Additional file 1 of GPR43 stimulation on TCRαβ+ intraepithelial colonic lymphocytes inhibits the recruitment of encephalitogenic T-cells into the central nervous system and attenuates the development of autoimmunity
Creators: name: Prado, Carolina
name: Espinoza, Alexandra
name: Martínez-Hernández, J. Eduardo
name: Petrosino, Joseph
name: Riquelme, Erick
name: Martin, Alberto J. M.
name: Pacheco, Rodrigo
Publisher: name: figshare
Year: 2023
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. Intestinal microbiome composition changes during EAE. Stool samples were collected from healthy miceand EAE mice at indicated time points and microbial composition was determined.. Alpha diversity metrics. Evaluation of microbial richness and diversity within each group was assessed using the Chao1 index and the Shannon Index.Analysis of beta diversity.Values represent the divergence of intestinal microbiome throughout EAE progression.PCoA analysis based on unweighted UniFrac throughout EAE progression.Intestinal microbiome composition at the Phylum level was analysed by mean percent ASV abundance and represented as pie chart for each time-point.Comparison between Bacteroidota and Firmicutes Phyla abundance throughout EAE progression.Intestinal microbiome composition at Family level of top three more abundant families was analysed by mean percent ASV abundance.In the box plots, error bars correspond to minimum and maximum data points, the horizontal line corresponds to the median, and the box contains 75% of the data points. Data were obtained from 4–11 mice per group. *, p &lt; 0.05; **, p &lt; 0.01 by one-way ANOVA followed by Tukey’s post-hoc test. Figure S2. GPR43 expression on IEL TCRαβ+ T-cells is upregulated by SCFAs.WT mice were non-treatedor treated with 250 mM SCFAs in the drinking water for two weeks and GPR43 expression was analysed in IEL CD4, CD8αβ and CD8αα subsets or total TCRαβ+ T-cells by flow cytometry. Scheme illustrating the experimental design.Quantification of GPR43 expression. Values are the MFI associated to GPR43 immunostaining.GPR43 expression was analysed in IEL CD4, CD8αβ and CD8αα subsets or total TCRαβ+ T-cells isolated from healthyor EAE mice at 25 dpi.Quantification of the clinical score throughout EAE development. The arrow indicates the time-point in which IEL were isolated.Quantification of GPR43 expression. Values are the MFI associated to GPR43 immunostaining.Representative histogram of GPR43 expression analysed by flow cytometry. Control, healthy miceand EAE mice.Each symbol represents data obtained from an individual mouse.Mean ± SD from 3–5 mice per group. *, p &lt; 0.05 by one-way ANOVA followed by Dunnett’s post-hoc testor unpaired Student’s t-test. Figure S3. Oral administration of SCFAs reduces EAE manifestation by increasing IL-10 and PD-1 expression on colonic lymphocytes. WT mice were treated with 250 mM SCFAs in the drinking water and two weeks later EAE was induced. SCFAs treatment was extended until 25 dpi. Treatments with acetate, propionate, butyrateor only waterare indicated.Disease severity was evaluated over time. Values represent mean ± SEM from a representativeof three independent experiments.The average water consumption per mouse was determined along EAE development. Data were obtained from four mice per group. Each symbol represents data obtained from an individual mouse per day. At 25 dpi IEL and LPL were isolated, re-stimulated wih PMA/Iono/BFA and then the absolute numbers of TCRαβ+ subsets, and the production of IL-10 and expression of PD-1 by CD4, CD8αβ and CD8αα TCRαβ+ T-cells was quantified. Data were obtained from 3–6 mice per group. Each symbol represents data obtained from an individual mouse. Mean ± SEM are indicated.*, p &lt; 0.05, **, p &lt; 0.01; ***, p &lt; 0.001; ****, p &lt; 0.0001 by one-way ANOVA followed by Dunnett’s post-hoc test. Purple asterisks correspond to comparisons between control and butyrate groups, whilst green asterisks correspond to comparisons between control and propionate groups and red asterisk correspond to comparisons between control and acetate groups. Figure S4. Donor colonic IEL are preferentially retained in the colonic epithelium of recipients. IEL were isolated from Cd45.1+/+ congenic mice and then i.v. transferred into Rag1−/− Cd45.2+/+recipient mice. Spleen, mesenteric lymph nodes, colonic intraepithelial lymphocytesand colonic lamina propria lymphocytes were isolated from recipients at different time points after the transfer and the percentage of total donor cellsand donor T-cells were quantified by flow cytometry. Schematic illustration of the experimental design. Frequency of CD45.1+ and TCRβ+ donor cells distributed in the different tissues analysed. Values represent mean ± SEM from 3 mice per group. Representative dot plots of CD45.1+and CD45.2+cells and TCRαβ+ and TCRγδ+ sub-populations of donor CD45.1+ cells isolated from the colonic intraepithelial compartment.*, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001; ****, p &lt; 0.0001 comparing with values obtained at time cero for each particular experimental group by one-way ANOVA followed by Dunnett’s post-hoc test. The asterisk colours indicate the experimental group with which they are associated. Figure S5. Dynamics of lymphocytes in the colonic lamina propria during EAE development. Colonic LPL were isolated from EAE mice at indicated time points and the frequency of CD19+ B-cells, TCRγδ+ and total TCRαβ+ T-cells was analysed.The absolute number of total or CXCR3+cells from the CD4+, CD8αα+ and CD8αβ+ TCRαβ subsets of colonic LPL isolated from EAE mice at indicated time points was determined. Percentage of total CD4+, CD8αα+ and CD8αβ+ TCRαβ T-cells from LPL or those expressing CD103, α4β7 and CCR9 were determined at different time points during EAE development.Values represent the mean ± SEM from 3–6 mice per group. *, p &lt; 0.05; **, p &lt; 0.01 by One-way ANOVA followed by Dunnett’s post-hoc test comparing frequencies of each cell type at different time points with the control condition.