IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r ^-/- and Il3r ^+/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

Keywords: CYTOKINES; INFLAMMATORY BOWEL DISEASE; INTESTINAL T CELLS.

Figure 1

IL-3 is upregulated in inflamed tissue of patients with IBD. (A) Microarray analysis of the publicly available data set GSE97012: mean values of selected cytokines plotted for patients with non-IBD (CON, n=27) versus UC (left, n=22) or patients with CD (right, n=19). Genes are ordered from low to high p values (top to bottom) with the black line separating genes with and without significantly different expression (above/below). (B) IL3 mRNA expression in colon tissue from patients with colonic CD (n=41), UC (n=68) and CON (n=21) donors as determined by qPCR. Comparison of levels between the entities (left) and comparison of levels between inactive and active disease (right); Kruskal-Wallis test with Dunn’s multiple comparisons post hoc test. (C,D) Immunofluorescence of cryosections from patients with CD (n=5–8), UC (n=5–6) and CON (n=5–8) for IL-3 (green) and CD4 (C, red) or CD14 (D, red); counterstaining with Hoechst (blue). Left: representative images, white arrowheads highlight double positive cells, scale bars – 25 µm; right: quantification of double positive cells per high power field (HPF); one-way analysis of variance with Tukey’s multiple comparisons post hoc test. CD, Crohn’s disease; CON, non-IBD control IL, interleukin; mRNA, messenger RNA; qPCR, quantitative PCR; UC, ulcerative colitis.

Figure 2

Il-3 alleviates T cell transfer colitis. (A) Il3 mRNA expression in tissue from various organs of unchallenged C57BL6/J mice as determined by qPCR. n=6–10 per group. (B) Il3 mRNA expression in tissue from various organs of unchallenged Rag1 ^−/− mice (pre transfer, left, n=5–6) and Rag1 ^−/− mice with established T cell transfer colitis (post transfer, right, n=3–11) as determined by qPCR. (C) Time course of Il3 mRNA expression in lamina propria mononuclear cells (LPMCs) of Rag1 ^−/− mice with T cell transfer colitis; n=3–11 per time point. (D) Concentration of Il-3 in supernatants of splenocytes and LPMCs re-stimulated with anti-CD3/28 antibodies at different time points of T cell transfer colitis as determined by ELISA; n=2–5 per time point. (E) Colitis in Rag1 ^−/− mice after transfer of naïve CD4⁺ T cells from Il3 ^+/+ and Il3 ^−/− mice. Upper panels: mini-endoscopy. Lower panels: histology of colon tissue. Left: representative images (scale bars – 12.5 µm), right: quantitative endoscopic and histological scores of disease severity. n=14–15 per group, unpaired t-test. (F–H) Colitis in Rag1 ^−/− mice after transfer of naïve CD4⁺ T cells from Il3r ^+/+ and Il3r ^−/− mice. (F) Mini-endoscopy (top) and histology of colon tissue (bottom). Left: representative images (scale bars – 12.5 µm), right: quantitative endoscopic and histological scores of disease severity. n=16–17 per group, Mann-Whitney (endoscopy) and unpaired t-test (histology). (G) Representative in vivo IVIS luminescence imaging of reactive oxygen species after i.p. injection of L-012. (H) Weight course. Normalisation to weight on the day adoptive T cell transfer was performed. n=16–17 per group, mixed-effects analysis with Sidak post hoc test. IL, interleukin; i.p., intraperitoneal; mLN, mesenteric lymph node; mRNA, messenger RNA; MPO, myeloperoxidase; pLN, peripheral lymph node; qPCR, quantitative PCR.

Figure 3

Il3r alters T cell recirculation. (A) Immunofluorescence staining for CD4 (left, red) and MPO (right, red) in colon tissue of Rag1 ^−/− mice after transfer of naïve CD4⁺ T cells from Il3r ^+/+ and Il3r ^−/− mice counterstained with Hoechst (blue). Left panels: representative images, white arrows highlight CD4⁺ or MPO⁺ cells. Right panels: quantification of CD4⁺ and MPO⁺ cells per high power field (HPF). n=11–12 per group, unpaired t-test (CD4) and Mann-Whitney (MPO); scale bars – 50 µm. (B) Migration of Il3r ^−/− or Il3r ^+/+ thymus T cells over porous membranes towards rm Ccl19. Upper panels: representative flow cytometry. Lower panel: quantification of Ccl19-specific transmigration; n=8–9 per group, Mann-Whitney test. (C) Recirculation of FarRed-stained Il3r ^−/− and CFSE-stained Il3r ^+/+ thymus T cells after i.p. injection. Upper panels: representative flow cytometry of peritoneal, blood and splenic cells. Lower panel: quantification; n=6–7 per group, paired t-test. i.p., intraperitoneal; MPO, myeloperoxidase.

