Human gut bacterial metabolism drives Th17 activation and colitis

Abstract

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity.

Keywords: T helper 17 cells; autoimmune disease; dietary supplementation; human gut microbiome; inflammatory bowel disease; microbial metabolism.

Figure 1.. E. lenta colonization promotes intestinal Th17 activity in the presence and absence of the gut microbiota.

(A-F) C57BL/6J SPF mice were gavaged every other day for 2 weeks with BHI media control or E. lenta 2243. (A) Representative ileal flow plots, (B) fold-change IL-17a+ CD4+ cells within the CD3+ gate, (C) IL-17a mean fluorescence intensity (MFI) in the ileal and colonic lamina propria (LP) (n=15). Data from three independent experiments. (D) Representative ileal flow plots, (E) fold-change of Rorγt+ CD4+ cells within CD3+ gate, (F) Rorγt MFI in the ileal and colonic LP (n=5). Fold-change is relative to BHI. Data from one experiment. (G-N) Germ-free (GF) mice were colonized with E. lenta 2243 for 2 weeks or remained GF. (G-I) Representative ileal flow plots, (H) fold-change of IL-17a+ CD4+ cells within the live CD3+ gate, (I) IL-17a MFI in the ileal and colonic LP (n=8–10). (J-L) Representative ileal flow plots, (K) fold-change of CD4+ Rorγt+ cells within the live CD3+ gate, (L) MFI of Rorγt in the ileal and colonic LP (n=8–10). Data from one representative experiment. Fold-change relative to GF. Each point represents an individual mouse, mean±SEM plotted in A-L. *p < 0.05; **p < 0.01; listed, Welch’s t-test. (M-N) RNA-seq of ileal LP CD4+ cells from GF or E. lenta monocolonized mice (n=4). (M) Volcano plot of Rorγt targets defined by transcription-target interactions (score > 1.5) (Ciofani et al., 2012). Red dots are significantly (p < 0.05; Wald test) differentially expressed between E. lenta monocolonized and GF mice. (N) Heatmap of significantly different Rorγt target transcripts (p < 0.05; Wald test). Transcripts in red are increased in Th17 versus Th0 as in (Ciofani et al., 2012) (fold increase of > 1.5 (Th17/Th0)). See Figures S1, S2, Data S1, S2.

Figure 2.. E. lenta induces IL-17a in an antigen-independent manner by lifting Rorγt inhibition.

(A-D) C57BL/6J SPF mice were gavaged for 2 weeks with BHI (control), E. lenta 2243 cell-free supernatant (CFS), or heat-killed E. lenta 2243 (dead). BHI and CFS were 3kDa filtered and 2X concentrated. (B) Representative flow plots, (C) fold-changes of IL-17a+ CD4+ cells within the live CD3+ gate, (D) IL-17a MFI in the ileum (relative to control) (n=4–5). Data from one experiment. (E-H) In vitro E. lenta CFS Th17 assay design. (F) Fold-change in IL-17a levels (relative to no treatment) measured via ELISA from Th17 skewed cells that received no treatment, BHI, E. lenta CFS, heat treated BHI or E. lenta CFS, <3kDa filtered BHI or E. lenta CFS (n=4–8). (G) Fold-change in IL-17a levels as measured by ELISA when treatments were added pre or post skewing. (n=14). (H) Fold-change in proliferation levels (relative to BHI) as quantified by MTT assay from the pre and post CFS treatment (n=9). Data from two independent experiments. p-values are Welch’s t-tests. (I-J) Rorγ luciferase assay where Rorγ luciferase reporter cells were treated with BHI or E. lenta 2243 CFS at 0.625X concentration (n=5), ursolic acid at 667nM as a positive control of a Rorγ inhibitor (n=2), or no treatment (n=2). (J) Rorγ activity as measured by luminescence - relative light unit (RLU). (K-N) Antigen-specificity experiments. (L) Relative IL-17a+ CD4+ levels within the live CD3+ gate as assessed by flow cytometry (levels relative to BHI T cell control). Data are from 2 independent experiments (n=10–12). (M) Fold-change in CD4+ IL-17a+ cells within the live CD3+ population from T cells isolated from SPF mice colonized with E. lenta and co-cultured with dendritic cells (DCs) loaded with lysates from BHI, E. lenta, B. adolescentis, no DCs or PMA/ionomycin (PI). Levels are relative to BHI. (N) Fold-change in CD4+ IL-17a+ cells within the live CD3+ population from T cells isolated from SPF mice colonized with B. adolescentis and co-cultured with DCs loaded with lysates from BHI, E. lenta, B. adolescentis, no DCs or PI. Levels are relative to the BHI control. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; listed; one-way ANOVA with Tukey or Holm Sidak tests unless otherwise stated. Mean±SEM is displayed. Model figures made with BioRender.com. See also Figures S2, S3, Data S3.

