Orai1 overexpression improves sepsis-induced T-lymphocyte immunosuppression and acute organ dysfunction in mice

Abstract

Immune paralysis induced by sepsis, especially dysfunction of CD4⁺ T cells, leads to an increased risk of infection. In sepsis, abnormal differentiation of T lymphocytes is associated with multiorgan dysfunction syndrome. In T lymphocytes, the Orai1/nuclear factor of activated T Cells (NFAT) pathway is a critical mediator of infection, inflammation, and autoimmunity. In this study, we confirmed immunosuppression of splenic CD4⁺ T cells and abnormal differentiation of T lymphocytes in septic mice. Furthermore, we found that the Orai1/NFAT signaling pathway was inhibited in septic mice; however, the overexpression of Orai1 not only improved immune function of T cells in sepsis but also reduced the mortality and organ damage in septic mice. Moreover, the overexpression of Orai1 could reverse the increases in the numbers of T regulatory and T helper 17 cells in septic mice. These data suggest that the Orai1-mediated NFAT signaling pathway can improve sepsis-induced T-lymphocyte immunosuppression and acute organ dysfunction.

Keywords: Immunosuppression; NFAT; Orai1; Sepsis; Th17; Treg.

Figure 1

Effects of ORAI1 overexpression on organ damage and mortality in septic mice. (A and B) CD4⁺ T cells were incubated for 30 min with anti-CD3 and anti-CD28 monoclonal antibodies. Expression levels of ORAI1 were measured by western blotting. Results are displayed as the mean ± SD (n = 3). ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group. (C) BALB/c mice (n = 60) were randomized into three groups and monitored daily for 7 consecutive days. A Kaplan–Meier curve was plotted to analyze the survival rates, and a log-rank test was used for comparison. ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group. (D–F) Renal and hepatic pathological damage was assessed using histology and scores. Representative pathological images are shown for results obtained based on a typical assay. ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.

Figure 2

Effects of ORAI1 overexpression on the proportions of CD3⁺ and CD8⁺ T cells in the spleen of septic mice. BALB/c mice were randomized into three groups. The expression of CD3, CD4, and CD8 was detected by flow cytometry. (A) First, CD3⁺ T cells were circled from total spleen cells, then CD4⁺ and CD8⁺ T cells were circled from CD3⁺ T cells, and finally, Th17 cells and Tregs were circled from CD4⁺ T cells. (B) Proportion of CD3⁺ T cells in total cells in each group. Results are displayed as the mean ± SD (n = 6). ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group. (C) Proportions of CD4⁺ and CD8⁺ cells in CD3⁺ T cells in each group. Results are displayed as the mean ± SD (n = 6). ^#P < 0.05 vs. the 24-h CLP group, ^&P > 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.

Figure 3

Effects of ORAI1 overexpression on the proportions of Th17 cells and Tregs in septic mice. BALB/c mice were randomized into three groups. The proportions of Th17 cells and Tregs in CD4⁺ T cells from the spleen were examined by flow cytometry. Results are displayed as the mean ± SD (n = 6). ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.

Figure 4

Effects of ORAI1 overexpression on NFAT1 translocation in splenic CD4⁺ T cells. BALB/c mice were randomized into three 3 groups. NFAT1 translocation was analyzed by laser scanning confocal microscopy. The yellow arrow represents NFAT1 intranuclear transfer. The mean optical density was recorded for statistics. Results are displayed as the mean ± SD. ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.

Figure 5

Effects of ORAI1 overexpression on immune dysfunction of septic splenic CD4⁺ T-cells. BALB/c mice were randomized into three groups. CD4⁺ T cells were collected 24 h after the operation. IL-4 and IFN-γ expression in CD4⁺ T cells was measured by ELISA after 24-h incubation with anti-CD3 and anti-CD28 antibodies. Results are displayed as the mean ± SD (n = 6). ∗P < 0.05. The CCK-8 assay was performed to measure splenic CD4⁺ T-cell proliferation. ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.

Figure 6

Effects of ORAI1 overexpression on calcium signaling in CD4⁺ T cells in sepsis. BALB/c mice were randomly divided into three groups. (A and B) [Ca²⁺]i was measured using Fluo-3. (C) [Ca²⁺]i was measured using a Fura-2 ratio at 340 and 380 nm. Results are displayed as the mean ± SD (n = 6). ^#P < 0.05 vs. the 24-h CLP group, ∗P < 0.05 vs. the sham group.