Figure 4

Il3r-deficiency alters structure and dynamics of the cytoskeleton. (A,B) Differentially expressed genes in RNA sequencing of Il3r ^−/− or Il3r ^+/+ thymus T cells (n=3 per group). (A) Volcano plot of the top 4245 regulated genes, grey line indicates significance threshold. Significantly differentially expressed cytoskeleton-related genes (as identified by Database for Annotation, Visualization and Integrated Discovery (DAVID) annotation analysis, n=90) highlighted in red, selected gene names indicated. (B) Heatmap of normalised log(fold change) of 90 cytoskeleton-related genes as identified by DAVID annotation analysis. (C) Coral Kinome Tree plotted with data from a Pamgene kinome analysis of Il3r ^−/− vs Il3r ^+/+ thymus T cells. Kinases with lower activity in Il3r ^−/− are highlighted in blue, kinases with higher activity in Il3r ^−/− are highlighted red. (D) Left panels: volcano plots showing all differently activated serin/threonine kinases (STK) and protein tyrosine kinases (PTK) in Il3r ^−/− versus Il3r ^+/+ thymus T cells. Cytoskeleton-associated kinases are highlighted in blue. Right panels: heatmaps of mean kinase statistics for cytoskeleton-associated STK and PTK. (E) Scanning electron microscopy (SEM) of the actin cortex in Il3r ^−/− or Il3r ^+/+ thymus T cells. Upper panels: representative SEM pictures (left, scale bars – 2 µm) and magnifications with representative FiNTA software-based quantification grids (right, scale bar – 1 µm). Lower panel: quantification of the mean mesh hole area; n=7–9 per group, unpaired t-test. (F) Fluorescence recovery after photobleaching (FRAP) of the actin cortex in Il3r ^−/− or Il3r ^+/+ thymus T cells. Schematic representation of FRAP experiment (upper panel) and quantification of the half-time recovery period (lower panel); n=6–8 per group, unpaired t-test.

Figure 5

Increased deformability of Il3r ^−/− lymphocytes. (A) Schematic representation of RT-DC measurement. Cells from a reservoir are pumped through a narrow constriction in a polydimethylsiloxane (PDMS)-based microfluidic chip (15 µm capillary) and deformation ensuing due to hydrodynamic stresses is analysed; figure drawn with licensed BioRender software. (B) Representative RT-DC scatter plot of deformation versus cell size (cross-sectional area) of thymus T cells from Il3r ^−/− and Il3r ^+/+ mice highlighting representative cells of the populations. (C) Representative kernel density estimate (KDE) plot (with grey isoelasticity lines) of thymus T cells from Il3r ^−/− and Il3r ^+/+ mice (left) and quantification of the calculated Young’s modulus of lymphocytes from thymus and pLN as well as pregated CD3⁺CD4⁺ lamina propria T cells from mice with transfer colitis. Quantification normalised to the mean of the Il3r ^+/+ cells in each independent experiment; n=7–12 per group, Mann-Whitney test; effect size: Cohen’s d=1.468 (Thymus), d=1.363 (CD3⁺CD4⁺ LP), d=0.065 (pLN). (D) Schematic representation of atomic force microscopy (AFM) cantilever and cell deformation (left). Quantification of the elasticity of CD4⁺ lamina propria lymphocytes from mice with established transfer colitis with Il3r ^−/− and Il3r ^+/+ T cells by AFM; n=212 each, Mann-Whitney test. (E) Histograms of Cd123 expression on CD3⁺CD4⁺ Treg (CD25⁺CD127⁻) or non-Treg (CD25⁻) Il3r ^+/+ thymocytes as determined by flow cytometry. Data are representative for six mice from four independent experiments. (F) Quantification of the calculated Young’s modulus of total CD4⁺ and regulatory CD4⁺ T cells (right) from Il3r ^−/− and Il3r ^+/+ mice by AFM; n=100–172 per group, Mann-Whitney test. (G) Representative RT-DC KDE plot (upper panel) and quantification of the calculated mean Young’s modulus (lower panel) of splenic CD3⁺CD4⁺ CD25⁺ and CD25⁻ lymphocytes from Il3r ^−/− and Il3r ^+/+ mice; n=6–10 per group, two-way analysis of variance with Tukey’s multiple comparison test; effect size: Cohen’s f=0.071. LP, lamina propria; pLN, peripheral lymph node; Treg, regulatory T cell; WT, wild-type.

