Peripheral and intestinal mucosal-associated invariant T cells in premature infants with necrotizing enterocolitis

Jiayi Tian, Chaoying Yan, Yanfang Jiang, Haohan Zhou, Liyuan Li, Jingjing Shen, Jian Wang, Hongyu Sun, Guang Yang, Wei Sun, Jiayi Tian, Chaoying Yan, Yanfang Jiang, Haohan Zhou, Liyuan Li, Jingjing Shen, Jian Wang, Hongyu Sun, Guang Yang, Wei Sun

Abstract

Background: Necrotizing enterocolitis (NEC) is a potentially fatal inflammatory gastrointestinal disease in preterm infants with unknown pathogenesis. Mucosal-associated invariant T (MAIT) cells primarily accumulate at sites where exposure to microbes is ubiquitous and regulate immunological responses. As the implications of these cells in NEC development in premature infants remain unknown, we investigated the role and characteristics of MAIT cells in NEC pathogenesis. Methods: The percentage of different MAIT cell subsets in peripheral blood samples of 30 preterm infants with NEC and 22 control subjects was estimated using flow cytometry. The frequency of MAIT cells in the intestinal tissues of five NEC patients and five control subjects was also examined. The level of serum cytokines was estimated using cytometric bead array. Potential associations between the different measurements were analyzed using the Spearman's correlation test. Results: Compared with controls, the NEC patients were found to have significantly reduced percentages of circulating CD161+ CD3+ CD8αα+ T cells and CD161+ CD3+ TCRγδ-TCRVa7.2+ MAIT cells. In the intestinal tissues, the percentage of MAIT cells was significantly higher in samples from the NEC patients than the controls. Furthermore, the percentage of circulating MAIT cells in the peripheral blood samples was inversely correlated with that in the intestinal tissues of the NEC patients. The percentage of CD8αα+ MAIT cells was found to be significantly reduced in both peripheral blood and intestinal tissues of NEC patients. Following treatment, the frequency of circulating MAIT cells significantly increased in NEC patients and reached a level similar to that in the control subjects. However, there was no difference in the percentage of circulating CD8αα+ MAIT cells before and after treatment in the NEC patients. Conclusion: Our results suggested that during the development of NEC MAIT cells accumulate in the inflammatory intestinal tissues, while the percentage of CD8aa+ MAIT cells is significantly decreased, which may lead to the dysfunction of MAIT cells in gut immunity.

Keywords: T cells; inflammatory cytokines; mucosal-associated invariant T cells; necrotizing enterocolitis; preterm.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Tian, Yan, Jiang, Zhou, Li, Shen, Wang, Sun, Yang and Sun.

