TRIM26 Promotes Antifungal Immunity By Regulating The Differentiation Of Th17 Cells

The HIV epidemic in today’s world,organ transplantation in modern medicine,chemotherapy for cancer patients,and widely used immunosuppressive therapies have led to the emergence of many immunocompromised clinical patients,and these immunodeficient individuals have very poor resistance to fungi,which can cause life-threatening invasive infections with extremely high mortality.However,there are only a few drugs available for the clinical treatment of Candida albicans infections,and the emergence of drug resistance in some invasive fungal patients with high clinical morbidity and mortality urgently requires us to develop new antifungal drugs and therapies.In contrast to the study of antiviral and antibacterial immune mechanisms,little is known about the immune signaling pathways of host antifungal responses,thus limiting the development of vaccines and drugs for fungal control.It is imperative to discover the molecules and mechanisms that regulate the pathways of host immune system against fungal infections,and to provide a scientific basis for the development of new approaches based on immunotherapy.Innate immunity is the body’s first line of defense against pathogenic invasion of the organism.The host relies on pattern recognition receptors(PRRs)on the surface of innate immune cells at the early stage of fungal infection to non-specifically recognize fungal cell wall surface glycoproteins,initiate activation of intracellular CLR signaling pathways,promote phagocytosis and clearance of Candida albicans by innate immune cells such as macrophages and neutrophils,and produce ROS and NO to inhibit the growth of the fungus,while activating NF-κB and MAPKs signaling pathways to promote the release of inflammatory cytokines,which can further strengthen innate immune cells and regulate the differentiation of Th cells to activate the host adaptive immune response,while adaptive immunity can amplify innate immunity through cytokines to recruit and enhance the function of certain innate immune cells.Post-translational modifications of proteins play an important role in antifungal innate immune signaling,while studies on ubiquitination modifications in the regulation of antifungal immunity are still in their infancy.The number of E3 ubiquitin ligases in the human genome is large,yet only the functions of several E3 ubiquitin ligases in the antifungal immune response have been reported,and whether other ubiquitin ligases are also involved in the complex antifungal immune response needs to be further explored.We investigated the role of TRIM family members in the antifungal immune response and found that TRIM26 KO mice had a higher mortality rate after fungal infection compared to WT.TRIM26 is an important member of the TRIM family of E3 ubiquitin ligases and regulates related signaling pathways through its ubiquitinylase activity in a variety of pathophysiological processes,and it has been reported that TRIM26 negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear IRF3.,but how it is involved in the antifungal immune response is currently unreported.Our subsequent study revealed that TRIM26 deficiency may lead to increased Th17 differentiation and more secretion of CXCL1,which recruits more neutrophils,resulting in disruption of normal renal physiological functions by hyperinflammatory responses,thus causing higher mortality than WT mice upon fungal infection in vivo.Study objectives:1.To investigate whether TRIM26 regulates the host antifungal immune response2.To elucidate the molecular mechanism of TRIM26 in the anti-fugal immunity responsee.Research methods:1.Construct of mouse systemic fungal infection modelTen TRIM26 KO mice and WT mice,each born in the same litter at 6-8 weeks of age and weighing approximately 21.5 g,were injected with a dose of 1×105 Candida albicans at a body weight of 21.5 g per mouse by tail vein injection,and the changes in body weight and the number of mice that died were observed and measured daily at the same time point of injection,and the change in body weight and survival curves were calculated and plotted.The body weight change curve and survival curve were calculated and plotted.2.Investigate the effect of TRIM26 on fungal growthFour TRIM26 KO mice and WT mice,each born in the same litter at 6-8 weeks of age and weighing approximately 21.5 g,were selected and injected with a dose of 1×105 Candida albicans by tail vein injection at a body Weight of 21.5g per mouse,and the kidney,liver and spleen were removed at day 5,and the ground and homogenized tissue leachate was applied to YPD plates,and the number of colonies grown Counting was performed to calculate the tissue load.The other kidney was sectioned and then the sections were processed for H&E and PAS staining.3.Investigate the effect of TRIM26 on the fungicidal function of innate immune cellsBMDMs and BMDCs from WT and TRIM26 KO mice were isolated and co-cultured with FITC-labeled HKCA-Y at a certain ratio for 1h,after which the cells were collected and the phagocytosis of Candida albicans by immune cells was detected by flow cytometry.Mouse peritoneal macrophages,BMDMs,BMDCs,and neutrophils,were co-cultured with Candida albicans for 4 h.The cells were lysed,and the lysate was coated on YPD plates,and the number of colonies grown was counted after incubation overnight at 37℃.BMDMs and BMDCs were stimulated by HKCA-Y to produce ROS,and ROS production was detected using flow cytometry after loading FITC-labeled detection probes inside the cells.The supernatants of BMDMs were collected after Oh,24h,48h and 72h of stimulation with HKCA-Y(MOI=10),and nitrite was detected in the supernatants using a nitrite detection kit to determine the amount of NO production.4.Investigate the effect of TRIM26 on innate immune cell developmentThe spleens and lymph nodes of 6-8 week littermate WT and TRIM26 KO mice were selected and collected in healthy condition,and after isolating them into individual cells,the cell surface markers CD45,CDllb,CDllc,Ly-6G,Ly-6C,F4/80,and MHC Ⅱ were stained using a combination of fluorescently labeled antibodies,and flow cytometry was applied to CD45+cells as gating to analyze the number of immune cells in each group.5.Investigate the effect of TRIM26 on