Additional file 1 of IL-22 alters gut microbiota composition and function to increase aryl hydrocarbon receptor activity in mice and humans
Creators: name: Mar, Jordan S.
name: Ota, Naruhisa
name: Pokorzynski, Nick D.
name: Peng, Yutian
name: Jaochico, Allan
name: Sangaraju, Dewakar
name: Skippington, Elizabeth
name: Lekkerkerker, Annemarie N.
name: Rothenberg, Michael E.
name: Tan, Man-Wah
name: Yi, Tangsheng
name: Keir, Mary E.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1: Figure S1. Supplement to IL-22Fc microbiota response in mice. (A) After a two week acclimatization period during which animals were co-housed, six- to eight-week-old, female C57BL/6J mice (n=16) were randomly re-caged on study day 0 into two treatment groups and separately housed for the remainder of the experiment: IL-22Fc treated (n=8, four mice per cage) and control (n=8, four mice per cage). Animals were treated with 50 ug/mouse of either IL-22Fc or anti-ragweed isotype control via intraperitoneal injection three times a week for two weeks (as indicated by the red arrow), and euthanized on day 14. See methods for additional details. (B) Richness following IL-22Fc or aRW treatment. P-values determined by T-test. (C-D) Normalized abundance in control and IL-22Fc treated animals of the top highly IL-22Fc responsive bacterial genera consistently enriched (C) or depleted (D). Statistical significance determined by DESeq2 and corrected for false discovery. * BH-corrected p-value &lt;0.05. For all plots, triangle, square, and circle markers indicate ileal, colonic, and fecal samples respectively. Teal and red markers indicate animals treated with (IL-22Fc (n=8) or isotype control (n=8), respectively, respectively. Figure S2. Tryptophan metabolism pathways. (A) Depiction of well established tryptophan metabolism pathways relevant to the GI microbiome. As the functional capacity of the gut microbiome is highly complex and yet to be fully characterized, additional, as yet unidentified microbial metabolic pathways involving L-Trp metabolism may exist. Predominantly microbiome derived enzymes and metabolites are highlighted in green [13, 20]. L-Trp: L-Tryptophan, 5-HT: Serotonin, 5-HIAAld: 5-Hydroxyindoleacetaldehyde, 5-HIAA: 5-Hydroxyindoleacetic acid, L-Kyn: L-Kynurenine, ILA: Indole Lactic Acid, IPA: Indole Propionic Acid, IAAld: Indole Acetaldehyde, IAA: Indole Acetic Acid, I3C: Indole-3-Carboxaldehyde. Figure S3. Supplement to IL-22Fc tryptophan metabolism response in mice. (A) Fecal concentrations of additional microbially derived L-Trp metabolites included in our panel that were not significantly altered by IL-22Fc treatment. Teal and red markers indicate animals treated with IL-22Fc (n=8) or isotype control (n=8), respectively. (B) Log2(Fold change from baseline) in fecal concentrations of additional microbially derived L-Trp metabolites included in our panel. Statistical significance determined by Rank-Sum Test (. p&lt;0.1, * p&lt;0.05, ** p&lt;0.01). (C) Predicted prevalence of tryptophan synthase, tryptophanase, and aldehyde dehydrogenase among ASVs representing the highly IL-22Fc enriched or depleted bacterial genera. Statistical significance determined by Barnard’s Test (* p&lt;0.05). (D) Predicted prevalence of tryptophan synthase, tryptophanase, and aldehyde dehydrogenase among ASVs representing the highly IL-22Fc enriched or depleted bacterial genera, stratified by genus. Teal and Red bars indicate bacterial genera enriched or depleted following IL-22Fc treatment, respectively. Figure S4. Supplement to Intestinal AhR Signaling following IL-22Fc treatment in mice. (A) Scatterplot of L-Trp, I3C, and 5-HIAA fecal concentration vs. fecal AhR activity. (B-C) Scatterplot of predicted ileal (B) or colonic (C) metagenomic counts of tryptophan synthase, tryptophanase, and aldehyde dehydrogenase vs. fecal AhR activity. For all panels, Spearman's rank correlation coefficient (rho) was calculated to determine correlation strength. Figure S5. Supplement to Proxy Strain AhR Activity and Metabolite Production. (A-B) AhR activity, reported as Log2(fold change over matched sterile culture media) of sterile filtered culture supernatant from bacterial isolates representing the highly IL-22Fc depleted (A) or enriched (B) genera, stratified by bacterial isolate. (C) Metabolite production, reported as Log2(fold change over matched sterile culture media) of sterile filtered culture supernatant from bacterial isolates that induced AhR activity, stratified by bacterial isolate. Culture supernatants were collected and measured in triplicate for all panels. Statistical significance indicates relative AhR activity or metabolite concentration is statistically greater than zero as tested by one sample T-test (* p&lt;0.05, ** p&lt;0.01), indicating an in vitro AhR activity or metabolite concentration is statistically greater than that of matched sterile culture media. Figure S6. Supplement to efmarodocokin alfa effect on fecal metabolite concentrations in humans. (A) Fecal concentrations of L-Kyn at screening in HV (n=69) compared to UC (n=46). P-value determined by Rank-Sum test. (B) Fecal AhR Activity (reported as fold change over unstimulated wells) at screening in HV (n=69) and UC (n=46). P-value determined by T-test. (C) Fecal concentrations of L-Trp, I3C, IPA, and L-Kyn at screening in HV (n=69), moderate UC (mMCS ≤ 5, n=10), and severe UC (mMCS &gt; 5, n=13). P-value determined by Rank-Sum test. (D) Fecal AhR Activity (reported as fold change over unstimulated wells) at screening in HV (n=69) moderate UC (mMCS ≤ 5, n=10), and severe UC (mMCS &gt; 5, n=13). P-value determined by T-test. (E) AUC of Log2(fold change from screening) for fecal concentrations of L-Trp, I3C, IPA, and L-Kyn in enrolled UC participants following administration of efmarodocokin alfa (n=9) or placebo (n=5). P-value determined by T-Test. For panel E, only enrolled UC participants with a complete sample collection history (screening, day 29, day 43, day 64, and day 85) were considered for AUC calculations. Figure S7. Supplement to efmarodocokin alfa effect on fecal microbiota in enrolled trial participants. (A) Mean Log2(fold change in abundance at screening between enrolled UC and HV) of the IL-22Fc depleted (n=8) and IL-22Fc enriched (n=5) genera present in the fecal microbiome of enrolled trial participants at screening. P-value determined by T-test. (B) Normalized abundance of highly IL-22Fc depleted (B) or enriched (C) genera in the fecal microbiome of enrolled HV (n=39) and UC (n=24) participants at screening. P-value determined by DESeq2 analysis. Figure S8. Supplement to efmarodocokin alfa effect on fecal microbiota AUC in humans. AUC of Log2(fold change from screening) for normalized abundance of IL-22Fc depleted (A) or enriched (B) genera detected in enrolled UC patients receiving either placebo (n=5) or efmarodocokin alfa (n=9) treatment. Only enrolled UC patients with a complete sample collection history (screening, day 29, day 43, day 64, and day 85) were considered for AUC calculations. Note that CAG-873 was not detected in any enrolled UC patients with a complete sampling history. P-value determined by T-Test.