Roseburia intestinalis stimulates TLR5-dependent intestinal immunity against Crohn's disease

Abstract

Background: Crohn's disease (CD) is a chronic inflammatory disorder characterized by intestinal immune dysfunction. Multiple factors, including gut dysbiosis, are involved in the pathogenesis of CD. However, the effect of commensal bacteria on controlling the inflammatory response in individuals with CD remains unclear.

Methods: We detected Toll-like receptor 2 (TLR2), TLR4, and TLR5 expression in Roseburia intestinalis (R. intestinalis)-treated mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Then, we quantified the signs of colonic inflammation, the proportion of regulatory T cells (Tregs) and the expression of thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β in TLR-5-deficient (Tlr5^-/-) mice, bone marrow chimera mice (generated using wild-type (WT) and Tlr5^-/- mice), and anti-TSLP/anti-TGFβ-treated C57BL/6 mice with colitis induced by TNBS. In vitro, we used the lipopolysaccharide (LPS)-stimulated human intestinal epithelial cell line Caco-2 as an inflammatory colon cell model treated with or without the TLR5-siRNA intervention in the presence of R. intestinalis and incubated human monocyte-derived dendritic cells (DCs) with the supernatant of Caco-2 cells. Then, we cocultured human CD4⁺ T cells with the aforementioned DCs to determine the differentiation of Tregs. Additionally, samples from patients with CD were collected to analyse the correlation between TLR5/TSLP/TGFβ expression and the percentage of R. intestinalis.

Findings: Here, we show that R. intestinalis inhibits the development of CD by increasing the differentiation of anti-inflammatory Tregs. Mechanistically, R. intestinalis stimulates TSLP production in intestinal epithelial cells (IECs) through TLR5 but not TLR2 or TLR4. TSLP produced by IECs specifically induces the secretion of interleukin-10 (IL-10) and TGFβ from DCs, which is necessary for subsequent Treg differentiation. Consequently, the depletion of TLR5 (using Tlr5^-/- mice) or inhibition of TSLP (using anti-TSLP neutralizing antibodies) attenuates the protective effect of R. intestinalis on experimental colitis in mice. Importantly, the expression of TSLP in patients with CD is positively correlated with the level of R. intestinalis.

Interpretation: These findings reveal the previously unknown mechanism of R. intestinalis-mediated intestinal immune regulation, which may provide the basis for new therapeutic strategies for CD.

Funding: This work was funded by the National Natural Science Foundation of China (81670504 and 81970494), the Key Project of Research and Development Plan of Hunan Province (2019SK2041) and the Changsha Municipal Natural Science Foundation (kq2014258).

Keywords: Crohn's disease; R. intestinalis; TSLP; Toll-like receptor 5; Tregs.

Figure 1

R. intestinalis-induced differentiation of Tregs requires TLR5 expression in vivo. (a and b) Western blots (a) and RT‒PCR (b) showing the expression of TLR2, TLR4, and TLR5 in the colon of different groups of mice treated with medium, TNBS, or TNBS plus R. intestinalis. Protein levels in Western blots were quantified using ImageJ software (a). Tubulin was used as a reference. (c and d) Tlr5^−/− mice were treated with TNBS to induce colitis and then administered R. intestinalis. The body weight change (c) and DAI score (d) were evaluated (n=6). (e and f) The histopathological scores of the Tlr5^−/− mice were assessed, and representative images of colon tissues stained with H&E are shown (n=6). (g and h) Representative flow cytometry plots (gated on CD4⁺ cells) and histograms of colonic CD4⁺CD25⁺FOXP3⁺ cells (Tregs) in Tlr5^−/− mice (n=6). *p<0.05, **p<0.01, ***p<0.001, and ns, not significant. Data represent the cumulative results from two or three independent experiments.