Figures

FIGURE 1
FIGURE 1
Flow cytometry for analyzing the frequency of circulating CD3+ CD161+ T cells in patients with NEC. Peripheral blood mononuclear cells (PBMCs) from 30 NEC patients and 22 controls were stained with fluorescent anti-CD3, anti-CD161, anti-CD8α, anti-CD8β, and anti-CD4 antibodies. The cells were gated initially on living lymphocytes, and on CD3+ CD161+ T cells, and then CD3+ CD161+ CD8αβ- cells. The percentage of CD3+ CD161+, CD3+ CD161+ CD8αα+, CD3+ CD161+ CD8αβ+, CD3+ CD161+ CD4+, and CD3+ CD161+ DN T cells in the subjects were analyzed by flow cytometry. (A) Flow cytometric analysis. (B–F) Quantitative analysis. Data are representative dot plots or expressed as the mean percentage of cells in individual subjects. The difference between the two groups was analyzed by Mann–Whitney U nonparametric test. The horizontal lines represent the median values.
FIGURE 2
FIGURE 2
Flow cytometric analysis of the frequency of circulating MAIT cells in NEC patients. Peripheral blood mononuclear cells (PBMCs) from NEC patients and controls were stained with fluorescent anti-CD3, anti-CD19, anti-CD161, anti-TCRγδ, anti-TCR Vα7.2, anti-CD8α, anti-CD8β, and anti-CD4 antibodies. The cells were gated sequentially on living lymphocytes, CD19−CD3 + T cells, CD3 + TCRγδ-cells, TCR Vα7.2 + CD161 +, and then TCR Vα7.2 + CD161 + CD8αβ- MAIT cells. The percentage of CD3 + TCRγδ-, CD3 + TCRγδ +, CD3 + TCRγδ-Vα7.2 + CD161 + MAIT, CD8αα +, CD8αβ +, CD4 + and DN MAIT cells was analyzed by flow cytometry. (A) Flow cytometry analysis. (B–H) Quantitative analysis. (I–J) Stratification analysis of the percentage of circulating MAIT cells in NEC patients. Data shown are representative dot plots or expressed as the mean percentage of cells in individual subjects. The difference between these two groups was analyzed using the Mann–Whitney U nonparametric test. The horizontal lines represent the median values.
FIGURE 3
FIGURE 3
Flow cytometric analysis of the frequency of CD161 + CD3 + cells in the intestinal tissues of NEC patients. Lamina propria mononuclear cells (LPMCs) from five NEC patients and five non-NEC controls were stained with fluorescent anti-CD3, anti-CD161, anti-CD8α, anti-CD8β, and anti-CD4 antibodies. The cells were gated sequentially on living lymphocytes, and on CD3 + CD161 + T cells, and then CD3 + CD161 + CD8αβ- cells. The percentage of CD3 + CD161 + T cells in lymphocytes and of CD3 + CD161 + CD8αα +, CD3 + CD161 + CD8αβ +, CD3 + CD161 + CD4 +, and CD3 + CD161 + DN T cells in total CD3 + CD161 + T cells were analyzed by flow cytometry. (A) Flow cytometry analysis. (B–F) Quantitative analysis. Data shown are representative dot plots or expressed as the mean percentage of cells in individual subjects. The difference between the two groups was analyzed using the Mann–Whitney U nonparametric test. The horizontal lines represent the median values.
FIGURE 4
FIGURE 4
Flow cytometric analysis of the frequency of MAIT cells in the intestinal tissues of NEC patients. Lamina propria mononuclear cells (LPMCs) from NEC patients and non-NEC controls were stained with fluorescent anti-CD3, anti-CD19, anti-CD161, anti-TCRγδ, anti-TCR Vα7.2, anti-CD8α, anti-CD8β, and anti-CD4 antibodies. The cells were gated sequentially on living lymphocytes, CD3 + CD19− T cells, CD3 + TCRγδ- cells, and TCR Vα7.2 + CD161 + MAIT cells, and then Vα7.2 + CD161 + CD8αβ- MAIT cells. The percentage of CD3 + TCRγδ-, CD3 + TCRγδ +, CD3 + TCRγδ-TCRVα7.2 + CD161 + MAIT, CD8αα +, CD8αβ +, and CD4 + and DN MAIT cells were analyzed by flow cytometry. (A) Flow cytometry analysis. (B–H) Quantitative analysis of cells in the LPMCs from the control subjects and NEC patients. (I) Quantitative analysis of the percentage of circulating and intestinal MAIT cells in the control and NEC groups. (J) Correlation between the percentage of MAIT cells in PBMCs and LPMCs of NEC patients. Data shown are representative dot plots or expressed as the mean percentage of cells in individual subjects. The difference between the two groups was analyzed by the Mann–Whitney U nonparametric test and their correlation was also analyzed. The horizontal lines represent the median values.
FIGURE 5
FIGURE 5
Levels of serum cytokines in the NEC patients and controls. The levels of serum IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ in individual subjects were determined by CBA. (A,D,G,J,M,P,S) The serum levels of IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ in the control subjects and NEC patients. Data shown are expressed as the mean values in individual subjects. The difference between the two groups was analyzed by the Mann–Whitney U nonparametric test. The horizontal lines represent the median values. Correlations among the percentages of circulating MAIT cells and the levels of serum inflammation cytokines in NEC patients. Potential correlations among the percentages of circulating MAIT cells and the levels of serum cytokines were analyzed by the Spearman correlation tests. (B,E,H,K,N,Q,T) The percentages of circulating MAIT cells showed no significant correlation with the levels of serum cytokines in NEC patients. Correlations among the percentages of circulating CD8αα + MAIT cells and the levels of serum inflammation cytokines in NEC patients. Potential correlations among the percentages of circulating CD8αα + MAIT cells and the levels of serum cytokines were analyzed by the Spearman correlation tests. (C,F,I,L,O,R,U) The percentages of circulating CD8αα + MAIT cells showed no significant correlations with the levels of serum cytokines in NEC patients.
FIGURE 6
FIGURE 6
Altered frequencies of MAIT cells and CD8αα + MAIT cells in NEC patients after treatment. The percentage of MAIT cells and CD8αα + MAIT cells were analyzed in NEC patients (n = 6) before and after treatment using the Wilcoxon test. The difference between the percentage of circulating MAIT cells and CD8αα + MAIT cells in the control and after-treatment groups was analyzed by the Mann–Whitney U nonparametric test. (A,B) The percentage of circulating MAIT cells and CD8αα + MAIT cells in individual patients before and after treatment. (C,D) Quantitative analysis of the percentage of circulating MAIT cells and CD8αα + MAIT cells in the controls and NEC patients after treatment. Horizontal lines represent the median values.

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