Dynamic Monitoring Of Immune Status And Antifungal Mechanism Of Innate Immune Cells In AIDS-associated Talaromycosis

Talaromycosis is a fatal deep infectious mycosis caused by Talaromyces marneffei(T.marneffei)infection.T.marneffei mainly affected immunosuppressed individuals,such as HIV/AIDS,rarely infected general population.Immunocytes and cytokines/chemokines derived from them are responsible for T.marneffei clearance.However,how the host innate immune system(innate immune cells and cytokines derived from them)defense against T.marneffei infection in immunosuppressed AIDS patient,especially the dynamic changes in immune parameters during both infection and treatment have not been reported.This study dynamic monitored immune parameters before and after treatment in AIDS-associated talaromycosis clinically.Then we explored that when host adaptive immunodeficiency,the molecular mechanisms of innate immune cells,especially macrophage and neutrophils defense against T.marneffei infection in vivo and in vitro.This research includes the following three parts:Part I:Dynamic Monitoring of Immune Status during Antifungal Therapy in AIDS-associated TalaromycosisObjective:To elucidate the clinical characteristics,dynamic immunological parameters changes during infection and treatment and identified the prognostic factors of AIDS-associated talaromycosis.Methods:Talaromyces marneffei coinfected AIDS patients were followed up,their immunocytes and cytokine profiles were obtained at different stages of antifungal therapy.In addition,Clinical manifestations and routine laboratory examinations were analyzed.Then explored the dynamic change of immunological mechanisms and the prognostic factors of AIDS-associated talaromycosis.Results:1.The main clinical symptoms of AIDS-associated talaromycosis were fever,cough/sputum,abdominal pain/diarrhea,skin lesions,weight loss and anemia.The common signs of the patients were lymphadenopathy,hepatomegaly,splenomegaly and abdominal/pleural effusion.The CD4+T cell count(14(9-28)cells/?L)and CD8+T cell count(169(103-331)cells/?L)were significantly lower than simple HIV infection patients CD4+ T cell count(351±46 cells/?L)and CD8+T cell count(899,[703,1522 cells/?L]).And the CD4+T and CD8+T cell counts gradually increased during antifungal therapy.However,the percentage of activated CD4+T and CD8+ T cells gradually decreased.2.The percentage and absolute value of neutrophils in talaromycosis were higher than those in healthy individuals and simple HIV patients,and reached peak on 3 and 7 days after antifungal treatment,respectively,then they gradually recovered to normal levels.The absolute count and percentage of monocytes in talaromycosis were lower than those in healthy individuals and simple HIV patients,and decreased significantly on 3 days after treatment,then they returned to normal levels on 15 days after treatment.3.Compared with the healthy individuals and simple HIV patients,pro-inflammatory cytokines(IL-12,IL-17A,TNF-?,IFN-?,IL-18,IL-1?)and anti-inflammatory cytokines(IL-10)and chemokine(IP-10)increased significantly before treatment(p<0.05)in talaromycosis patients.Among these,TNF-?,IL-6,IL-17A,IL-1?,IL-7,IL-8,SDF-1?,and IP-10 continued to increase and reached peak levels on 3 days after treatment.Thereafter,these levels gradually decreased on 7 days after treatment.IL-18 significantly increased at baseline,3 days,and 7 days,and then sharply decreased to normal levels on 15 days after treatment.However,plasma levels of IL-12 and IFN-? had peak levels at baseline and gradually decreased after treatment,returning to normal levels on 7 days.4.Furthermore,the levels of TNF-?,IL-6? IL-8,IL-1?,and IP-10 in dead patients were significantly higher than survived patients(p<0.05)and simple HIV patients(p<0.05).Conclusion:The adaptive immune function of patients with AIDS-associated talaromycosis was severely impaired.Timely and effective antifungal therapy combined with ART can improve the outcomes.The findings indicated that innate immune cells and cells-derived cytokines were critical for host defense against T.marneffei infection;furthermore,due to deficiency of T lymphocyte result in an imbalance immune response,excessive inflammatory cytokines("cytokine storm")may be associated with poor outcomes.Part II:Talaromyces marneffei Induce Macrophage Pyroptosis by Upregulating NLRP3/Caspase-1/gasdermin D pathwayObjective:We established the models of T.marneffei invasive infection in immunodeficient mice and macrophage cell line,to elucidate the mechanism of macrophage defenses against T.marneffei infection,and explore preliminary the effect of antifungal in combination with immunomodulator therapy on the prognosis of talaromycosis.Methods:By established the models of T.marneffei invasive infection in immunodeficient BALB/c mice and macrophage cell line,we investigated macrophage pyroptosis in vitro and in vivo.We adopt Western Blotting,immunohistochemistry,immunofluorescence,ELISA and other technology to detect spleen and macrophage pyroptosis