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Basic Research
Overactivation of the ILC2-AREG-Treg axis promotes the progression of cervical cancer by shaping an immunosuppressive microenvironment
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Abstract:
[Abstract] Objective: To investigate the role and mechanism of the group II innate lymphoid cell (ILC2)-amphiregulin (AREG)-regulatory T (Treg) cell axis in the regulation of the immune microenvironment in cervical cancer. Methods: Tumor tissue samples were obtained from patients with stage I-IIA cervical cancer (n = 8) who were admitted to the First Affiliated Hospital of Xinjiang Medical University between May 2021 and May 2022. Normal cervical tissues from patients undergoing surgery for uterine fibroids served as controls (n = 8). Additionally, peripheral blood samples were collected from patients with cervical cancer at all stages (n = 30), with peripheral blood from healthy individuals serving as controls (n = 30). The mRNA expression levels of AREG and forkhead box protein P3 (Foxp3) were analyzed via the GEPIA database. Multiplex immunofluorescence and flow cytometry were performed to detect the infiltration of ILC2s and Treg cells in tissues and peripheral blood. ELISA, immunohistochemistry (IHC) and WB were carried out to verify the expression of AREG, Foxp3 and IL-10, followed by correlation analysis among ILC2s, AREG, Treg cells and IL-10. For in vitro experiments, ILC2s and peripheral blood mononuclear cells (PBMC) were isolated from cervical cancer patients and treated with recombinant human IL-33 (rhIL-33), anti-IL-33 antibody (α-IL-33), recombinant human AREG (rhAREG) and anti-human AREG antibody (α-AREG), respectively. CCK-8 assay and flow cytometry were used to evaluate the effects of different concentrations of rhAREG on the proliferation and apoptosis of HeLa and SiHa cells. The concentrations of AREG and IL-10 in cell culture supernatants were measured by ELISA, and the proportion of Treg cells were determined by flow cytometry. In addition, this study also compared the differences in the levels of ILC2s, AREG, Treg cells, and IL-10 in the peripheral blood of cervical cancer patients before and after surgery. Results: The infiltration of ILC2 and expression of AREG in tissues and peripheral blood of cervical cancer patients were markedly higher than those in the normal control group. The proportion of Treg cells and expression levels of Foxp3 and IL-10 were also significantly upregulated in cervical cancer patients (P < 0.05). Correlation analysis confirmed a positive correlation among ILC2s, AREG, Treg cells and IL-10. In vitro experiments showed that different concentrations of rhAREG had no significant effects on the proliferation or apoptosis of HeLa and SiHa cells (P > 0.05). rhIL-33 effectively activated ILC2s and upregulated AREG secretion (P < 0.01), which was reversed by α-IL-33 treatment (P < 0.05). rhAREG significantly promoted Treg cell differentiation and IL-10 secretion (P < 0.001), whereas α-AREG markedly reversed these biological effects (P < 0.01). In addition, the levels of ILC2s, Treg cells, AREG and IL-10 in peripheral blood of cervical cancer patients were significantly decreased after surgery (P < 0.0001). Conclusion: The aberrant activation of the ILC2-AREG-Treg immunoregulatory axis may participate in cervical cancer progression by shaping an immunosuppressive tumor microenvironment.
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