An Intestinal Th17 Subset is Associated with Inflammation in Crohn's Disease and Activated by Adherent-invasive Escherichia coli

Abstract

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role.

Figure 1.

pTh17 cells express CCR5 and produce high levels of pro-inflammatory cytokines. A] Gating strategy to track pTh17 cells according to chemokine receptor expression. B] Frequencies of IL-17+, IFN-γ+, and IFN-γ/IL-17 co-producing cells among ex vivo-stimulated Th1, Th1/17, and Th17 subsets according to CCR5 expression from peripheral blood of HD [n = 20]. C] RORC/γt expression was measured ex vivo by intracellular staining of gated peripheral T cell subsets [HD, n = 6].T-bet protein expression was measured ex vivo by intracellular staining of gated peripheral T cell subsets [HD, n = 4]. MFI of T-bet in CD4⁺CD45RA⁺ naïve T cells are indicated as a dotted line as negative control. D] Percentage of T cells in sorted circulating T cell subsets that expressed a functional IL-23R, as assessed by STAT3 phosphorylation in response to brief [30 min] stimulation with IL-23. E] Frequencies of IL-17+, IFN-γ+, and IFN-γ/IL-17 co-producing cells by ex vivo-stimulated T cell subsets isolated from the lamina propria of CD [n = 6] and UC [n = 6] patients. Statistical significance was calculated using one-way ANOVA and reported as *p <0.05, **p <0.01, ***p <0.001. HD, healthy donors; CD, Crohn’s disease; MFI, Mean Fluorescence Intensity; UC, ulcerative colitis.

Figure 2.

RNA sequencing of circulating and intestinal Th17 subsets indicates that pTh17 cells are highly differentiated Th17 effector cells. A/B] Volcano plots of differentially expressed genes in pTh17 cells as compared with cTh17 cells and CCR5⁺Th1/17 cell in peripheral blood [A] or the intestinal lamina propria [B]. Selected, up-regulated Th17-effector genes and down-regulated, stemness-associated genes are indicated. C] Surface expression of PD1, CD27, CCR7, IL-1R1, and CCR4 proteins was validated by flow cytometry in CCR6⁺Th subsets in unrelated healthy donors, and compared with those from peripheral blood and the intestinal lamina propria of Crohn’s disease [CD] patients.

Figure 3.

Intestinal pTh17 cells are associated with inflammation in Crohn’s disease [CD]. A] Percentages of Th1/17 and Th17 subsets gated according to CCR5 expression in blood [n = 23], lymph nodes [LN; n = 7], and lamina propria [LPMC; n = 22] of CD patients. B] Frequencies of Th1/17 and Th17 subsets gated according to CCR5 expression in the lamina propria of CD [n = 22], ulcerative colitis [UC] [n = 29] and colorectal cancer [CRC] patients [non-inflamed areas, n = 6]. A, B] Statistical significance was calculated using one-way ANOVA and reported as *p <0.05, **p <0.01, ***p <0.001. C] Percentages of Th1/17 and Th17 subsets subdivided according to CCR5 expression isolated from paired, intestinal, inflamed, and non-inflamed areas of CD patients [n = 10]. Dots and bars represent individual patients and mean values, biopsies from the same patients are connected with a line. Statistical significance was calculated using paired t test and reported as *p <0.05, **p <0.01, ***p <0.001. D] Correlation between changes in the percentage of intestinal Th1/17 and Th17 subsets [CCR5[+] violet dots, CCR5[-] grey dots], and the Simple Endoscopic Score of disease Severity [SES-CD] before and after infliximab treatment in CD patients [n = 6]. Variation of frequencies of T cell subsets was calculated setting to 100% their percentage in the lamina propria of CD patients before infliximab treatment; ΔSES-CD score were calculated as the difference between SES-CD scores at baseline and after infliximab treatment. Correlations were determined using Spearman’s correlation coefficient.

Figure 4.

IL-23 promotes pTh17 cell generation in vitro and in vivo. A] Expression of CXCR3 [left panel] and CCR5 [right panel] on cTh17 cells [CCR6⁺CXCR3^-CCR5^-] isolated from HD [n = 4-8] after in vitro stimulation with anti-CD3/CD28 antibodies in the absence [–] or presence of IL-23 or IL-12. B] Concentrations of IL-23, IL-1β, IL-12, and IL-6 per gram of homogenised intestinal biopsies of CD [n = 10] and UC [n = 7] patients. C] Frequencies of Th1/17 and Th17 subsets among CD4⁺T cells in the intestinal lamina propria at baseline [before] and after 24 weeks [after] of risankizumab treatment of CD patients [n = 5]. Dots and bars represent individual patients and mean values, biopsies from the same patients are connected with a line. A–C] Statistical significance was calculated using paired t test and reported as *p <0.05, **p <0.01, ***p <0.001. HD, healthy donods; CD, Crohn’s disease; UC, ulcerative colitis.

Figure 5.

Adherent-invasive E. coli [AIEC] selectively activate pTh17 cells and induce high levels of IL-23 from DC. A] Experimental strategy to measure antigen specificities in intestinal Th subsets. B] Mean percentages of CD40L induction on the indicated intestinal T cell lines, derived from CD patients [n = 3–6], upon activation with autologous monocytes pulsed with CMV- [pp65, left], peptides derived from Candida albicans- [MP65, centre], or heat-killed E. coli [HKEB, 0111:B4 strain right]. C] CD40L induction on intestinal pTh17 cell line, derived from CD [n = 7] or UC patients [n = 3] after activation with autologous monocytes incubated with the E. coli strains AIEC-LF28 or Nissle 1917. D] Cytokine [left panel] and chemokine [right panel] production by monocyte-derived dendritic cells in response to AIEC-LF82 or E. coli Nissle 1917 strains was measured by ELISA [n = 9]. Statistical significance was calculated using Mann–Whitney’s test and reported as *p <0.05, **p <0.01, ***p <0.001. DC, dendritic cells; CD, Crohn’s disease; CMC, cytomegalovirus; UC, ulcerative colitis.

Figure 6.

T cells co-expressing CCR6 and CCR5 are enriched in E.coli-colonised areas. A] Representative image of CCR6 + CCR5 + T cells and E. coli localisation within CD-intestinal tissue. Sections were stained with DAPI [blue], CD3 [green], CCR6 [grey], CCR5 [red], and E. coli [orange]. B] E. coli cell abundance among CD patients subdivided into high and low groups of CCR6 + CCR5 + T cells. Statistical significance was calculated using unpaired Student’s t test for comparison within two groups.