The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries

Abstract

Specific microbial signals induce the differentiation of a distinct pool of RORγ⁺ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ⁺ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ⁺ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues.

Keywords: IL-17; NASH; RORγ; Treg; colon; liver; microbiota; muscle; stem cell; tissue regeneration.

Figure 1:. Accumulation of RORγ⁺ Treg cells in skeletal muscle early after acute injury

(A–C) scRNA-seq of Treg cells from hindlimb muscles of Foxp3^GFP mice 3 days after CTX-induced injury. A) 2D UMAP plot. B, C) Density plots of the expression of the indicated genes and signatures. (D) Flow-cytometry of RORγ⁺ Treg cells at steady-state and 3 days after CTX-induced injury. Upper panel: representative dot-plots (2–3 independent experiments); lower panel: summary data. (E) Time course of fractions (top) and numbers (bottom) of Helios⁺ and RORγ⁺ Treg cells in CTX-injured muscles. Unpaired t-test (D). See also Figure S1.

Figure 2:. A colonic provenance for muscle RORγ⁺ Treg cells

(A) Transcriptomic fold-change/fold-change (FC) plots comparing muscle RORγ⁺ vs spleen Treg cells at day 4 after CTX-induced injury vis-a-vis colon RORγ⁺ vs spleen Treg cells. Red: colonic RORγ⁺ Treg up-signature. (B) Spleen or colon CD4⁺ T cells were transferred into Tcrb^−/− mice followed by CTX injection. Experimental design (left), representative dot-plots (middle), and summary data (right) of RORγ⁺ Treg cells in colon and muscle. (C-E) Colonic photoconversion (PhC) of Kaede mice ± CTX-induced injury. C) Experimental design (left) and representative dot-plots (right) of colon PhC efficiency at day 0. D, E) emigration of the indicated colonic cells to hindlimb muscles after 48 hr. Representative dot-plots (left) and summary data (right). (F-J) Paired scRNA-seq and scTcr-seq of muscle and colon Treg cells on day 3 after CTX injection. F) Left: UMAP of muscle data; right: pie-charts of scTcr-seq data showing the proportion of clonally expanded cells in each cluster. Different colors depict individual clones; non-expanded clones in gray. G) Same as panel F for colon Treg cells. H) Upset plot of intra-organ and inter-muscle-colon clonotype sharing. Set size indicates total number of expanded clones for each subtype. On top, the total number of shared clones is displayed. Incidences of clone sharing between any muscle Treg subtype and colonic RORγ⁺ (red) or Helios⁺ (blue) Treg cells are highlighted. I) Pie-charts depicting numbers and frequencies of clonotypes shared between muscle Treg cell subtypes and colonic RORγ⁺ or Helios⁺ Treg cells. Different colors depict individual clones; non-expanded clones in gray. J) Clonal overlap score between muscle Treg subtypes and colonic RORγ⁺ vs Helios⁺ Treg cells in individual mice. Representative dot-plots are from 2–3 independent experiments. Unpaired t-test (B, D), paired t-test (E), or two-way ANOVA (J). See also Figure S2.

Figure 3:. Mechanisms of RORγ⁺ Treg cell accumulation in regenerating muscle

(A) Egress of diverse lymphocyte populations from the colon, and (B) their emigration to spleen after 24 hr of colonic PhC of Kaede mice. Top: representative dot-plots; bottom: summary data. (C) Muscle RORγ⁺ Treg cell numbers after CTX-induced injury in vehicle- or FTY720-treated mice. (D) Colon PhC coupled with CTX-induced injury as per Figure 2C. Representative dot-plots (left) and summary data (right) of emigration of colonic cells to hindlimb muscles after 48 hr. (E) RORγ⁺ Treg cell fraction (left) and number (right) in hindlimb muscles 2 days after CTX-induced injury in in control- (Ctl), αCCL2-, or CP-105696-treated mice. (F, G) Mice were treated with isotype (IgG) or αMHC-II antibody. F) Muscle RORγ⁺ Treg cell fraction (left) and number (right), and G) Ki-67 expression 3 days after CTX-induced injury. Representative dot-plots are from 2–3 independent experiments. Unpaired t-test (A, B, F, G), two-way ANOVA (C), or one-way ANOVA (E). See also Figure S3.

