Tnfaip6 Secreted by Bone Marrow-Derived Mesenchymal Stem Cells Attenuates TNBS-Induced Colitis by Modulating Follicular Helper T Cells and Follicular Regulatory T Cells Balance in Mice.

Guangli Gu,Shiquan Li,Fei Lu,Ruizhi Zeng,Lan Chen,Lingling Zhan,Zhaoliang Liang,Xiaoping Lv,Gengfeng Liu,Huiqin Wang,Xiaodan Lv

doi:10.3389/fphar.2021.734040

Abstract

Objective: To investigate the immunological mechanism of bone marrow–derived mesenchymal stem cells (BM-MSCs) in inflammatory bowel disease (IBD). Methods: Mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)–induced colitis were intraperitoneally injected with phosphate-buffered saline, BM-MSCs, BM-MSCs with tumor necrosis factor–induced protein 6 (Tnfaip6) knockdown mediated by RNA interference recombinant adenovirus, and BM-MSCs–infected with control adenovirus or recombinant mouse Tnfaip6. The disease activity index, weight loss, and histological scores were recorded. Serum levels of Tnfaip6 and pro- and anti-inflammatory cytokines, including interleukin (IL)-21, tumor necrosis factor-alpha (TNF-α), IL-10 were measured by enzyme-linked immunosorbent assay. The relative expression levels of these cytokines, B-cell lymphoma 6 (BCL-6) and fork-like transcription factor p3 (Foxp3) in the colon were determined by real-time quantitative PCR (RT-qPCR). BCL-6 and Foxp3 are the master regulators of follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr), respectively. The infiltration of Tfh and Tfr in mesenteric lymph nodes (MLNs) and spleens was analyzed by flow cytometry. Results: Compared to the normal control group, the expression levels of BCL-6 and IL-21 in the colon, Tfh infiltration, and ratios of Tfh/Tfr in the MLNs and spleen, and the serum concentrations of IL-21 and TNF-α increased significantly in the colitis model group (p < 0.05). Intraperitoneal injection of BM-MSCs or Tnfaip6 ameliorated weight loss and clinical and histological severity of colitis, downregulated the expression of BCL-6, IL-21, and TNF-α, upregulated the expression of Foxp3, IL-10, and Tnfaip6 (p < 0.05), increased Tfr and reduced the infiltration of Tfh in the MLNs and spleen, and downregulated the Tfh/Tfr ratio (p < 0.05). On the other hand, BM-MSCs lost the therapeutic effect and immune regulatory functions on Tfh and Tfr after Tnfaip6 knockdown. Conclusion: Tfh increase in the inflamed colon, Tfh decrease and Tfr increase during the colitis remission phase, and the imbalance of the Tfh/Tfr ratio is closely related to the progression of IBD. Tnfaip6 secreted by BM-MSCs alleviates IBD by inhibiting Tfh differentiation, promoting Tfr differentiation, and improving the imbalance of Tfh/Tfr in mice.

Highlights

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by an abnormal inflammatory response
Slight green fluorescence was detected at the multiplicity of infection (MOI) of 50 and 100, while strong green fluorescence was observed at MOI of 200 and 400, and morphology retraction was detected in some of the cells at the MOI of 400 (Figure 1)
The levels of tumor necrosis factor–induced protein 6 (Tnfaip6) in the transfected bone marrow–derived (BM)-Mesenchymal stem cells (MSCs) were significantly lower than those measured from the naïve BM-MSCs and BM-MSCs infected with control adenovirus (Figures 4B,C). These findings suggested that Tnfaip6 RNAi recombinant adenovirus has been successfully transfected into BM-MSCs, leading to a significant knockdown of Tnfaip6 but did not affect the phenotype of MSCs

Summary

Introduction

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by an abnormal inflammatory response. The global incidence and prevalence of IBD has been rising with highest prevalence in Europe and North America and rising incidence in newly industrialized countries in Africa, Asia, and South America (Ng et al, 2017; Jones et al, 2019; Xu et al, 2021). Studies have shown a greater predominance of CD in women than in men, but a greater predominance of UC in men than in women (Goodman et al, 2020). IBD severely affects the life quality of patients with a long course and recurrence. Current studies have shown that genetic susceptibility, immune response disorders, and environmental factors are involved in the pathogenesis of IBD. The precise etiology and pathogenesis of IBD are yet unclear. Since no curative treatments have been developed for IBD, it is important to study the pathogenesis of IBD and explore effective treatment methods

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Conclusion