Interleukin-6-mediated CCR9+ interleukin-17-producing regulatory T cells polarization increases the severity of necrotizing enterocolitis

Abstract

Background: Increased frequency of CCR9⁺ CD4⁺ T cells in peripheral blood is linked to several gastrointestinal inflammatory diseases; however, its relationship with necrotizing enterocolitis (NEC) is not understood. We investigated whether the frequencies of CCR9⁺ CD4⁺ T cells and related subsets were increased in peripheral blood of both patients and mice with NEC.

Methods: CCR9⁺ CD4⁺ T cells and related subsets were evaluated by flow cytometry in peripheral blood collected from both patients and mice with NEC and controls. The suppressive function of CCR9⁺ regulatory T (Treg) cells in NEC was assessed via in vitro suppression assay. An in vitro T cell polarization assay was performed to investigate the role of proinflammatory cytokines in Treg cell polarization. In vivo Treg cell polarization analysis was performed using NEC mice treated with anti-interleukin-6 (IL-6) receptor antibody.

Findings: A higher proportion of CCR9⁺ CD4⁺ T cells occurred in peripheral blood of both patients and mice with NEC than in controls. Elevated CCR9⁺ CD4⁺ T cells were primarily CCR9⁺ IL-17-producing Treg cells, possessing features of conventional Treg cells, but their suppressive activity was seriously impaired and negatively correlated with the severity of intestinal tissue injury. IL-6 promoted polarization of CCR9⁺ Treg cells to CCR9⁺ IL-17-producing Treg cells, and blocking IL-6 signalling inhibited this conversion in vitro and ameliorated experimental NEC in vivo.

Interpretation: Collectively, these data suggested that CCR9⁺ IL-17-producing Treg cells may be a biomarker of severity and highlight the possibility that antibodies targeting IL-6R could ameliorate NEC by modulating lymphocyte balance. FUND: This work was supported by the Science and Technology Planning Project of Guangdong Province, China (2017A020215100), the Science and Technology Foundation of Guangzhou, China (201704020086 and 201604020154), the Medical Scientific Research Foundation of Guangdong Province, China (A2017304 and A2014704), and the Social Science and Technology Development Foundation of Dongguan, China (2016108101037).

Keywords: CCR9; IL-17-producing Treg cell; Interleukin-6; Necrotizing enterocolitis.

Fig. 1

CCR9⁺ CD4⁺ T cells and related subset in peripheral blood from NEC patients and controls. (a and b) CCR9 expression in lymphocytes was assessed by flow cytometry in peripheral blood mononuclear cells from NEC patients (n = 77) and controls (CTRL; n = 80). (a) Representative flow cytometric plots of CCR9 expression in gated CD3⁺ T cells (top), CD4⁺ T cells (middle) and CD8⁺ T cells (bottom). Numbers on the representative flow cytometry graph indicate the percentage of CCR9⁺ cells in that subset. (b) The absolute numbers (left) and frequencies (right) of CCR9⁺ CD3⁺ T cells (top), CCR9⁺ CD4⁺ T cells (middle), and CCR9⁺ CD8⁺ T cells (bottom). P-values were calculated using Mann-Whitney U–test. (c) The expression of IL-17 and Foxp3 in CCR9⁺ CD4⁺ T cells from patients with NEC (n = 6) and controls (CTRL; n = 7) was evaluated by quantitative real-time PCR (qRT-PCR). ns: not significant, *, P < .05. (D - F) The expression of IL-17 and Foxp3 in CCR9⁺ CD4⁺ T cells from patients with NEC and controls (CTRL) was evaluated by intracellular cytokine staining and flow cytometry. (d) Representative flow cytometric plots showing IL-17 expression in CCR9⁺ CD4⁺ Foxp3⁺ Treg (CCR9⁺ Treg) cells in patients with NEC and controls. (e) The frequency of CCR9⁺ IL-17⁺ Treg cells in patients with NEC (n = 65) and CTRL (n = 66). ***, P < .001. (f) Spearman correlation (r) between the frequencies of CCR9⁺ CD4⁺ T cells and CCR9⁺ IL-17⁺ Treg cells in NEC patients (n = 65) and CTRL (n = 66).

Fig. 2

CCR9⁺ IL-17⁺ Treg cells are phenotypically similar to Treg and Th17 cells. (a–b) Three subsets of CD4⁺ T cells defined by the expression of Foxp3, CD127, IL-17, and CCR9: conventional Treg cells (Treg), CCR9⁻ Foxp3⁺ CD127^dim/− cells; conventional Th17 cells (Th17), CCR9⁻ Foxp3⁻ IL-17⁺ cells; CCR9⁺ IL-17⁺ Treg cells (CCR9⁺ IL-17⁺ Treg), CCR9⁺ Foxp3+ CD127^dim/− IL-17⁺ cells. Surface markers or cytokines of CD4⁺ Foxp3⁺ Treg cells, CCR9⁺ IL-17⁺ Treg cells, and Th17 cells were examined by flow cytometry after stimulation for 5 h with Leukocyte Activation Cocktail in the presence of brefeldin A protein transport inhibitor. Representative histograms (a) and mean fluorescence intensity (b) showing the expression of CTLA-4, CD127, CCR6, and CD161; the expression of the intracellular cytokines IL-10 and IL-17 was measured in the above samples. n = 8; *, P < .05; **, P < .01; ***, P < .001; ns: not significant; MFI, mean fluorescence intensity. P-values were calculated using Kruskal-Wallis with paired comparisons test.