Figure 4:. Microbiota-dependence of muscle RORγ⁺ Treg cells

(A) Comparison of RORγ⁺ Treg cells from hindlimb muscles 3 days after CTX-induced injury of specific-pathogen-free (SPF) or germ-free (GF) mice. Left: representative dot-plots (3 independent experiments); right: summary data. (B) Same as panel A except SPF mice were treated or not with the Abx cocktail, VMNA (vancomycin, metronidazole, neomycin, ampicillin). (C, D) scRNA-seq comparison of muscle Treg cells from vehicle- and VMNA-treated Foxp3^GFP mice 3 days after CTX-induced injury. C) 2D UMAP plot of the two conditions combined (left), each individual condition (middle) and their differential (right). (D) Bubble-plot of the average transcript expression of key marker genes in muscle Treg cells of vehicle- vs VMNA-treated mice. (E, F) Monocolonization. E) Experimental design: GF mice were orally gavaged with Peptostreptococus magnus (Pm) or Clostridium ramosum (Cr) before CTX-induced injury. F) RORγ⁺ Treg cells in colonic lamina propria (left) or muscles (right). Unpaired t-test (A, B) or one-way ANOVA (F). See also Figure S4.

Figure 5:. Impacts of RORγ⁺ Treg cell depletion on immunocytes and tissue repair

Comparisons of hindlimb muscles from Maf^wt and Maf^ΔTreg littermates 0, 3, or 7 days after CTX-induced injury. Quantification of (A) Total, and B) RORγ⁺ Treg cells. (C) scRNA-seq of muscle Treg cells 3 days after CTX-induced injury. 2D UMAP plot of combined data from the two genotypes (left); each individual condition (middle) and their differential (right). (D) Quantification of muscle RORγ⁺CD4⁺Foxp3⁻ (Tconv) cells, and (E) production of IFNγ and IL-17A by Tconv and γδT cells. (F-J) Analysis of muscle repair efficiency 7 days after CTX-induced injury. F) Quantification of muscle neutrophils (NFs). G) Representative images of H&E staining (left); distribution of cross-sectional areas of individual centrally nucleated fibers (middle); average fiber areas for individual mice (right). H) Quantification of fibrotic areas via picrosirius red (PSR) staining. (I, J) Transcriptional analyses of whole muscle (n=3). I) Volcano plot illustrating FC differences between the two genotypes. Repair-related signatures are highlighted. J) Gene-signature scores of inflammatory gene sets. Representative dot-plots and images are from 2–4 independent experiments. t-test of weighted sums (G middle), Chi-squared test (I), otherwise unpaired t-test. See also Figure S5.

Figure 6:. Impacts of IL-17A on muscle stem cells and repair

(A-D) B6 mice were treated with vehicle or rIL-17A early, late or continuously (cont.) after CTX-induced injury. Muscles were analyzed 7 days post-injury. A) Treatment schematic. B) Distribution of cross-sectional areas of individual centrally nucleated fibers (left), and average fiber areas for individual mice (right). C) Quantification of muscle fibrotic areas via PSR staining. D) Quantification of muscle NFs. (E-G) RNA-seq analysis of sorted MuSCs from PBS- or rIL-17A-treated mice on days 0, 1 and 3 after CTX-induced injury. Treatment schedule as per panel A. E) Il17ra transcript quantification. F) k-means clustering of dynamically differential transcripts. Only select clusters are depicted. G) FC/FC plots for PBS- vs rIL-17A-treated mice on day 3 versus 1 after injury. MuSC differentiation signature genes highlighted in red. (H-J) Freshly isolated MuSCs were cultured in vitro with or without rIL-17A. Representative images (left, 2–3 independent experiments) and summary data (right) of H) EdU incorporation, I) myogenin (MyoG), and J) Myosin Heavy Chain (MyHC) expression after 2, 3 and 4 days of culture, respectively. Kruskal-Wallis test (B left), Chi-squared test of the number of signature genes falling on either side of the diagonal (G), Unpaired t-test (H-J), otherwise one-way ANOVA. See also Figure S6.

Figure 7:. Generality of RORγ⁺ Treg cells’ role in regulating tissue inflammation

(A) RORγ⁺ Treg cell fractions in diverse tissues of SPF and GF mice. (B, C) scRNA-seq of cardiac muscle Treg cells after myocardial infarction. B) 2D UMAP plot. C) Density plot of the indicated genes and signatures. (D) Quantification of liver RORγ⁺ Treg cells after one week of feeding with control diet (Ctl) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) ± VMNA treatment. (E-J) Maf^ΔTreg or Maf^wt mice were fed with CDAHFD for one week. Quantification of liver E) total Treg cells; F) RORγ⁺ Treg cells; G) RORγ⁺ Tconv (left) and γδT cells (right); and H) IL-17A expression. I) Volcano plot illustrating the transcriptional differences in livers from the two genotypes. J) Pathway analysis of transcripts significantly up-regulated ≥ 1.5-fold in Maf^ΔTreg livers. (K) Mice were fed with CDAHFD for 8 weeks. Representative images (left) and summary data (right) of liver PSR staining. Representative dot-plots and images are from 2 independent experiments. Unpaired t-test (A, D-F, K), or paired t-test (G, H). See also Figure S7.