Figure 2

TLR5-expressing IECs are required for R. intestinalis-mediated amelioration of colitis in TNBS-induced mice. (a) Schematic illustrating the experimental procedure of BMT. Irradiated WT mice and Tlr5^−/− mice were reconstituted with BM from WT or Tlr5^−/− donors. After bone marrow reconstitution and SPF standardization, the chimeric mice were presensitized with TNBS administered cutaneously on day 0, administered TNBS solution intrarectally on days 8 and 10, followed by the intragastric administration of the R. intestinalis suspension or 3 consecutive days and then sacrificed for analysis on day 15. Abbreviations: Abx, antibiotics; R.I, R. intestinalis; TNBS, 2,4,6‑trintirobenzenesulfonic acid. (b and c) Changes in body weight (b) and DAI score (c) over the last 7 days of administration of TNBS and R. intestinalis in reconstituted mice (n=6). The solid line refers to the TNBS group compared with the control group (WT→WT (TNBS) vs. WT→WT (control), WT→Tlr5^−/− (TNBS) vs. WT→ Tlr5^−/− (control); Tlr5^−/−→WT (TNBS) vs. Tlr5^−/−→WT (control); Tlr5^−/−→ Tlr5^−/− (TNBS) vs. Tlr5^−/−→Tlr5^−/− (control)); the dotted line refers to TNBS + R.I group compared with the TNBS group (WT→WT (TNBS + R.I) vs. WT→WT (TNBS); WT→Tlr5^−/− (TNBS + R.I) vs. WT→Tlr5^−/− (TNBS); Tlr5^−/−→WT (TNBS + R.I) vs. Tlr5^−/−→WT (TNBS); Tlr5^−/−→Tlr5^−/− (TNBS + R.I) vs. Tlr5^−/−→Tlr5^−/− (TNBS)). ^&&&p<0.001, WT→Tlr5^−/− (TNBS + R.I) vs. WT→WT (TNBS + R.I); ^†p<0.05, Tlr5^−/−→WT (TNBS + R.I) vs. WT→WT (TNBS + R.I); ^‡p<0.05, ^‡‡p<0.01, WT→Tlr5^−/− (TNBS + R.I) vs. Tlr5^−/−→Tlr5^−/− (TNBS + R.I); ^$$$p<0.001, Tlr5^−/−→WT (TNBS + R.I) vs. Tlr5^−/−→Tlr5^−/− (TNBS + R.I). All control or TNBS groups (WT→WT (control) vs. WT→ Tlr5^−/− (control) vs. Tlr5^−/−→WT (control) vs. Tlr5^−/−→Tlr5^−/− (control); WT→WT (TNBS) vs. WT→ Tlr5^−/− (TNBS) vs. Tlr5^−/−→WT (TNBS) vs. Tlr5^−/−→Tlr5^−/− (TNBS)) showed no significant differences. (d and e) Representative images of colon tissues stained with H&E and colon histopathological score (n=6). (f and g) Histograms of Tregs in the reconstituted mice and representative colon flow cytometry plots (gated on CD4⁺ cells) (n=6). *p<0.05, **p<0.01, ***p<0.001, and ns, not significant. Data represent the cumulative results from two or three independent experiments.

Figure 3

R. intestinalis-mediated expression of TSLP and TGFβ requires TLR5 signalling in IECs. (a) Caco-2 cells transfected with the TLR5-siRNA or scrambled RNA were treated with PBS, LPS, LPS plus R. intestinalis in different amounts. ELISAs of TSLP (left panel) and TGFβ (right panel) levels in the supernatant of Caco-2 cells. R.I1, R.I2 and R.I3: bacteria to cell ratios of 10: 1, 5: 1 and 1: 1, respectively. (b and c) Colon TSLP (b) and TGFβ (c) mRNA expression was detected in the reconstituted mice using RT‒PCR (n=6). (d and e) ELISAs of the blood serum concentrations of TSLP (d) and TGFβ (e) in the reconstituted mice (n=6). *p<0.05, **p<0.01, ***p<0.001, and ns, not significant. Data represent the cumulative results from two or three independent experiments.

