IL-17 And NF-κB Signaling Pathways In Immune Responses To Fungal Infections

Objective:Fungal infection is a pathophysiological process that occurs in the interaction between pathogenic microorganisms and the human body,and its occurrence and development involve numerous molecules and signaling pathways.It has been shown that interleukin-17(IL-17),which is mainly secreted by T-helper 17(Th17)cells,plays a role in immunity against fungal pathogens,but its role in human Cryptococcus neoformans infection remains unclear.The classical nuclear factor-κB(NF-κB)signaling pathway,which belongs to the downstream of IL-17 signaling,is involved in the host response against fungal infection,but the mechanism of fungal infection caused by NF-κB gene deficiency is still not fully understood.In this study,the pathogenesis of Cryptococcus neoformans meningitis and fungal conjunctivitis was studied,the molecular mechanism of IL-17 production in human Cryptococcus neoformans meningitis was investigated,and the effect of NF-κB-related molecular mutations on the immune response to fungal infection in patients with fungal conjunctivitis was explored.Methods:The clinical data,cerebrospinal fluid(CSF)and serum samples of patients with Cryptococcus neoformans meningitis were collected,and the expression levels of IL-17 in CSF and serum were detected by the enzyme-linked immunosorbent assay(ELISA).Subsequently,the peripheral blood mononuclear cells(PBMCs)were isolated from the whole blood of some healthy individuals,and an in vitro experimental model stimulated by Cryptococcus neoformans was constructed to explore IL-17-producing immune cells via flow cytometry.The STAT3-related signaling pathway produced by IL-17 was detected by phospho-specific flow cytometry and Western blot(WB).After the signal transducer and activator of transcription 3(STAT3)inhibitors were added,the expression of phosphorylated STAT3 protein was detected by phospho-specific flow cytometry,and the changes of IL-17 production were identified by flow cytometry and ELISA.Venous blood was collected from patients with fungal conjunctivitis,and the mutant gene RelA related to NF-κB signaling was identified by whole exome sequencing.The immunophenotype of cells isolated from patients were determined by flow cytometry,then CD14~+cells were sorted and monocyte-derived dendritic cells(MDDC)were cultured.Fungal stimulants were added on the 6th day,and cells were collected by flow cytometry to detect the efficiency of cell differentiation.Real-time Quantitative polymerase chain reaction(RT-q PCR)was used to detect the m RNA expression of tumor necrosis factor alpha(TNF-α),and the expression of RelA in cytoplasm and nucleus were observed using confocal microscopy.The changes of NF-κB signaling pathway in PBMCs were analyzed by WB and co-immunoprecipitation(Co-IP).The Seahorse analyzer was manipulated to assess the glycolytic capacity of cells in patients with RelA mutations.Results:In the early stage of Cryptococcus neoformans meningitis,the expression of IL-17 in CSF increased significantly,while decreased after antifungal treatment.After stimulation with Cryptococcus neoformans in vitro,the results of ELISA assay showed that IL-17 was continuously expressed in the cell supernatant.The results of flow cytometry displayed that IL-17-producing CD4~+T cells increased strongly after stimulation.The data of WB and phospho-specific flow cytometry demonstrated that the phosphorylation level of STAT3 in CD4~+T cells induced by Cryptococcus neoformans was significantly increased.And the level of phosphorylated STAT3 as well as the production of IL-17 in both intracellular and supernatant were decreased after treatment with STAT3 inhibitor.The whole-exon sequencing revealed that there was a new mutation of RelA gene on exon 11(located in the TAD region,RelA-N453T)in patients with fungal conjunctivitis.Compared with the control group,the RelA-N453T mutation was associated with a decrease in CD3~+T cells,CD14 cells,and an increase in B cells.The examination results of confocal microscopy indicated that the RelA-N453T mutation caused abnormal nuclear translocation of RelA protein in MDDCs.The expression levels of inflammatory factors such as TNF-αand IL-1βin the serum of patients were not elevated.RT-q PCR assay showed that stimulation with lipopolysaccharide(LPS)could not increase the expression of TNF-αin cells isolated from patient.According to the WB and Co-IP results,phosphorylation of RelA were generated with LPS stimulation.However,RelA could not bind to P300 protein,and the acetylation of RelA protein would be inhibited.Analysis of Seahorse demonstrated that RelA mutation can significantly inhibit the glycolytic response of immune cells.Conclusion:IL-17 may be a potential biomarker for Cryptococcus neoformans infection,and the RelA-N453T mutation can suppress host immunity against Aspergillus.Our study contributes to bridging the gap in the research on the correlation between immune responses against fungal pathogens and IL-17 and NF-κB signaling pathways,and provides a theoretical basis for the development of new therapeutic targets for antifungal infection.