Notch 1 Is Involved in CD4+ T Cell Differentiation Into Th1 Subtype During Helicobacter pylori Infection

Abstract

Helicobacter pylori infection induces CD4⁺ T differentiation cells into IFN-γ-producing Th1 cells. However, the details of mechanism underlying this process remain unclear. Notch signal pathway has been reported to regulate the differentiation of CD4⁺ T cells into Th1 subtype in many Th1-mediated inflammatory disorders but not yet in H. pylori infection. In the present study, the mRNA expression pattern of CD4⁺ T cells in H. pylori-infected patients differed from that of healthy control using Human Signal Transduction Pathway Finder RT2 Profiler PCR Array, and this alteration was associated with Notch signal pathway, as analyzed by Bioinformation. Quantitative real-time PCR showed that the mRNA expression of Notch1 and its target gene Hes-1 in CD4⁺ T cells of H. pylori-infected individuals increased compared with the healthy controls. In addition, the mRNA expression of Th1 master transcription factor T-bet and Th1 signature cytokine IFN-γ was both upregulated in H. pylori-infected individuals and positively correlated with Notch1 expression. The increased protein level of Notch1 and IFN-γ were also observed in H. pylori-infected individuals confirmed by flow cytometry and ELISA. In vitro, inhibition of Notch signaling decreased the mRNA expression of Notch1, Hes-1, T-bet, and IFN-γ, and reduced the protein levels of Notch1 and IFN-γ and the secretion of IFN-γ in CD4⁺ T cells stimulated by H. pylori. Collectively, this is the first evidence that Notch1 is upregulated and involved in the differentiation of Th1 cells during H. pylori infection, which will facilitate exploiting Notch1 as a therapeutic target for the control of H. pylori infection.

Keywords: CD4+ T cell; Helicobacter pylori; NOTCH1; Th1 cell; differentiation.

Figure 1

The results of PCR array analysis showing that the signal pathway of Helicobacter pylori–infected subjects was altered compared with healthy controls. (A) Volcano plot: the red and green circles indicate upregulated and downregulated genes (>2-fold), respectively. The black circles represent no change. The blue line indicates the desired 0.05 threshold for p-value of t-test. (B) Scatter plot. The center line represents genes with no difference in expression. The upper left point represents genes with upregulated expression, and the lower right point represents genes with downregulated expression.

Figure 2

Increase in mRNA expression levels of Notch1 and Hes1 in CD4⁺ T cells of H. pylori–infected patients. CD4⁺ T cells were isolated from H. pylori–infected subjects (n = 52) and the control group (n = 39), and Notch 1 and Hes1 mRNA expression was assessed by qPCR. The results were normalized to β-actin. The data are presented as the mean ± SD of three replicates. (A) Notch1 expression; (B) Hes-1 expression; (C) Notch1 expression in different H. pylori–associated gastrointestinal disorders; (D) Hes1 expression in different H. pylori–associated gastrointestinal disorders. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Upregulated mRNA expression of T-bet in CD4⁺ T cells of H. pylori–infected patients. CD4⁺ T cells were isolated from H. pylori–infected subjects (n = 52) and control group (n = 39), and T-bet mRNA expression was measured by qPCR. The results were normalized to that of β-actin. The data are expressed as the mean ± SD of three replicates. (A) T-bet mRNA expression. (B) The correlation of mRNA expression between T-bet and Notch1 was evaluated by Pearson correlation analysis. (C) The mRNA expression of T-bet in different H. pylori–associated gastrointestinal diseases. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Upregulated mRNA expression of IFN-γ in CD4⁺ T cells of H. pylori–infected subjects. CD4⁺ T cells were isolated from H. pylori–infected subjects (n = 52) and the control group (n = 39), and IFN-γ mRNA expression was measured by qPCR. The results were normalized to that of β-actin. The data are presented as the mean ± SD of three replicates. (A) The mRNA expression of IFN-γ. (B) The correlation between the mRNA expression of IFN-γ and Notch1 was evaluated by Pearson correlation analysis. (C) The mRNA expression of IFN-γ in different H. pylori–associated gastrointestinal diseases. *p < 0.05, **p < 0.01.

Figure 5

Upregulation of Notch 1 and IFN-γ protein expression in CD4⁺ T cells of H. pylori–infected patients. CD4⁺ T cells were isolated from H. pylori–infected subjects (n = 10) and the control group (n = 10) and stained with PE-Notch1 and FITC-IFN-γ antibodies. The protein levels of Notch 1 and IFN-γ were determined by flow cytometry. Cells were stained with an isotype-matched antibody as control. The representative flow cytometric result was shown, and the mean fluorescence intensity (MFI) of Notch 1 (A) and IFN-γ (B) was evaluated. (C) The concentration of IFN-γ in the culture supernatant of CD4⁺ T cells from H. pylori–infected subjects (n = 10) and healthy controls (n = 10) was assessed by ELISA. The data are presented as the mean ± SD of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Inhibition of Notch signaling decreases Th1 response in H. pylori–infected subjects. CD4⁺ T cells were isolated from H. pylori–infected subjects (n = 10) and incubated with 20 μmol/L DAPT for 24 h before stimulation with H. pylori (MOI = 50). Cells were incubated with DMSO as control. (A) The mRNA expression of Notch1, Hes-1, T-bet, and IFN-γ in CD4⁺ T cells of H. pylori–infected patients was detected by qPCR. The results were normalized to that of β-actin. The data were presented as the mean ± SD of three replicates. (B) Cells were stained with PE-Notch1 antibody with an isotype antibody as control, and the mean fluorescence intensity (MFI) of Notch 1 was evaluated with and without DAPT treatment. (C) Cells were stained with FITC-IFN-γ antibody with isotype antibody as control. A representative flow cytometric result was shown, and the MFI of Notch 1 was evaluated with and without DAPT treatment. (D) The concentration of IFN-γ in the culture supernatant of CD4⁺ T cells from H. pylori–infected subjects (n = 10) with and without DAPT treatment was detected by ELISA. The data are presented as the mean ± SD of three experiments. *p < 0.05, **p < 0.01.