Figure 4

R. intestinalis induces Treg differentiation through IEC-mediated DC activation in a TLR5-dependent manner in vitro. (a) Schematic of the induction of Tregs. Caco-2 cells were stimulated with LPS (24 h), followed by treatment with R. intestinalis (24 h). Human monocyte-derived DCs (from human-derived monocytes) were incubated with Caco-2 supernatants for 48 h. Then, extensively washed DCs were cocultured with human naive CD4⁺ T cells (Th0) for 48 h to induce Treg differentiation. Human-derived monocytes and Th0 cells were collected from peripheral blood samples from healthy participants. FCM, Flow Cytometry. (b) Caco-2 cells were treated with PBS, LPS, R. intestinalis, and LPS plus R. intestinalis. DCs were stimulated with the supernatant from Caco-2 cells (48 h). The DCs were washed and cocultured with CD4⁺ Th0 cells, and the supernatant of Caco-2 cells without DCs was used as a control. The proportion of Tregs was evaluated using flow cytometry analysis. (c) The proportion of Tregs was analysed statistically. (d) The levels of IL-10 and TGFβ in the supernatant of the DCs mentioned above in (b) were detected using ELISAs. (e) Caco-2 cells were transfected with the TLR5-siRNA or scrambled RNA. Transfected Caco-2 cells were treated with different concentrations of PBS, LPS, and LPS plus R. intestinalis. Western blot analysis of TLR5 levels in Caco-2 cells. The quantification of the TLR5 protein levels in Western blots using ImageJ software is shown. Tubulin was used as a reference. (f and g) DCs were treated with supernatants from the different groups of Caco-2 cells mentioned above in (e), and then washed DCs were cocultured with CD4⁺ Th0 cells. A flow cytometry analysis of the number of Tregs was performed (gated on CD4⁺ cells), and data were processed for statistical analysis. *p<0.05, ***p<0.001, and ns, not significant. Data represent the cumulative results from three independent experiments.

Figure 5

R. intestinalis promotes DC-induced differentiation of Tregs to inhibit inflammation through a mechanism requiring TSLP expression in IECs. (a) Caco-2 cells transfected with the TSLP-siRNA or TGFβ siRNA were stimulated with LPS+R.I in the presence or absence of the recombinant TSLP/TGFβ proteins. The supernatant of the Caco-2 cells was used to induce human monocyte-derived DCs. TGFβ and IL-10 levels in DC culture supernatants were measured using ELISAs. (b and c) DCs were treated with the supernatants described above in (a) and then extensively washed and cocultured with human naive CD4⁺ T cells (Th0). The proportion of Tregs was analysed using flow cytometry (gated on CD4⁺ cells), and a statistical analysis was performed. (d) Schematic of C57BL/6 WT mice treated with the anti-TSLP (neutralization of TSLP) mAb, TNBS and R. intestinalis. (e and f) Changes in body weight (e) and DAI score (f) of each group (n=6). ***p<0.001, control vs. TNBS; ^#p<0.05, TNBS vs. TNBS+R.I; ^&p<0.05, anti-TSLP+TNBS vs. anti-TSLP+TNBS+R.I; ^$p<0.05, TNBS+R.I vs. anti-TSLP+TNBS+R.I. (g and h) The colon histopathological score and representative images of colon tissues stained with H&E (n=6). (i and j) Representative flow cytometry plots of Treg cells (gated on CD4⁺ cells) in colon tissues and statistical analysis (n=6). (k) ELISAs of TSLP, TGFβ and IL-10 levels in colon tissue homogenates (n=6). *p<0.05, **p<0.01, ***p<0.001, and ns, non‑significant. Data represent the cumulative results from two or three independent experiments.

Figure 6

TLR5, TSLP, TGFβ and IL-10 levels in the intestine are positively related to R. intestinalis in patients with CD. (a-d) Representative images of IHC detection of colonic TLR5, TSLP, TGFβ and IL-10 expression in healthy controls (ctrl) or patients with CD. (e-h) Each section stained using IHC was evaluated, and five images from each slide were randomly selected and measured using ImageJ. The mean densities of TLR5, TSLP, TGFβ and IL-10 were statistically analysed (ctrl: n=22; patients: n=24). (i) Comparative analysis of R. intestinalis in the faeces of the healthy controls or the patients with CD. (j-m) The correlations between the levels of TLR5, TSLP, TGFβ and IL-10 with R. intestinalis in controls and patients with CD were analysed (Spearman's correlation analysis with two-tailed p values). **p<0.01 and ***p<0.001. Data represent the cumulative results from two or three independent experiments.

Figure 7

A proposed model illustrating the inhibitory effect of R. intestinalis on inflammation and colitis. R. intestinalis increases TSLP expression in IECs expressing TLR5 and induces IL-10 production and TGFβ expression in DCs. These cytokines then promote the differentiation of Tregs to inhibit colonic inflammation. TLR5, Toll-like receptor 5; IECs, intestinal epithelial cells; DCs, dendritic cells; Treg, regulatory T cell.