enzyme important in health and disease. *Physiol Rev* 2018;98(2):641–665. https://doi.org/10.1152/physrev.00037.2016

[32] Carrera L, Gazzinelli RT, Badolato R, et al. *Leishmania* promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. *J Exp Med* 1996; 183(2):515–526. https://doi.org/10.1084/jem.183.2.515

[33] Bussotti G, Piel L, Pescher P, et al. Genome instability drives epistatic adaptation in the human pathogen *Leishmania*. *Proc Natl Acad Sci U S A* 2021;118(51):e2113744118. https://doi.org/10.1073/pnas.2113744118

[34] Späth GF, Beverley SM. A lipophosphoglycan-independent method for isolation of infective *Leishmania* metacyclic promastigotes by density gradient centrifugation. *Exp Parasitol* 2001;99(2):97–103. https://doi.org/10.1006/expr.2001.4656

[35] Xie M, Zhang D, Dyck JR, et al. A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway. *Proc Natl Acad Sci U S A* 2006;103(46):17378–17383. https://doi.org/10.1073/pnas.0604708103

[36] Xing Y, Yao X, Li H, et al. Cutting edge: TRAF6 mediates TLR/IL-1R signaling-induced nontranscriptional priming of the NLRP3 inflammasome. *J Immunol* 2017;199(5):1561–1566. https://doi.org/10.4049/jimmunol.1700175

[37] Chen Y, Wang H, Shen J, et al. Gasdermin D drives the nonexosomal secretion of galectin-3, an insulin signal antagonist. *J Immunol* 2019; 203(10):2712–2723. https://doi.org/10.4049/jimmunol.1900212

[38] Mao K, Chen S, Chen M, et al. Nitric oxide suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock. *Cell Res* 2013;23(2):201–212. https://doi.org/10.1038/cr.2013.6

[39] Yu S, Zheng X, Zhou B, et al. SARS-CoV-2 spike engagement of ACE2 primes S2' site cleavage and fusion initiation. *Proc Natl Acad Sci U S A* 2022;119(1):e2111199119. https://doi.org/10.1073/pnas.2111199119

[40] Melo GD, Goyard S, Lecoeur H, et al. New insights into experimental visceral leishmaniasis: real-time in vivo imaging of *Leishmania donovani* virulence. *PLoS Negl Trop Dis* 2017;11(9):e0005924. https://doi.org/10.1371/journal.pntd.0005924

[41] Jain SK, Sahu R, Walker LA, Tekwani BL. A parasite rescue and transformation assay for antileishmanial screening against intracellular *Leishmania donovani* amastigotes in THP1 human acute monocytic leukemia cell line. *J Vis Exp* 2012;70:4054. https://doi.org/10.3791/4054

[42] Yao X, Zhang C, Xing Y, et al. Remodelling of the gut microbiota by hyperactive NLRP3 induces regulatory T cells to maintain homeostasis. *Nat Commun* 2017;8(1):1896. https://doi.org/10.1038/s41467-017-01917-2

**How to cite this article:** Cao X, Kokou KC, Yu S, Chen M, Niu J, Lecoeur H, Prina E, Späth GF, Meng G. TAK1 deficiency in macrophages increases host susceptibility to *Leishmania* infection. *Infect Microb Dis* 2023;5(3):127–136. doi: 10.1097/IM9.0000000000000127
[25] Toepp AJ, Petersen CA. The balancing act: immunology of leishmaniosis. *Res Vet Sci* 2020;130:19–25. https://doi.org/10.1016/j.rvsc.2020.02.004

[26] Müller AJ, Filipe-Santos O, Eberl G, Aebischer T, Späth GF, Bousso P. CD4+ T cells rely on a cytokine gradient to control intracellular pathogens beyond sites of antigen presentation. *Immunity* 2012;37(1):147–157. https://doi.org/10.1016/j.immuni.2012.05.015

[27] Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. *Nat Rev Immunol* 2016;16(9):581–592. https://doi.org/10.1038/nri.2016.72