Figure 3.. E. lenta is associated with inflammatory bowel disease and contributes to colitis in a Rorc-dependent manner.

(A-B) Abundance of E. lenta was assessed using Metagenomic Intra-Species Diversity Analysis System (MIDAS) to map metagenomic reads to isolate genomes and metagenome assembled genomes from the human gut environment (Almeida et al., 2019; Nayfach et al., 2016). (A) Relative abundance of E. lenta from studies of ulcerative colitis (UC) (n=52), Crohn’s disease (CD) (n=53), and controls (n=100) (Qin et al., 2010; Weng et al., 2019). Species reads were normalized by the total number of reads across species in the sample then set relative to control. (B) Relative abundance of E. lenta in IBD (Crohn’s disease and ulcerative colitis patients combined) and control samples from (p=0.04, t-test on the glm coefficient). (C-G) Germ-free (GF) or E. lenta strain 2243 monocolonized mice (colonized for 2 weeks) were treated with 2% Dextran Sulfate Sodium (DSS) for 6 days and (D) disease was scored based on published scoring metrics (Chassaing et al., 2014). p-values are two-way ANOVA with Sidak test. Data are from 2 independent experiments (n=7–8). (E) Relative colon length (relative to GF). (F) Representative histology from GF or E. lenta monocolonized colons with H&E staining at 10X magnification. (G) Relative lipocalin levels as measured via ELISA in colon content. (H-K) IL-10^−/− SPF mice were gavaged with a BHI media or E. lenta 2243 3X a week for 6 weeks and (I) percentage of initial weight was tracked (n=11). p-values are two-way ANOVA with Sidak test. (J) Percent survival of IL-10^−/− mice gavaged with BHI or E. lenta 2243 three times a week for 10 weeks (n=19–26). Data are from 2 independent experiments. p-value is a Log-rank Mantel-Cox test. (K) Lipocalin levels were measured via ELISA in colon content after 6–10 weeks of gavage. Data are from 2 independent experiments and set relative to BHI (n=11). (L-P) SPF Wt or Rorc^−/− mice were gavaged with a BHI media or E. lenta 2243 every other day for 2 weeks then treated with 2% DSS for 7 days. (M) Disease scores over time in Wt SPF mice treated with BHI or E. lenta. p-values are two-way ANOVA with Sidak test (n=4). (N) Day 7 colon lengths. (O) Disease scores over time in Rorc^−/− SPF mice treated with BHI or E. lenta. (P) Day 7 colon lengths. Data are from 1 experiment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; listed; Welsh’s t-tests unless otherwise stated. Mean±SEM is displayed. Each dot represents an individual mouse. Model figures made with BioRender.com. See also Figures S2, S4.

Figure 4.. Th17 activation and colitis severity varies between E. lenta strains.

(A-D) Representative flow plots and fold-change relative to germ-free (GF) of the IL-17a+ CD4+ within the live TCRβ+ gate in the (A-B) ileal and (C-D) colonic lamina propria from GF mice or E. lenta 2243, 15644, 1356, or AB12 monocolonized mice. Data from 2 independent experiments (n=8–11). (E-H) GF mice or mice monocolonized with E. lenta 2243 or E. lenta 1356 for 2 weeks were treated with 2% DSS and (F) disease was tracked for 7 days (n=8–10). p-values are two-way ANOVA with Sidak test. (G) Day 7 colon lengths. (H) Lipocalin levels as quantified via ELISA in the colon content. G-H are relative to GF. *p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001; listed; one-way ANOVA with Holm Sidak or Tukey test unless otherwise noted. Mean±SEM is displayed. Each dot represents an individual mouse. Model figures made with BioRender.com. See also Figures S2, S5, Data S1, S2.

Figure 5.. The gut bacterial enzyme Cgr2 is associated with and sufficient for IL-17a induction and associated with autoimmune disease states.