Fig. 3

The suppression activity of CCR9⁺ IL-17⁺ Treg cells is impaired in patients with NEC. (a) Representative intracellular staining for CCR9⁺ IL-17⁺ Treg cells in gated freshly isolated CCR9⁺ CD4⁺ CD25⁺ CD127^dim/− Treg (CCR9⁺ Treg) cells in NEC patients (n = 4) and controls (CTRL; n = 4). (b) The frequencies of CCR9⁺ IL-17⁺ Treg cells in freshly isolated CCR9⁺ Treg cells in NEC patients (n = 4) and CTRL (n = 4). *, P < .05. (c) A representative T cell proliferation and suppression assay of freshly sorted CCR9⁺ Treg cells (5 × 10⁴) isolated from a NEC patient and a CTRL co-cultured with purified CD4⁺ CD25⁻ responder T cells at a 1:1 ratio. Responder CD4⁺ T cells were stained with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) and stimulated with anti-CD3/CD28-coated microbeads, and their proliferation in 5 days was determined by flow cytometry. Activated (Teff) and nonactivated (unstimulated) CD4⁺ T cells without Treg were used as controls. (d) The proliferation of responder CD4⁺ T cells in different situations. n = 4. P-values were calculated using Student's t-test or one-way ANOVA with Bonferroni multiple comparison test. (e) Distribution of CCR9⁺ IL-17⁺ Treg cells in CD127^dim/− CCR9⁺ Treg fractions defined based on their expression of CD45RA and Foxp3. The numbers on the representative flow cytometry graph shows the number of CCR9⁺ IL-17⁺ Treg cells in each CCR9⁺ Treg fraction; percentages (shown in round brackets) indicate the frequencies of CCR9⁺ IL-17⁺ Treg cells within each fraction of the total CCR9⁺ Treg cells from a representative sample. (f) The percentage distribution of CCR9⁺ IL-17⁺ Treg cells within each fraction of the total CCR9⁺ Treg cells. n = 5. Fr: Fraction; Fr I: CD45RA⁺ Foxp3^low resting Treg cells; Fr II: CD45RA⁻ Foxp3^hi activating Treg cells; Fr III: CD45RA⁻ Foxp3^low non-Treg cells. P-values were calculated using Mann-Whitney U–test or Kruskal-Wallis with paired comparisons test.

Fig. 4

Inducing peripheral CCR9⁺ Treg cells towards CCR9⁺ IL-17⁺ Treg cells polarization under NEC inflammatory conditions. (a–d) CCR9⁺ CD4⁺ CD25⁺ CD127^dim/− Treg (CCR9⁺ Treg) cells were cultured with IL-1β, IL-6, or both in the presence of anti-CD3/CD28 antibody-coated microbeads and IL-2 for 4 days. (a) IL-17 and Foxp3 expression levels in CCR9⁺ Treg cells from patients with NEC and controls (CTRL). (b) The frequency of CCR9⁺ IL-17⁺ Treg cells in patients with NEC and CTRL. Representative histograms (c) and mean fluorescence intensity (d) showing the expression of RORγt in CCR9⁺ Treg cells in the presence or absence of IL-1β or IL-6. n = 5; MFI, mean fluorescence intensity. (e and f) CCR9⁺ Treg cells were stimulated with IL-1β, IL-6, IL-2, and anti-CD3/CD28 antibody-coated microbeads in the presence of antibodies targeting IL-6 receptor (aIL-6R), IL-1β, or both for 4 days. (e) Representative flow cytometric plots showing IL-17 and Foxp3 expression levels in CCR9⁺ Treg cells from patients with NEC. (f) The frequency of CCR9⁺ IL-17⁺ Treg cells in the presence or absence of neutralizing antibodies to IL-6R, IL-1β, or both in vitro. n = 5; *, P < .05; **, P < .01; ns: not significant. P-values were calculated using one-way ANOVA with Bonferroni multiple comparison test.

