IL-17 signalling is critical for controlling subcutaneous adipose tissue dynamics and parasite burden during chronic murine *Trypanosoma brucei* infection

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a neglected tropical disease caused by the parasite *Trypanosoma brucei*. HAT is endemic in sub-Saharan Africa, causing significant morbidity and mortality, particularly in rural communities. A major clinical manifestation of HAT is weight loss, often severe, accompanied by wasting of adipose tissue. This adipose tissue wasting is observed in both human and animal models of infection, but the underlying mechanisms remain poorly understood. Previous research has indicated a correlation between *T. brucei* infection and adipose tissue dysfunction, particularly in the gonadal white adipose tissue (gWAT). Studies have shown that *T. brucei* colonizes gWAT, leading to reduced tissue mass and contributing to overall weight loss. However, the precise immune mechanisms driving this adipose tissue wasting remain unclear. Cytokines, such as tumor necrosis factor (TNF) and interleukin-17A (IL-17A), are known to play crucial roles in regulating adipose tissue structure and function. IL-17A, in particular, has been implicated in regulating adipogenesis and thermogenesis in both white and brown adipose tissue. Furthermore, elevated IL-17 levels have been reported in other parasitic infections, suggesting a potential role in the immune response to *T. brucei*. This study focuses on the subcutaneous white adipose tissue (scWAT) as another potential site of parasite colonization and a key player in the weight loss observed in HAT. Due to its proximity to the skin, scWAT represents an important potential reservoir for *T. brucei*, offering the parasite access to nutrients and facilitating transmission to the vector. The researchers aim to investigate the immune response in the scWAT during *T. brucei* infection, specifically focusing on the role of IL-17 signaling. The study utilizes a mouse model, focusing on inguinal white adipose tissue (iWAT) as an analog of human scWAT, to investigate the impact of *T. brucei* infection on adipose tissue structure, function, and the local immune response.

The literature review section of the paper discusses previous research on the impact of *Trypanosoma brucei* infection on adipose tissue and the role of IL-17 in various contexts. It highlights prior studies demonstrating weight loss and adipose tissue wasting in mice and humans infected with *T. brucei*, with a particular focus on the gonadal white adipose tissue (gWAT). The review covers the known effects of TNF and IL-17A on adipose tissue structure and function, including their roles in limiting tissue expansion and inhibiting adipogenesis. The involvement of IL-17A signaling in thermogenesis in both white and brown adipose tissues is also reviewed. Furthermore, the literature review summarizes existing knowledge on the elevation of IL-17 in infections caused by *T. cruzi* and *T. congolense*, and its importance in controlling resistance to these infections. The review underscores the existing gap in understanding the precise role of the immune response to *T. brucei* infection in influencing adipose tissue structure and function, particularly in the context of scWAT, which is a clinically relevant site of infection. This gap is the impetus for the current study, which aims to address the lack of detailed analysis of the scWAT response to *T. brucei* infection.

The study employed a multi-faceted approach, combining several complementary techniques to comprehensively analyze the effects of *T. brucei* infection on murine iWAT. The methodology included: 1. **In vivo infection model:** Mice were infected with *T. brucei brucei* Antat 1.1 strain, and the clinical symptoms (piloerection, hunching), parasitemia, body weight, food intake, and adipose tissue mass (iWAT and gWAT) were monitored over time. 2. **Histological analysis:** H&E staining was used to assess iWAT morphology and lipid droplet size, while HSP70 staining was employed to determine parasite load in iWAT and gWAT. 3. **Bulk transcriptomics:** Bulk RNA sequencing (RNA-seq) was performed on iWAT samples to analyze gene expression profiles in both male and female mice, comparing naive and infected states. This analysis involved differential gene expression analysis using DESeq2 and pathway enrichment analysis using DAVID, focusing on KEGG pathways. 4. **Mass cytometry (CyTOF):** CyTOF was employed to perform high-dimensional analysis of immune cell populations in the iWAT. This allowed for detailed characterization of immune cell subsets, including macrophages, granulocytes, dendritic cells, B cells, and various CD4+ T cell subsets, and measurement of cytokine production (IFNγ, IL-17A). 5. **Single-cell RNA sequencing (scRNA-seq):** ScRNA-seq was performed on the stromal vascular fraction (SVF) of iWAT at an early time point (7 dpi) to examine cellular heterogeneity and transcriptional responses to infection at a higher resolution than with bulk RNA-seq. Specific cell types were identified based on marker gene expression. NicheNet analysis was employed to predict ligand-receptor interactions among cells. 6. **Genetic models:** Global *Il17a/f* knockout mice and mice with adipocyte-specific deletion of the *Il17ra* (AdipoqCreIl17rafl/fl) were utilized to investigate the specific role of IL-17 signaling in the observed phenotypes. This allowed researchers to directly assess the impact of IL-17 deficiency on body weight, iWAT mass, lipid droplet size, and parasite burden. 7. **RT-qPCR:** RT-qPCR was performed to measure the expression of specific genes, including *Il17ra*, *Dpp4*, *Pi16*, and *Pparg*, in iWAT samples. 8. **Cytokine and glycerol quantification:** Enzyme-linked immunosorbent assay (ELISA) or multiplex assays were used to determine circulating levels of cytokines (IL-17A, TNFα, IFNγ) in both mouse and human serum samples. A glycerol assay kit was used to measure circulating glycerol levels. Statistical analyses involved appropriate tests (t-tests, ANOVA, etc.) depending on data distribution. All experiments were conducted with appropriate ethical approvals.