Figure 6

Enhanced recirculation of Il3r ^−/− regulatory T lymphocytes from the inflamed colon. (A) Schematic representation of recirculation studies in T cell transfer colitis in vivo (cf. main text for details); figure drawn with licensed BioRender software. (B) Representative lightsheet microscopy of Il3r ^+/+ mLN after staining with vesseldye-CF770 (red) and with antibodies against Lyve-1-eFluor570 (green), CD4-AF647 (light blue) and counterstaining with Hoechst (dark blue); three-dimensional reconstruction with Imaris software V.9.9; scale bar – 200 µm. (C) Representative virtual sections of mLNs from Rag1 ^−/− mice with transfer colitis induced by Il3r ^−/− or Il3r ^+/+ T cells and treated with ozanimod/DATK32 or placebo control analysed by lightsheet microscopy. Whole-organ staining with antibodies against Lyve-1-eFluor570 (white) and CD25-AF488 (scale from black to light magenta); scale bars – 400 µm, dashed square highlights the mLN centre, white arrowheads highlight representative CD25 signal. (D) Representative flow cytometry of mLNs (left, gated on CD3⁺CD4⁺) and quantification of the CD25⁺Foxp3⁺ regulatory T cell (Treg) index (right, Treg fraction in ozanimod/DATK32 per fraction in placebo) in mLNs (upper panel) and spleens (lower panel). n=4–5 per group, Mann-Whitney test. (E) Immunofluorescence staining for Foxp3 in colon tissue of Rag1 ^−/− mice with transfer colitis induced by Il3r ^−/− or Il3r ^+/+ T cells: Representative confocal microscopy (upper panels) and quantification of Foxp3⁺ cells as well as the ratio of Foxp3⁺ to CD4⁺ cells (stained on sequential sections, lower panels). n=11–12 per group, Mann-Whitney (Foxp3) and unpaired t-test (ratio); scale bars – 50 µm, white arrowheads highlight Foxp3⁺ cells. HPF, high power field; mLN, mesenteric lymph node; S1P, sphingosine-1-phosphate; Treg, regulatory T cell.

Figure 7

IL-3 stiffens human CD4⁺ T cells and correlates to Tregs in the inflamed gut. (A) Representative RT-DC kernel density estimate plot (with grey isoelasticity lines) of CD4⁺ peripheral blood T cells stimulated with anti-CD3/CD28 antibodies and treated with or without rh IL-3 (upper panel) and quantification of the calculated mean Young’s modulus normalised to values of untreated samples (lower panel); n=8 per group, Wilcoxon signed-rank test; effect size: Cohen’s d=1.323. (B) Representative atomic force microscopy force curve (upper panel) and quantification of the calculated Young’s modulus relative to the mean of untreated cells (lower panels) of CD4⁺ peripheral blood T cells stimulated with anti-CD3/CD28 antibodies and treated with or without rh IL-3; n=59–62 per group, paired Mann-Whitney test. (C) Representative western blot analysis (upper panels) and quantification (lower panels) of G-Actin and F-Actin (as well as GAPDH control) in CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies and treated with or without rh IL-3; n=5–6 per group, Wilcoxon matched pairs signed-rank test. (D) Migration of CD4⁺ peripheral blood T cells over porous membranes towards rh CCL19 or rh CCL21. Left panels: Representative flow cytometry. Right panels: Quantification of transmigration; n=6 per group, paired t-test. (E) Flow cytometry of lamina propria mononuclear cells isolated from biopsies of patients with IBD. Representative histogram of CD123 staining on CD4⁺CD25⁺Foxp3⁺ Treg cells and CD4⁺CD25⁻Foxp3⁻ non-Treg cells (left). Data are representative for five independent experiments. (F) Correlation of IL3 mRNA with IL10 and TGFB1 mRNA expression as determined by qPCR in colon tissue from patients with IBD (n=21–45). Spearman’s R, significance levels and a regression line are indicated. (G) Immunofluorescence of cryosections from patients with IBD (CD or UC, each n=5) for CD4 (green) and Foxp3 (magenta) or IL-3 (green); counterstaining with Hoechst (blue). Left panels: representative images, scale bars – 25 µm; right panel: correlation of IL-3⁺ cells and CD4⁺Foxp3⁺ per total CD4⁺ cells; Spearman’s R, significance levels and a regression line are indicated. CD, Crohn’s disease; F-Actin; filamentous actin; G-Actin, globular actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBD, inflammatory bowel disease; IL, interleukin; mRNA, messenger RNA; qPCR, quantitative PCR; rh, recombinant human; RT-DC, real-time deformability cytometry; TGF, transforming growth factor; Tregs, regulatory T cells; UC, ulcerative colitis.