Fig. 5

Inhibiting IL-6R ameliorates NEC by restoring the balance of Treg/Th17 cells in NEC mice. (a) Experimental design (top) and Kaplan-Meier analysis of the survival rate for dam-fed pups (CTRL) and NEC pups treated with control IgG (NEC + cIgG) or anti-IL-6 receptor (NEC + aIL6R) antibodies from the first day of induction of NEC. Data are pooled from three independent experiments; *, P < .05; **, P < .01. (b) Representative intestinal histological changes in CTRL, NEC + cIgG, and NEC + aIL6R pups. Ileal tissues were stained with hematoxylin and eosin. Magnification × 200. (c) NEC severity scores of the histopathological evaluation of mouse ilea (n = 15 for CTRL, 18 for NEC + cIgG, and 22 for NEC + aIL6R groups), graded microscopically by two independent pathologists. *, P < .05; **, P < .01. (d) Incidence of NEC (damage scores >2) in NEC + cIgG and NEC + aIL6R groups. Columns represent the average values of each group (three independent experiments, n = 30); *, P < .05. (e) Representative flow cytometric plots from the analysis of Foxp3 and IL-17 expression in gated CD4⁺ T cells in CTRL, NEC + cIgG, and NEC + aIL6R groups. (f) The percentages of Treg (left), Th17 (middle) and IL-17 producing CD4⁺ Foxp3⁺ Treg cells (IL-17⁺Treg) (right) in CTRL (n = 6), NEC + cIgG (n = 5), and NEC + aIL6R (n = 6) groups. Data are shown as the mean ± SD; *, P < .05; ***, P < .001. P-values were calculated using one-way ANOVA with Bonferroni multiple comparison test. (g) Representative immunoblot analysis of Foxp3, RORγt, STAT3, p-STAT3, STAT5, and p-STAT5 in ilea of CTRL, NEC + cIgG, and NEC + aIL6R mice. One of three independent experiments is shown. (h) Immunoblot results showing the expression of Foxp3, RORγt, STAT3, p-STAT3, STAT5, and p-STAT5; β-actin was used as an internal control. Data are pooled from three independent experiments (n = 6 per group)**, P < .01; ***, P < .001; ns: not significant. P-values were calculated using one-way ANOVA with Bonferroni multiple comparison test.

Fig. 6

Negative correlation of peripheral CCR9⁺ IL-17⁺ Treg cells inversely correlate with histological scoring in NEC mice. (a) The frequencies of circulating CCR9⁺ IL-17⁺ Treg cells in normal neonatal mice (CTRL; n = 5) and mice with different grades of intestinal injury (NEC; n = 17). P-values were calculated using Student's t-test. (b) Spearman correlation (r) between the frequencies of circulating CCR9⁺ IL-17⁺ Treg cells and histological scoring in NEC mice (n = 17). (c) Spearman correlation (r) between the histological scoring and the frequencies of Th17 cells (left), IL-17⁺ Treg cells (middle), and Treg cells (right) of intestine in NEC mice (n = 17). (d) Spearman correlation (r) between the frequencies of circulating CCR9⁺ IL-17⁺ Treg cells and the frequencies of Th17 cells (left), IL-17⁺ Treg cells (middle), and Treg cells (right) of intestine in NEC mice (n = 17). (e) Spearman correlation (r) between the histological scoring and the frequencies of CCR9⁺ Th17 cells (left), CCR9⁺ IL-17⁺ Treg cells (middle), and CCR9⁺ Treg cells (right) of intestine in NEC mice (n = 17). (f) Spearman correlation (r) between the frequencies of circulating CCR9⁺ IL-17⁺ Treg cells and the frequencies of CCR9⁺ Th17 cells (left), CCR9⁺ IL-17⁺ Treg cells (middle), and CCR9⁺ Treg cells (right) of intestine in NEC mice (n = 17).

Fig. 7

Negative correlation of CCR9+ IL-17+ Treg cells with clinical severity of NEC patients. (a) Spearman correlation (r) between the frequencies of CCR9⁺ IL-17⁺ Treg cells and Bell stage in NEC patients (n = 65) and CTRL (n = 66). (b) The frequencies of CCR9⁺ IL-17⁺ Treg cells in surgical (n = 11) and nonsurgical (n = 8) patients of Bell stage III. P-values were calculated using Mann-Whitney U–test. (c) The frequencies of CCR9⁺ IL-17⁺ Treg cells in two NEC IIIA and two NEC IIIB patients who experienced the course of moderate feeding intolerance (FI), NEC I (I), NEC II (II), NEC IIIA (IIIA), and NEC IIIB (IIIB) (only for NEC IIIB patients). **, P < .01; ***, P < .001. P-values were calculated using one-way ANOVA with Bonferroni multiple comparison test. (d) The frequency of CCR9⁺ IL-17⁺ Treg cells were negatively correlated with intestinal barrier integrity biomarkers [TFF3 (left), I-FABP (middle), and zonulin (right)] in NEC patients (n = 65) compared to controls (CTRL; n = 66) analysed by Spearman's rank correlation test. (e–f) Spearman correlation (r) between the frequencies (left) and absolute numbers (right) of CCR9⁺ IL-17⁺ Treg cells and gestational age, age and postmenstrual age in NEC patients (n = 65) and CTRL (n = 66).