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

Trypanosoma brucei is an extracellular protozoan parasite of humans and livestock that disseminates to multiple tissues, forming extravascular reservoirs and causing weight loss with loss of white adipose tissue (WAT) mass. Subcutaneous WAT (scWAT), adjacent to the skin, is a potential transmission-relevant niche yet its immunometabolic response to infection is poorly defined. Prior work implicated immune mediators such as TNF and IL-17A in regulating adipose tissue structure, and identified dermal γδ T cells producing IL-17 in local responses to T. brucei, but detailed profiling of scWAT was lacking. The study aims to define how T. brucei infection alters inguinal WAT (iWAT; a murine scWAT analog) structure and function and to test whether IL-17 signalling in adipocytes influences adipose wasting, systemic weight loss, and local parasite burden.

Previous studies show that T. brucei colonizes adipose depots and that infection associates with WAT mass loss and cachexia-like weight loss in mice. Cytokines such as TNF and IL-17A regulate adipose expansion and adipogenesis, and IL-17 family signalling can induce thermogenesis in adipose tissues. IL-17 responses are also elevated during infections with related trypanosomatids (T. cruzi, T. congolense) and can contribute to infection resistance. The skin has been identified as a significant reservoir for T. brucei with adjacent scWAT colonization; γδ T cells, particularly Vγ6+ IL-17-producing cells, modulate cutaneous inflammation and subcutaneous adipose wasting during T. brucei infection. However, the specific role of IL-17 signalling within adipocytes in scWAT during T. brucei infection remained unknown.

• Animals and infection: Male and female C57BL/6J mice, Il17af-/- (global IL-17A/F knockout), Adipoq-Cre x Il17rafl/fl (adipocyte-specific IL-17RA deletion), and IL-17A-GFP reporter mice were infected intraperitoneally with ~2×10^3 T. brucei brucei AnTat 1.1. Parasitaemia was monitored by microscopy using the rapid matching method. Experiments typically ended at 25 days post-infection (dpi); single-cell analyses were performed at 7 dpi.
• Phenotyping: Bodyweight trajectories and gross food intake (cage- or single-housed) were recorded. iWAT and gWAT pad masses were normalized to body weight.
• Histology: H&E staining for adipocyte lipid droplet morphometry; DAB HSP70 immunostaining to visualize tissue parasites; imaging and adipocyte size quantification with QuPath/Fiji Adiposoft.
• Serum assays: Glycerol quantified in mouse and human sera; mouse cytokines (IL-17A, IFNγ, TNFα) by MSD U-PLEX; human IL-17 by Luminex.
• Bulk RNA-seq: iWAT RNA at 25 dpi (male/female, naïve vs infected) processed by Novogene; Hisat2 alignment to GRCm39; DESeq2 for differential expression; KEGG enrichment by DAVID; heatmaps and PCA in R.
• Single-cell RNA-seq: iWAT stromal vascular fraction (SVF) isolated (adipocyte-depleted) at 7 dpi; 10x Genomics Chromium v3.1; Cell Ranger alignment to combined mm10/T. brucei reference; Seurat v4 integration, clustering, and marker-based annotation; subclustering of T cells and preadipocytes; gene module scoring (lipolysis, glycolysis, TCA, PPP) and Il17ra expression analysis; NicheNet for ligand-receptor predictions.
• Cytometry: Mass cytometry (CyTOF) panel for immune subsets and intracellular IFNγ/IL-17A; flow cytometry to validate IL-17A+ populations and γδ T cell CD27-defined subsets.
• qPCR: RT-qPCR for Dpp4, Pi16, Pparg in iWAT; genomic qPCR for parasite burden (Pfr2 copies/ng DNA) as tissue parasite load proxy.
• Statistics: Normality by Shapiro–Wilk; t-tests, Mann–Whitney, one-/two-way ANOVA with appropriate post hoc tests (p<0.05 significant). Human sample ethics approvals and animal welfare compliance detailed.