immune cell recruitment after infectionKidneys from WT and TRIM26 KO mice on the fifth day after infection with Candida albicans were excised,kidneys were digested with collagenase,processed into individual cells,stained for cell surface markers CD45,CD11b,Ly-6G,and F4/80 using a combination of fluorescently labeled antibodies,and immune cell types and numbers were detected and analyzed by flow cytometry,and spleen cells from mice five days after systemic infection were isolated and analyzed using the the same method for flow assay.6.Investigate the effect of TRIM26 onpost-infection helper T-cell differentiationSpleens from TRIM26 KO mice and WT mice five days after infection were taken,ground into individual cells and continued to be cultured in vitro,and splenocytes were secondarily stimulated with heated lethal C.albicans(MOI=1),and after the end of stimulation,the cell surface markers CD3 and CD4 and cell internal markers IL-17A and IFN-γ were stained with a combination of fluorescently labeled antibodies,followed by the ratio of Thl and Th17 was detected by flow-through assay,while cell culture supernatants were collected and the secretory levels of IL-17A and IFN-y were detected by ELISA.Results:1.TRIM26 KO mice were more susceptible to Candida albicans infection than WT mice in vivoFirst we constructed a Candida albicans systemic infection model for different members of the TRIM family KO mice available in the laboratory,and the results showed that compared to WT mice,TRIM26 KO mice showed significantly lower survival and more weight loss after infection with Candida albicans,while TRIM65 KO mice showed comparable survival as well as weight changes after infection with Candida albicans compared to WT mice.This suggests that TRIM26 positively regulates the antifungal immune response in vivo.2.TRIM26 KO mice have reduced ability to clear Candida albicansThe kidney,liver and spleen of the mice were removed on the fifth day after systemic infection,and their tissue grinds were taken and coated on YPD plates.After counting the number of colonies grown and calculating the tissue load,we found that the fungal load in the kidney,liver and spleen of the TRIM26 KO mice was significantly higher than that of the WT group.The same results were verified when the other kidneys were sectioned and treated with H&E and PAS staining,and we found that the kidneys of the TRIM26 KO mice had more severe tissue damage and a large number of mycelia were visible in the renal pelvis area.3.TRIM26 does not rely on phagocytic killing of innate immune cells for fungal clearance.We examined the phagocytosis of FITC-labeled Candida albicans by BMDM and BMDC in TRIM26 KO and WT mice using flow assays,and the killing ability of for immune cells against Candida albicans by co-culture with C.albicans.,Given that ROS and NO play essential roles in the killing of fungi by immune cells,we assessed ROS and NO production by BMDMs after HKCA-Y stimulation,and found that TRIM26 deletion did not affect the phagocytosis and killing of the fungi by innate immune cells.4.TRIM26 does not affect the development of innate immune cells.The spleens and lymph nodes of TRIM26 KO and WT healthy mice were isolated,and the number of immune cells in each group was measured by flow assay,and it was found that there was no difference in the number of innate immune cells such as neutrophils,monocytes,macrophages and dendritic cells in the spleens of TRIM26 KO mice and WT mice.5.Increased neutrophils recruitment in the tissues of TRIM26 KO mice after fungal infection.Because the kidneys is the most affected organs in systemic fungal infection and the recruitments innate immune cells in kidneys is vital for control fungal infection,we assessed the type and number of immune cells recruited to the kidney after infection.There was a higher proportion of neutrophils in the kidney of TRIM26 KO mice compared to WT mice,and no significant difference in the proportion of macrophages,and Ly-6G immunohistochemical staining of kidney sections illustrated the same results.We also evaluated the numbers of neutrophils in the spleens and found that TRIM26 KO mice had more neutrophils in the spleens.Owing to CXCL1 plays vital roles in the recruitment of neutrophils to sites of infection,we assessed the mRNA levels and secretion of CXCL1,and the results showed that TRIM26 deficency resulted in a a significant elevation of CXCL1.6.TRIM26 deletion enhances Th17 cell response after fungal infection in mice.Because the adaptive immune responses are also important in host defense against fungal infection and Th17 cells are essential in antifungal immunity,we isolated the spleens of TRIM26 KO and WT mice at five days upon Candida albicans infection,and the cells of spleens were stimulated with Candida albicans secondarily.The flow cytometry assays showed that the percentage of Th17 cells in splenocytes of TRIM26 KO mice was significantly higher than that of WT mice,while the percentage of Th1 cells was comparable between TRIM26 KO and WT mice.We also evaluated the secretion of IL-17A and IFN-y in the supernatants of spleen cells stimulated with Candida albicans and showed that the levels of IL-17A in spleen cells of TRIM26 KO mice were significantly increased as compared with WT mice,while the secretion of IFN-y was unchanged.7.TRIM26 KO mice infected with fungi caused more severe renal injury.qRT-PCR was performed for the expression of inflammatory factors and renal injury factors in the kidney of TRIM26 KO and WT mice after infection,and the mRNA levels of inflammatory factors IL-6,TNF-α and renal injury factors P-SELECTIN,KIM-1 and ICAM-1 were found to be significantly increased in TRIM26 KO mice as compared with that in WT mice.Next,the levels of creatinine and urea nitrogen,indicators of kidney function,were examined and found to be elevated in TRIM26 KO mice compared to WT mice,indicating that normal kidney function was disrupted,resulting in higher mortality in TRIM26 mice in the systemic infection model.Conclusions:1.TRIM26 postively regualtes antifungal immune response in vivo and inhibits fungal growth.2.TRIM26 suppresses the differentiation of Th17 cells,and inhibits the excessive CXCL1 production to recruit excessive neutrophils,and leads to protection the kidney from hyperinflammatory responses.