Mechanism of cGAS-STING regulation of immune responses against Cryptosporidium parvum infection in intestinal epithelial cells

doi:10.12140/j.issn.1000-7423.2025.03.001

Abstract

Abstract:

Objective To investigate the mechanism of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) in regulation of immune responses against Cryptosporidium parvum infections in intestinal epithelial cells. Methods Human colorectal cell line HCT-8 was infected with C. parvum oocysts (oocysts to cell ratio of 2:1) for 0, 4, 8, 12, 24, 36 and 48 h. Then, total protein was extracted from cells, and the relative STING protein expression was determined using Western blotting. Cell models of cGAS and STING knockdown were generated using small interfering RNA (siRNA). The relative expression of cGAS, STING, TANK-binding kinase 1 (TBK-1), p-TBK-1, interferon regulatory factor (IRF3), p-IRF3, nuclear factor-κB (NF-κB), p-NF-κB, interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) was determined using Wester blotting in HCT-8 cells 24 h post-infection, and the apoptosis of HCT-8 cells was detected using flow cytometry before and after STING knockdown. In addition, the relative expression level of C. parvum 18S mRNA was quantified using quantitative fluorescent PCR (qPCR) before and after STING knockdown. Statistical analysis of the data was conducted using the independent samples t-test. Results Western blotting assay determined that the relative STING protein expression was 3.000 ± 0.743, 2.756 ± 0.847, 2.397 ± 0.701, 3.645 ± 0.306, 3.773 ± 0.471, 3.982 ± 0.468 in HCT-8 cells 4, 8, 12, 24, 36 and 48 h post-infection with C. parvum, which was all higher than in uninfected cells (0 h) (1.000 ± 0.039) (t = 4.655, 3.587, 3.448, 14.870, 10.160, 11.000; all P < 0.05). The relative expression of cGAS, STING, p-NF-κB/NF-κB and TNF-α, p-TBK-1/TBK-1, p-IRF3/IRF3, IFN-β proteins was 1.024 ± 0.093, 1.042 ± 0.160, 1.060 ± 0.108, 0.665 ± 0.297, 0.929 ± 0.207, 0.740 ± 0.104 in C. parvum infected HCT-8 cells with STING knockdown, which was all lower than in infected cells (1.757 ± 0.332, 2.329 ± 0.336, 1.522 ± 0.230, 1.339 ± 0.088, 1.332 ± 0.036) (t = 10.25, 3.360, 6.231, 3.949, 3.159, 9.362; all P < 0.05), and the relative expression of cGAS and STING proteins was 0.771 ± 0.038 and 0.696 ± 0.094 in C. parvum infected HCT-8 cells with cGAS knockdown, which was all lower than in infected cells (1.231 ± 0.074, 1.238 ± 0.023) (t = 9.608, 9.674; both P < 0.05). Flow cytometry detected a lower apoptotic rate in C. parvum infected HCT-8 cells with STING knockdown [(18.90 ± 0.75)%] than in infected cells [(23.72 ± 2.55)%] (t = 3.141, P < 0.05), and qPCR assay quantified higher relative expression of C. parvum 18S mRNA in C. parvum infected HCT-8 cells with STING knockdown (1.335 ± 0.037) than in infected cells (1.002 ± 0.071) (t = 7.195, P < 0.05). Conclusion cGAS-STING contributes to host immune responses against C. parvum infections via activation of TBK1 and NF-κB signaling pathways.

Key words: Cryptosporidium parvum, Cyclic GMP-AMP synthase, Stimulator of interferon gene, Type Ⅰ interferon, Host immune response

CLC Number:

R531.5

CUI Lijun, LI Teng, SHEN Yujuan, ZHANG Jing, ZHOU Hao, CAO Jianping. Mechanism of cGAS-STING regulation of immune responses against Cryptosporidium parvum infection in intestinal epithelial cells[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(3): 311-316.

Figures/Tables 4

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