The Role And Mechanisms Of PGLYRP1 On Macrophage Innate Immune Response Against Cryptococcus Neoformans Through NF-κB Signaling Pathway

BackgroundCryptococcus neoformans is an important pathogenic fungus which can cause serious fatal diseases such as cryptococcal meningitis.Cryptococcal meningitis is difficult to treat and has a high mortality,the clinical outcome depends on the immune state of the host.As the first line of defense of innate immunity,macrophages are closely related to the protective immunity of the host.After exposure to Cryptococcus neoformans,the state of macrophages can show a high degree of plasticity after being stimulated by specific factors,such as cytokines,cellular indirect contacts and metabolites.Peptidoglycan recognition protein 1(PGLYRP1),as a multifunctional innate immune protein,not only has direct inhibition or killing effect on microorganisms,but plays an important role in regulating the immune response of the body.It plays an anti-inflammatory and pro-inflammatory role in different diseases.Based on the differential m RNA expression profile of preclinical microarray data,we hypothesized that the protein PGLYRP1 may be involved in the immunomodulatory process of macrophages against Cryptococcus neoformans infection.Methods1.To study the expression of PGLYRP1 protein in host Cryptococcus neoformans infection: the m RNA expression profiles of peripheral blood mononuclear cells(PMBC)from patients with cryptococcal meningitis and healthy people were analyzed by microarray,and then the m RNA and protein levels of PGLYRP1 in PBMC and plasma of patients with cryptococcal meningitis were verified by real-time quantitative PCR and Elisa.Then the mouse model of Cryptococcus neoformans infection was further constructed,and the spatiotemporal expression of PGLYRP1 in Cryptococcus neoformans infection model was detected by Elisa and immunofluorescence to determine whether PGLYRP1 was involved in the host Cryptococcus neoformans infection process.2.To study the direct effect of PGLYRP1 protein on Cryptococcus neoformans:through the experiment of direct co-incubation of PGLYRP1 protein with Cryptococcus neoformans,the proliferation curve of Cryptococcus neoformans was determined by enzyme labeling instrument,the activity of Cryptococcus neoformans was detected by trypan blue,the capsule size of Cryptococcus neoformans was detected by ink staining,and the colonyforming unit of Cryptococcus neoformans was detected by monoclonal formation test,in order to determine whether PGLYRP1 protein has direct inhibition or killing effect on Cryptococcus neoformans.3.To study the effect of PGLYRP1 protein on the phenotype of macrophages after Cryptococcus neoformans infection in vitro: by using PGLYRP1 protein,macrophages and Cryptococcus neoformans co-incubation system.The intracellular killing effect of macrophages on Cryptococcus neoformans was detected by monoclonal formation test,the phagocytic function of macrophages was detected by Giemsa staining,the release of cytokines and chemokines in the supernatant of macrophages was detected by Elisa,the levels of intracellular cytokines m RNA and protein were detected by real-time fluorescence quantitative PCR and WB,and the production of reactive oxygen species in supernatants was detected by flow cytometry.To determine the specific function of PGLYRP1 on macrophages against Cryptococcus neoformans infection.4.To study the effect of PGLYRP1 on the infection process of Cryptococcus neoformans in vivo: to further construct PGLYRP1-KO mice.Establish PGLYRP1-KO and WT Cryptococcus neoformans infection model in mice: observe the survival time,tissue colony load(lung,brain),tissue HE and PAS staining sections(lung,brain)and tissue supernatant cytokine detection(lung,brain,spleen)to explore the effect of PGLYRP1 on the infection process of Cryptococcus neoformans.5.To study the effect of PGLYRP1 protein on the potential pathway of macrophages after Cryptococcus neoformans infection: then we detected the changes of PGLYRP1 on macrophages in the transcriptome level of anti-Cryptococcus neoformans infection by highthroughput sequencing,and analyzed the enrichment of differential genes by GO and KEGG to identify the regulatory pathways that changed significantly.6.To study the mechanism of PGLYRP1 protein on the key pathways in macrophages after Cryptococcus neoformans infection: finally,we used real-time fluorescence quantitative PCR to verify the m RNA level of significantly changed pathway-related molecules;WB and immunofluorescence were used to detect the expression of key molecules in the pathway;and by adding this pathway inhibitor,the phenotypic changes of macrophages were detected to clarify the effect of PGLYRP1 protein on this pathway.Results1.The results of PGLYRP1 expression in Cryptococcus neoformans infection showed that: m RNA microarray analysis showed that PGLYRP1 gene was significantly increased in PBMC of patients with cryptococcal meningitis.Real-time quantitative PCR and Elisa methods showed that the levels of PGLYRP1 m RNA and protein in PBMC and plasma were significantly increased.In the mouse infection model,compared with the PBS control group,PGLYRP1 in Cryptococcus neoformans infection group increased at different time points in different tissues.2.The results in vitro showed that PGLYRP1 protein inhibited the intracellular replication of Cryptococcus neoformans in macrophages,PGLYRP1 protein promoted the production of pro-inflammatory cytokines and chemokines in macrophages against Cryptococcus neoformans,and PGLYRP1 protein promoted the production of reactive oxygen species in macrophages against Cryptococcus neoformans infection.However,PGLYRP1 protein had no direct effect on Cryptococcus neoformans,and PGLYRP1 had no significant effect on nitride production and phagocytosis of macrophages against Cryptococcus neoformans infection.3.In vivo,PGLYRP1-KO Cryptococcus neoformans infected mice significantly shortened survival compared with WT mice;HE staining and PAS staining showed that the pulmonary infection was more serious and there was more inflammatory cell infiltration.;lung and brain tissue showed heavier colony load and cytokine profiles in tissue supernatants showed that PGLYRP1-KO Cryptococcus neoformans infected mice were more inclined to unprotected cellular immunity.4.RNA-seq sequencing and GO and KEGG enrichment analysis showed that the highly expressed genes in macrophages were significantly enriched in NF-κB signal pathway after the addition of PGLYRP1 protein.The results of real-time fluorescence quantitative PCR showed that the key genes of NF-κB pathway,such as MMP9,TNF,ICAM1 and CD40,were significantly increased in PGLYRP1 group.WB results showed that the key molecule of NF-κB signal pathway,p65 and phosphorylated p65,were increased in PLGYRP1 group.The results of immunofluorescence showed that nuclear translocation of p65 occurred in PLGYRP1 group.WB results showed that the expression of NF-κB in lung tissue of PGLYRP1-KO mice was not different from that of WT mice.After adding NF-κB inhibitor,the ability of macrophages to resist Cryptococcus neoformans infection decreased.ConclusionPGLYRP1 was highly expressed in cryptococcal meningitis.High expression of PGLYRP1 protein can promote the production of pro-inflammatory cytokines,chemokines and reactive oxygen species in macrophages against Cryptococcus neoformans infection.PGLYRP1 plays a protective role in the process of infection in mice.In mechanism,PGLYRP1 may enhance the innate immune response of macrophages against Cryptococcus neoformans infection by regulating NF-κB signal pathway.This study reveals the mechanism of PGLYRP1 protein in the process of macrophage anti-Cryptococcus neoformans,provides a new idea for immunotherapy of Cryptococcus neoformans,and has potential clinical transformation value.