(A) Th17 skewed CD4+ T cells were treated with CFS from E. lenta strains (14A, 28B, 15644, 1160AU, 1356, Valencia, 11C, AB12, AB8, 2243), or a BHI media control. Fold-change in IL-17a levels (relative to BHI) as measured via ELISA. E. lenta strains were classified as not significantly different from BHI (P > 0.05) or significantly higher than BHI (P < 0.05) by a one-way ANOVA Dunnett’s test compared to BHI (listed above each bar). With these classifications we performed comparative genomics using ElenMatchR (Bisanz et al., 2020) (n=8–12). Genes in the red box correspond to the (B) cardiac glycoside reductase (cgr)-associated gene cluster (cac) (Koppel et al., 2018). (C) Fold-change of IL-17a relative to no treatment in Th17 skewed cells treated with Rhodococcus erythropolis CFS with induced (+Thiostrepton) or uninduced (−Thiostrepton) expression of cgr2:wt (n=14), a partial loss-of-function variant cgr2:Y333N (n=12), no treatment (n=14), or BHI (n=8–12). p-values are one-way ANOVA with Holm Sidak test. (D) C57BL/6J SPF mice were gavaged with CFS from R. erythropolis with induced or uninduced expression of cgr2:wt, cgr2:Y333N, or a BHI+Thiostrepton. Fold-change of CD4+ IL-17a+ cells within the live CD3+ gate in the ileal lamina propria relative to BHI (n=10). Data represents a combination of at least two independent experiments for A-D. p-values are one-way ANOVA with Tukey test. (E) Shotgun metagenomic analysis from the PRISM IBD study (Franzosa et al., 2019). cgr2 presence was determined when ≥ 0.35 gene copies per cell were present within an E. lenta+ sample. Crohn’s disease (n=68), ulcerative colitis (n=53), and controls (n=34). A chi-squared test of independence determined no significant differences in cgr2 carriage. (F) The correlation of Mayo Clinic disease activity scoring with cgr2+ E. lenta (black n = 9) or cgr2− E. lenta (white n = 8) levels in E. lenta+ samples from ulcerative colitis stool samples. Rho values, p-values are one-tailed Pearson correlations. (G) Ratio of cgr2 to the universal E. lenta marker elnmrk1 in the same ulcerative colitis subjects in F. (H) cgr2 copies/μg DNA in healthy (n=11) and rheumatoid arthritis (RA) (n=12) stool samples. p-values are Mann-Whitney. (I) cgr2 prevalence in healthy and RA stool samples with the number of positive samples over total sample numbers on top of bars. p=0.15, one-sided Fisher’s exact test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; listed. Mean±SEM is displayed. Each point represents an individual subject. See also Figures S2, S5, Data S3.

Figure 6.. Characterization of Th17-modulatory factors metabolized by cgr2+ E. lenta.

(A-B) BHI media control, cgr2+ E. lenta 2243, and cgr2− 1356 3kDa filtered cell-free supernatant (CFS) were fractionated using RP-HPLC for our Th17 assay. Differentially active fractions were analyzed with LCHRMS. (B) RP-HPLC fractions 2–6 from BHI, 2243, and 1356 CFS were administered to Th17 skewed cells. Fold-change in IL-17a levels (relative to no treatment) as assessed via ELISA. (C-D) LCHRMS was performed on RP-HPLC fraction 5 from BHI, 2243, and 1356 CFS. Top 50 differentially abundant features (by p-value, one-way ANOVA) Z-scores displayed in a hierarchical clustered heatmap. (D) Log₂ fold-change of putative Cgr2 substrates (Cgr2S1–2) and putative Cgr2 metabolites (Cgr2M1–15) (n=4/group). (E) Feature-based molecular networking analysis of Cgr2S1 - m/z 963.5286 (yellow), which is in the lipids/lipid-like molecules, steroidal glycoside cluster, cosine score of 0.3889 for steroidal glycoside parent class assignment for Cgr2S1. (F) Intensity values of Cgr2S1 - m/z 963.5286 from LCHRMS analysis of RP-HPLC fraction 5. p-values are one-way ANOVA with Holm Sidak tests. (G) Steroidal glycosides (1: HILIC-pos_Cluster_1532, 2: C8-pos_Cluster_0588, 3: HILIC-pos_Cluster_1531) fold-change relative to controls in the PRISM and NLIBD/LLDeep IBD studies (Franzosa et al. 2019). Crohn’s disease (n=88), ulcerative colitis (n=76) and controls (n=56). (H-J) Correlation of steroidal glycosides relative abundance from the PRISM and NLIBD/LLDeep IBD studies to fecal calprotectin levels (μg/g). Spearman Rho, p-values listed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; listed Data are displayed as heatmaps, Tukey box and whisker plots, individual points, or as mean±SEM. Model figures made with BioRender.com. See also Data S4.

Figure 7.. Dietary arginine inhibits Th17 activation and colitis induction by cgr2+ E. lenta.

(A-C) Analysis of Th17 cells in germ-free (GF), E. lenta 2243 or 15644 monocolonized mice on a 1% or 3% arginine (Arg) diet. (B) Representative flow plots (C) fold-change of CD4+ IL-17a+ cells within the live CD3+ in the ileal lamina propria relative to the GF 1% Arg. Data are from 2 independent experiments (n=8–11). (D-G) SPF mice on a 1% or 3% Arg diet were gavaged with BHI, 2243, or 15644 for two weeks then treated with 2% DSS. (E) Disease scores over 7 days. p-values are two-way ANOVA with Sidak test (1% vs. 3% 2243) (n=5–10). (F) Relative colon lengths. (G) Relative lipocalin in colon content. Levels are relative to the BHI control diet in F-G. Data are from 2 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 one-way ANOVA with Tukey test unless otherwise stated. Mean±SEM is displayed. Each point represents an individual mouse. Model figures made with BioRender.com. See also Figures S2, S7.