Additional file 1 of Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation
Creators: name: Sargsian, Shushan
name: Mondragón-Palomino, Octavio
name: Lejeune, Alannah
name: Ercelen, Defne
name: Jin, Wen-Bing
name: Varghese, Alan
name: Lim, Yvonne A. L.
name: Guo, Chun-Jun
name: Loke, P’ng
name: Cadwell, Ken
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Physiology
subject: Ecology
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file 1: Figure S1. Enzymatic profile of the OA isolates in vivo. Representative images of α-chymotrypsin and β-glucuronidase enzymatic activity measured in fresh stool from mice monocolonized with the indicated OA isolates. GF, germ-free; SPF, Specific pathogen free. Figure S2. Bacterial burden and absolute numbers of Treg and Th17 cells induced by the OA isolates. (A) Colony forming units (CFUs) per mg of stool from mice colonized with indicated bacteria on the day cells were collected for analysis by flow cytometry. Each dot corresponds to one mouse. (B-D) Absolute numbers of Rorγt+ Helios− Foxp3+ Tregs (B), Rorγt+ Foxp3+ Tregs (C) and Rorγt+ Foxp3− Th17 cells (D) in the colon lamina propria of mice colonized with the indicated bacteria. SPF, Specific pathogen free; MF, Minimal flora mice; MF stool, GF mice gavaged with stool from Minimal flora mice; KH mix, Mice gavaged with consortium of 17 Clostridia strains from Atarashi et al., 2013; SFB, Segmented filamentous bacteria; GF, Germ-free. Each dot corresponds to one mouse. Each group was tested in at least 2 independent experiments consisting of 2–5 mice. Kruskal–Wallis with Dunn’s multiple comparisons test was used to compare between each group and GF in B-D. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S3. Host transcriptional response to colonization by OA isolates. (A) PCA plot showing the overall transcriptional response (RNAseq) of cecal cells from GF mice and mice monocolonized with OA2, OA6 and OA8. Each dot corresponds to one mouse. (B-F) Heat maps of differentially expressed genes between pairs of groups. Genes with base mean &gt; 50 and absolute value of logFC ≥ 2 are shown. (G-O) Expression of indicated genes between GF controls and monocolonized groups. Each dot represents one mouse. Bar graphs show mean ± SEM. Kruskal–Wallis with Dunn’s multiple comparisons test was used to compare between all groups. *p &lt; 0.05. Figure S4. Genes correlated with Foxp3 expression. (A) Scatter plot of transcripts correlated with Foxp3 expression. Figure S5. Monocolonization with a subset of OA isolates increases total bile acids in stool. Quantification of total bile acids per g of stool from mice monocolonized with the indicated bacteria. PBS, PBS as a technical control; GF, germ-free mouse; MF, minimal flora mouse. Kruskal–Wallis with Dunn’s multiple comparisons test was used to compare between each group and GF. **p &lt; 0.01, ****p &lt; 0.0001. Figure S6. Spearman correlation matrix depicting relationships between OA isolate properties. Heatmap showing Spearman correlations between all measured variables for the OA isolates including Treg and Th17 cell induction, Trichuris egg hatching, and production of SCFAs, BAs, H2S, and enzymes. Variables with insufficient data points to complete the correlation analysis were removed from the heatmap. Figure S7. dbRDA analysis to determine effect size of bacterial enzyme production on lymphocyte differentiation induced by the OA isolates. (A) Enzyme production data used to perform the distance-based redundancy analysis (dbRDA) and statistical significance of the F statistic for each variable. (B) PCA biplot depicting dbRDA results for Treg and Th17 cell induction and enzyme production. (C) Proportion of variance explained by 10 significant variables on the variance of Treg and Th17 cell induction. Figure S8. Treg and Th17 cell induction is not correlated with Trichuris egg hatching ability by the OA isolates. (A) Relationship between T. muris egg hatching rate and the induction of Rorγt+ Foxp3+ Tregs for the OA isolates. (B) Relationship between T. muris egg hatching rate and the induction of Rorγt+ Helios− Foxp3+ Tregs for the OA isolates. (C) Relationship between T. muris egg hatching rate and the induction of Rorγt+ Foxp3− Th17 cells for the OA isolates. (D) Relationship between T. trichiura egg hatching rate and the induction of Rorγt+ Foxp3+ Tregs for the OA isolates. (E) Relationship between T. trichiura egg hatching rate and the induction of Rorγt+ Helios− Foxp3+ Tregs for the OA isolates. (F) Relationship between T. trichiura egg hatching rate and the induction of Rorγt+ Foxp3− Th17 cells for the OA isolates. Spearman r and p values are depicted on each plot. Each dot corresponds to the average value per OA isolate and the red line depicts the linear regression slope.