[28] Chatelain R, Varkila K, Coffman RL. IL-4 induces a Th2 response in *Leishmania major*-infected mice. *J Immunol* 1992;148(4):1182–1187. https://doi.org/10.4049/jimmunol.148.4.1182

[29] Shapouri-Moghaddam A, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization, and function in health and disease. *J Cell Physiol* 2018;233(9):6425–6440. https://doi.org/10.1002/jcp.26429

[30] Schleicher U, Paduch K, Debus A, et al. TNF-mediated restriction of arginase 1 expression in myeloid cells triggers type 2 NO synthase activity at the site of infection. *Cell Rep* 2016;15(5):1062–1075. https://doi.org/10.1016/j.celrep.2016.04.001

[31] Caldwell RW, Rodriguez PC, Toque HA, Narayanan SP, Caldwell RB. Arginase: a multifaceted enzyme important in health and disease. *Physiol Rev* 2018;98(2):641–665. https://doi.org/10.1152/physrev.00037.2016

[32] Carrera L, Gazzinelli RT, Badolato R, et al. *Leishmania* promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. *J Exp Med* 1996; 183(2):515–526. https://doi.org/10.1084/jem.183.2.515

[33] Bussotti G, Piel L, Pescher P, et al. Genome instability drives epistatic adaptation in the human pathogen *Leishmania*. *Proc Natl Acad Sci U S A* 2021;118(51):e2113744118. https://doi.org/10.1073/pnas.2113744118

[34] Späth GF, Beverley SM. A lipophosphoglycan-independent method for isolation of infective *Leishmania* metacyclic promastigotes by density gradient centrifugation. *Exp Parasitol* 2001;99(2):97–103. https://doi.org/10.1006/expr.2001.4656

[35] Xie M, Zhang D, Dyck JR, et al. A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway. *Proc Natl Acad Sci U S A* 2006;103(46):17378–17383. https://doi.org/10.1073/pnas.0604708103

[36] Xing Y, Yao X, Li H, et al. Cutting edge: TRAF6 mediates TLR/IL-1R signaling-induced nontranscriptional priming of the NLRP3 inflammasome. *J Immunol* 2017;199(5):1561–1566. https://doi.org/10.4049/jimmunol.1700175

[37] Chen Y, Wang H, Shen J, et al. Gasdermin D drives the nonexosomal secretion of galectin-3, an insulin signal antagonist. *J Immunol* 2019; 203(10):2712–2723. https://doi.org/10.4049/jimmunol.1900212

[38] Mao K, Chen S, Chen M, et al. Nitric oxide suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock. *Cell Res* 2013;23(2):201–212. https://doi.org/10.1038/cr.2013.6

[39] Yu S, Zheng X, Zhou B, et al. SARS-CoV-2 spike engagement of ACE2 primes S2' site cleavage and fusion initiation. *Proc Natl Acad Sci U S A* 2022;119(1):e2111199119. https://doi.org/10.1073/pnas.2111199119

[40] Melo GD, Goyard S, Lecoeur H, et al. New insights into experimental visceral leishmaniasis: real-time in vivo imaging of *Leishmania donovani* virulence. *PLoS Negl Trop Dis* 2017;11(9):e0005924. https://doi.org/10.1371/journal.pntd.0005924

[41] Jain SK, Sahu R, Walker LA, Tekwani BL. A parasite rescue and transformation assay for antileishmanial screening against intracellular *Leishmania donovani* amastigotes in THP1 human acute monocytic leukemia cell line. *J Vis Exp* 2012;70:4054. https://doi.org/10.3791/4054

[42] Yao X, Zhang C, Xing Y, et al. Remodelling of the gut microbiota by hyperactive NLRP3 induces regulatory T cells to maintain homeostasis. *Nat Commun* 2017;8(1):1896. https://doi.org/10.1038/s41467-017-01917-2

How to cite this article: Cao X, Kokou KC, Yu S, Chen M, Niu J, Lecoeur H, Prina E, Späth GF, Meng G. TAK1 deficiency in macrophages increases host susceptibility to *Leishmania* infection. *Infect Microb Dis* 2023;5(3):127–136. doi: 10.1097/IM9.0000000000000127