proteins,inflammasome,and cytokines(cleaved caspase-1,gasdermin D-NT,NLRP3,ASC,IL-18,IL-1?,TNF-a)at different infection and treatment stages.We combined antifungal with immunomodulator therapy in a mouse models expect to improve outcomes.Results:1.When T.marneffei co-cultured with macrophages,T.marneffei induced the expression of Cleaved caspase-1,NLRP3,ASC,and TNF-? elevation in macrophages that differentiated from THP-1 cells(p<0.05),and the IL-18,IL-1? and TNF-?levels were significantly increased(p<0.05)in the culture supernatant.With the prolong of co-cultured time,the inflammasome and pyroptosis proteins expression in macrophages were more obvious.When T.marneffei act as a trigger for pyroptosis,the immunomodulator of thalidomide could further promote the macrophages pyroptosis.Moreover,thalidomide could promote intracellular TNF-? synthesis and inhibit extracellular TNF-? secretion(p<0.05).2.By intraperitoneal injected yeast phase of T.marneffei into the BALB/c immunodeficient mice,we successfully constructed systemic infection model of T.marneffei.3.T.marneffei could induce macrophages pyroptosis in mice spleen.With the progression of the disease,spleen macrophages pyroptosis protein and inflammasome(cleaved caspase-1,gasdermin D-NT,NLRP3,TNF-?)expression levels gradually increased,especially in dead mice.4.Thalidomide had no effect on talaromycosis,when combined with amphotericin B could alleviate the inflammatory infiltration in organs,and the weight of the mice trended to increase,but they did not improve the overall survival rate of the mice.Conclusion:We found that T.marneffei induced macrophage pyroptosis through the NLRP3/caspase-1/gasdermin D pathway.Moreover,thalidomide could further promote the macrophages pyroptosis when T.marneffei act as a trigger for pyroptosis.Thalidomide could promote pyroptosis and then enhance antimicrobial activity of macrophage.Combined with amphotericin B treatment can alleviate the inflammatory infiltration in mice organs,but they did not improve overall survival of the mice.Part III:Proteomics and Phosphoproteomics Analysis of Neutrophil Secretory Vesicles Induced by Talaromyces marneffeiObjective:We found that neutrophils chemotaxis to T.marneffei and produced secretion vesicles,the neutrophils dead after secretory depletion finally.In order to explore neutrophils anti-T.marneffei mechanisms and the death models of neutrophils after secretory depletion,we used high-throughput omics techniques to clarify the molecular mechanism of neutrophils defense against T.marneffei at protein and phosphorylation modification level.Methods:The neutrophil cell line HL-60 were co-cultured with T.marneffei or inoculated in fungi-free medium for 24 h.And the two groups cells were collected for total protein extraction,we applied high-throughput 4D-label free proteomics and phosphoproteomics technology,combined with bioinfonnatics to identify the mechanism of neutrophil defense against T.marneffei systematically.Results:1.T.marnefei can induce immature neutrophils differentiate into mature neutrophils.The neutrophils intracellular granule increased and secreted vesicles continuously.The neutrophils dead with T marneffei after secretory depletion finally.2.The proteomic analysis identified 165 differential expressed proteins,of which 70 proteins were up-regulated and 95 proteins were down-regulated.Differential expression proteins are mainly located at nucleus,cytoplasm,extracellular matrix and mitochondria.In the T marneffei infection group,the ribosomal metabolism and mitochondrial oxidative phosphorylation metabolism pathway were active,and the NAD(P)H oxidase system was up-regulated.And a series of secretory vesicles-related proteins and antibacterial-related proteins were identified Further functional analysis and pathway enrichment analysis showed that the differential expression proteins mainly enriched in vesicle secretion,mitochondrial oxidative phosphorylation,and ferroptosis pathway.3.Further analysis of the phosphoproteomics identified a total of 248 differential phosphorylation sites corresponding protein,of which 111 proteins expression levels up-regulated and 137 proteins expression levels down-regulated.Differential phosphorylation proteins are mainly located at the nucleus,cytoplasm,plasma membrane,and extracellular matrix.Further functional analysis and pathway enrichment analysis showed that differential phosphorylation proteins were mainly enriched in vesicle secretion and ferroptosis pathways.Conclusion:Combined proteomics and phosphoproteomics analysis,T.marneffei could induce neutrophils intracellular granule increased and produce secretory vesicles,which were rich in antimicrobial proteins.When neutrophils were co-cultured with T.marneffei,it can activate metabolic pathways of ribosome and mitochondrial oxidative phosphorylation.Neutrophils may defense against T.marneffei depending on NAD(P)H oxidase pathway to produce reactive oxygen species.However,the over-released reactive oxygen species by neutrophils can also lead itself ferroptosis.