γδ T cells control murine skin inflammation and subcutaneous adipose wasting during chronic Trypanosoma brucei infection

African trypanosomes colonise the skin to ensure parasite transmission. However, how the skin responds to trypanosome infection remains unresolved. Here, we investigate the local immune response of the skin in a murine model of infection using spatial and single cell transcriptomics. We detect expansion of dermal IL-17A-producing Vγ6+ cells during infection, which occurs in the subcutaneous adipose tissue. In silico cell-cell communication analysis suggests that subcutaneous interstitial preadipocytes trigger T cell activation via Cd40 and Tnfsf18 signalling, amongst others. In vivo, we observe that female mice deficient for IL-17A-producing Vγ6+ cells show extensive inflammation and limit subcutaneous adipose tissue wasting, independently of parasite burden. Based on these observations, we propose that subcutaneous adipocytes and Vγ6+ cells act in concert to limit skin inflammation and adipose tissue wasting. These studies provide new insights into the role of γδ T cell and subcutaneous adipocytes as homeostatic regulators of skin immunity during chronic infection.

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Juan F. F. Quintana 22/06/2023 Single cell RNA sequencing was conducted on on an an lllumina Novaseq 6000 sequencers by by Glasgow polyomics.Spatial RNA sequencing was conducted on on a NextSeq 550 lllumina instrument at at GenomeScan.Fastq sequence files were de-multiplexed, aligned, and annotated using a reference combined mouse (mmu10; https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/) and and Cell Ranger software (vCR6.1;single cell RNA sequencing) or or Space Ranger (vSR1.3;spatial RNA sequencing) softwares.Gene expression was counted using unique molecular identifier barcodes, and gene-cell matrices were constructed.FACS DIVA software (v9.0) was used for acquisition of of flow cytometry data.
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The data generated in this study have been deposited in the Gene Expression Omnibus database under accession code GSE226113 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE226113).A reviewer's token in included here: mlmroswwnraxbwn.The processed transcript count data and cell metadata generated in this study are available at Zenodo (DOI: 10.5281/zenodo.7677469).The flow cytometry data generated in this study are provided in the Supplementary Information/ Source Data file.
No human samples or subjects were used in this study No human samples or subjects were used in this study No human samples or subjects were used in this study No human samples or subjects were used in this study The maximum sample size was used for all the transcriptomics experiments presented in this study, within the limitations of the technology implemented (~10,000 cells per experiment, and 4 tissue sections per spatial transcriptomic slide).For in vivo experiments, we used a sample size of 4-5 animals per experimental condition.This sample size is based on measures of variance from historical data in the laboratory, and is enough to detect differences between experimental and control groups at a 5% significance level and 90% power.
No data were excluded from the analysis All experiments were conducted using independent biological replicates (at least n = 2 for single cell transcriptomics).The data obtained from the single cell experiments were succesfully replicated in independent in vivo experiments (e.g., expansion of CD27-gdT cells in the skin of female BALB/c mice).Spatial transcriptomics datasets were not replicated due to costs and logistics, but were independently validated using in situ hybridisation approaches.All flow cytometry measurements were conducted in at least two independent experiments with similar results.
Randomisation of scRNAseq or spatial transcriptomics was not appropriate as both naive and infected animals were processed in parallel and required additional labelling for downstream analysis (e.g., flow cytometry analysis, H&E analysis).Randomisation of in vivo experiments was not appropriate as the infected animals required to be closely monitored for the development of clinical adverse effects.Animals used for flow cytometry analysis described here as validation for single cell and spatial transcriptomics were randomly allocated into cages.
Blinding of scRNA was not appropriate as data processing and analysis need to be carried out appropriately and differentially for biological between experimental conditions.Measurements associated with parasitaemia and clinical scoring were conducted by a trained scientist blinded to the study.For flow cytometry analysis, blinding was not carried out in order to allow inclusion of appropriate measurements of background intensity and signal adjustments during acquisition.
The following anti-mouse antibodies were used for flow cytometry experiments: Fixable viability dye eFluor780 (Thermo, 1/1,000 All antibodies for flow cytometry were used at the recommended concentration by the manufacturer and tested in pilot titration experiments.The antibodies used for imaging were used exactly as recommended by the manufacturer, including the concentration.National Jewish Health) were backcrossed to FVB/n background N10.Female mice aged (6-8 weeks old) were used for infection.Mice were age and weight-matched within experiments.Animals were housed on a 12 h light-dark cycle and fed ad libitum.Room temperature was between 20-24C and humidity was between 50-70%.No wild animals were used in this study Only female mice were used in this study.Historical data in the laboratory suggests that both sexes respond similarly to infection by Trypanosoma brucei.No field samples were used in this studyAll animal experiments were approved by the University of Glasgow Ethical Review Committee and performed in accordance with the home office guidelines, UK Animals (Scientific Procedures) Act, 1986 and EU directive 2010/63/EU.All experiments were conducted under SAPO regulations and UK Home Office project licence number PC8C3B25C to Dr. Jean Rodgers.The in vivo work related to the single cell and spatial transcriptomic experiments were conducted at 21 post-infection (dpi) and correlated with increased clinical scores and procedural severity.