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.