The study revealed several key findings regarding the role of IL-17 signaling in the response to *T. brucei* infection: 1. **Sex-dependent weight loss:** Male mice exhibited significant weight loss during infection, whereas female mice did not show significant changes in body weight. This difference was associated with variations in food intake and iWAT mass. 2. **iWAT wasting:** *T. brucei* infection led to a significant reduction in iWAT mass in male mice, accompanied by adipocyte hypotrophy (reduction in lipid droplet size). Female mice also showed a reduction, though not significant. 3. **Impaired adipose tissue function:** Both male and female mice exhibited reduced circulating glycerol levels upon infection, reflecting impaired adipose tissue function. This was corroborated by similar observations in human HAT patients. 4. **Transcriptional changes:** Bulk transcriptomic analysis revealed upregulation of immune and inflammatory pathways and downregulation of metabolic pathways, including lipolysis and thermogenesis, suggesting a shift towards energy conservation in infected mice. Upregulation of TH17-related genes was more prominent in male mice. 5. **Immune cell expansion:** Mass cytometry showed an expansion of macrophages, granulocytes, and dendritic cells in both sexes. A significant increase in IL-17A-producing CD4+ Teff cells was observed in both sexes. Notably, the iWAT of infected male mice showed upregulation of the *Il17ra* gene, indicating increased responsiveness to IL-17A signalling in males. 6. **Critical role of IL-17A/F:** Global IL-17A/F deficiency protected against infection-induced weight loss and iWAT wasting in male mice but not females. IL-17A/F deficiency led to increased clinical symptom severity. 7. **Single-cell analysis insights:** Single-cell RNA-seq revealed that Treg cells and a cluster of IL-17A-producing Vγ6+ cells are the primary sources of IL-17A. The IL-17A receptor was upregulated specifically in preadipocytes during infection. Increased frequency of interstitial preadipocytes was observed. 8. **Adipocyte IL-17 signalling and preadipocyte development:** Adipocyte-specific deletion of the *Il17ra* also prevented weight loss and iWAT wasting in male mice, resulting in an accumulation of smaller adipocytes and an increase in the number of DPP4+ PI16+ interstitial preadipocytes. Importantly, this adipocyte-specific deletion also resulted in a significantly increased parasite burden in the iWAT. These findings highlight the critical role of IL-17A/F signaling, particularly through the adipocyte IL-17 receptor, in controlling iWAT dynamics, preadipocyte differentiation, and local parasite control during chronic *T. brucei* infection. The sex-specific differences observed emphasize the need for sex-specific considerations in the study of HAT.

This study provides significant new insights into the complex interplay between *T. brucei* infection, the immune system, and adipose tissue metabolism. The findings clearly establish a previously unappreciated role for IL-17 signaling in the control of adipose tissue dynamics during HAT. The sex-dependent effects observed highlight the need to consider sex as a biological variable in future studies of this neglected tropical disease. The observation of elevated IL-17A in human HAT patients further strengthens the translational relevance of these findings. The unexpected finding that abrogation of adipocyte IL-17 signaling leads to increased parasite burden suggests that adipocytes play a more active role in the host's immune response than previously thought. The study challenges the traditional view of adipose tissue as a passive energy storage depot and reveals its important role as a coordinator of immune responses and tissue dynamics during infection. The data support a model in which IL-17 signaling contributes to adipocyte maturation, potentially promoting an effective immune response and parasite clearance. This study's comprehensive approach utilizing diverse experimental methods strengthens the validity of its conclusions and opens avenues for future research exploring the detailed mechanisms underlying these interactions and the potential for therapeutic interventions targeting IL-17 signaling pathways in the treatment of HAT.

This research demonstrates a crucial role for IL-17 signaling in controlling adipose tissue dynamics and parasite burden during *T. brucei* infection. The study highlights a sex-dependent response, with male mice exhibiting more significant weight loss and iWAT wasting linked to increased IL-17 signaling and receptor expression in the iWAT. Adipocyte-specific IL-17 receptor deletion revealed that adipocytes are essential for regulating preadipocyte development and parasite control. These findings suggest that therapeutic interventions targeting IL-17 signaling may offer potential strategies for managing the weight loss and adipose tissue wasting associated with HAT. Future research should investigate the specific molecular mechanisms by which IL-17 signaling influences adipocyte maturation, immune cell function, and parasite control, and explore potential sex-specific therapeutic strategies.

While the study provides valuable insights, it has some limitations. The use of a mouse model may not fully recapitulate the complexities of human HAT. The study primarily focused on male mice, and additional research is required to explore the female response in more detail. The sample size for some experiments could be considered relatively modest, although it was sufficient to show statistical significance in most key findings. Further investigation of the underlying molecular mechanisms, for example, by evaluating metabolites, could provide deeper insights into how IL-17 signaling affects adipose tissue metabolism. The potential impact of other cytokines and immune cell types beyond the scope of the study should also be explored in future work.