• Sex-specific weight and feeding changes: Infected male mice lost significant body weight over 25 dpi (two-way RM ANOVA, p<0.0001 at multiple time points), with decreased food intake early (p=0.0052), while females showed no significant weight change and minimal feeding alterations.
• iWAT wasting and dysfunction: iWAT mass was significantly reduced in infected males (normalized to body weight), with adipocyte hypotrophy and immune infiltration. Lipid droplet size distributions shifted toward smaller droplets in males (majority 10–200 µm^2 vs 51–900 µm^2 in naïve), less pronounced in females. Circulating glycerol was reduced in infected mice of both sexes and in human HAT patients, indicating impaired adipose function.
• Parasite presence in adipose depots: Trypanosomes were detected histologically in both iWAT and gWAT; in males, iWAT had fewer parasites than gWAT, while females showed no depot difference.
• Bulk transcriptomics at 25 dpi (iWAT): In males, 3828 genes upregulated and 3332 downregulated (|log2FC|>0.5, Padj<0.01); in females, 3177 up and 2606 down. Upregulated pathways were immune/inflammatory (antigen presentation, cytokine–receptor interactions, complement). Downregulated pathways were metabolic, including lipolysis (KEGG mmu04923) and thermogenesis; key lipolytic genes (Pnpla2, Fabp4, Lipe) and Ucp1 were decreased, suggesting energy conservation.
• IL-17 signatures and cytokines: TH17 differentiation transcripts (e.g., Irf4, Il21r, Il6ra) were elevated in male iWAT. CyTOF showed increased CD4+ Teff cells, with elevated IFNγ and IL-17A-producing Teff cells in both sexes. Serum IL-17A increased in infected mice and total IL-17 increased in HAT patients. Notably, Il17ra mRNA was upregulated in male iWAT (not female), suggesting sex-biased IL-17 responsiveness.
• Role of IL-17A/F (global knockout): Il17af-/- mice developed earlier and more severe clinical signs, but parasitaemia dynamics were largely unchanged in males (some differences in females). Il17af-/- males did not lose weight; instead, they gained weight and retained more iWAT mass and larger adipocyte droplets during infection versus WT, indicating IL-17A/F drives iWAT wasting and lipid utilization in males.
• scRNA-seq at 7 dpi (46,546 high-quality cells): Expansion of IL-17A-expressing T cell subsets was detected, including TH17 and Vγ6+ γδ T cells (CD27+), corroborated by IL-17A-GFP reporter flow cytometry. CD27+ IL-17A+ γδ T cells increased, while IFNγ+ CD27− γδ T cells decreased.
• Preadipocyte responsiveness to IL-17: Il17ra expression was selectively upregulated in preadipocyte clusters (interstitial Dpp4+ Pi16−/+, committed preadipocytes), not in mature adipocytes, indicating these progenitors are IL-17 targets. Early infection increased lipolysis module scores in committed preadipocytes and adipogenesis-regulatory cells and upregulated glycolysis, TCA, and PPP genes across preadipocyte subsets.
• Adipocyte-specific IL-17RA deletion (Adipoq-Cre x Il17rafl/fl): Mice were protected from infection-induced weight loss, had higher food intake, and experienced less iWAT wasting at 25 dpi. They exhibited a higher frequency of small adipocytes at baseline and during infection. iWAT showed increased expression of Dpp4 and Pi16 and lack of Pparg induction, indicating accumulation of DPP4+ PI16+ interstitial preadipocytes with impaired maturation. Strikingly, iWAT parasite burden (Pfr2 copies/ng DNA) was significantly higher in Adipoq-Cre x Il17rafl/fl mice versus WT, demonstrating that adipocyte IL-17 signalling contributes to local parasite control.

The study addresses how IL-17 signalling shapes scWAT responses during chronic T. brucei infection. Findings indicate that infection induces IL-17A production by multiple T cell types (TH17 and Vγ6+ γδ T cells), with male iWAT exhibiting higher Il17ra expression, aligning with sex-specific weight loss and adipose wasting. IL-17A/F promotes iWAT mass loss and adipocyte lipid utilization in males, while adipocyte-intrinsic IL-17RA signalling is crucial for proper preadipocyte maturation towards Pparg+ adipocytes and for limiting local parasite burden. Early transcriptional activation of lipolysis and metabolic pathways in preadipocytes suggests a temporal shift: early metabolic activation followed by chronic suppression of lipolysis and thermogenesis at 25 dpi, consistent with an energy conservation state and diminished serum glycerol. The unexpected increase in iWAT parasite load upon adipocyte IL-17RA deletion implies adipocytes actively orchestrate local immunity and tissue microenvironments that constrain parasites, potentially via metabolic support of immune effectors or via adipocyte-derived immunoregulatory signals. Differences from global Il17af deficiency suggest contributions from IL-17C/IL-17RA signalling and complex cytokine network effects. Overall, adipocyte IL-17 signalling emerges as a central coordinator of adipose dynamics, immune responses, and parasite control in scWAT during trypanosomiasis, with notable sexual dimorphism.

This work establishes that chronic T. brucei infection elicits IL-17-driven responses in murine subcutaneous adipose tissue and that adipocyte IL-17RA signalling regulates preadipocyte fate, limits iWAT wasting, and constrains local parasite burden. Global loss of IL-17A/F prevents weight loss and iWAT wasting (especially in males), while adipocyte-specific Il17ra deletion protects against wasting but compromises parasite control and fosters accumulation of DPP4+ PI16+ interstitial preadipocytes with impaired maturation. The study highlights adipocytes as key organizers of tissue and immune responses in infection and reveals sex-specific differences in IL-17 responsiveness. Future work should dissect IL-17C contributions, define mechanistic links between adipocyte metabolism and antiparasitic immunity, elucidate hormonal drivers of sex differences, and identify precise metabolic biomarkers of adipose state across infection stages and in human disease.

• Human cohorts were small and not stratified by sex for cytokine and glycerol analyses, limiting generalizability and assessment of sexual dimorphism in patients.
• scRNA-seq used pooled iWAT SVF from five male mice per condition with one technical replicate per condition, potentially limiting biological variability assessment.
• Time points emphasized early (7 dpi) and chronic (25 dpi) stages; intermediate dynamics and causality between IL-17 signalling, metabolism, and parasite control remain inferential.
• Adipocyte Il17ra deletion affects IL-17RA heterodimer signalling (including IL-17C), complicating attribution solely to IL-17A/F.
• The study focuses largely on iWAT; extrapolation to other adipose depots and to human adipose physiology needs confirmation.