MicroRNA-mediated Inflammatory Responses Induced By Cryptococcus Neoformans Depend On The NF-κB Pathway In Human Monocytes

Cryptococcosis is a significant invasive fungal infection with noteworthy morbidity and mortality, which is usually caused by either Cryptococcus neoformans(C. neoformans)or Cryptococcus gattii(C. gattii). Epidemiological studies from China showed that the species, caused by C.neoformans are more often reported in immunocompromised and immunocompetent patients. While what’s puzzling is that these patients need longer treatment and it is more difficult to treat them. The urgent need for us to explore a more effective treatment and seek new targets for drug treatment.The innate immune system, including cellular components, monocytes, macrophages and many other effectors, is the first line of defense against pathogens, and broadly protects against invading microorganisms. Macrophages are important components with versatile functions prominently involved in host defense and immunity against foreign microorganisms, including bacteria, viruses, fungi and parasites.Against cryptococcal infection requires an appropriate innate immune response induced, and excessive immune activation may result in body injury or acute and chronic inflammatory diseases. It is an important role in the pathogenesis of Cryptococcus that the immune regulation is too effective to avoid improper immune response in cryptococcal infection. Therefore, the study of the interaction between the innate immune macrophages and C. neoformans is very importan to understand the body in the early stages of C. neoformans infection.Micro RNAs(mi RNAs) are a class of highly conserved small noncoding RNAs, 19-24 nucleotides in length, widely present in a variety of viruses, nematodes, plants and animals. Which post-transcriptionally regulate gene expression by targeting the 3′ untranslated region(3′ UTR) of target m RNAs. mi RNAs always prevent protein synthesis by degrading m RNAs and inhibit their translation. In general, mi RNAs act as key regulators in development, differentiation, homeostasis and cancers. An accumulation of evidence has shown that mi RNAs have a novel role in the regulation of the immune system, including the development and differentiation of immune cells, antibody production, and innate immune regulation.mi RNAs regulates cell differentiation and function in innate and adaptive immune cells by specific expression profiles, and its expression and functional disorders will lead to infection of various pathogens and immune diseases.In this study, in order to explore the mechanisms of mi RNA-related genes in pathogenic C. neoformans, we act effect and pathogenicity mechanism of mi RNA targeting related genes of MAPK ∕ NF-κB pathway in monocyte-macrophage induced by C. neoformans as research content. Firstly, we screened the differences expression of mi RNAs in THP-1 cell induced by C. neoformans, searched and collected the related bioinformatics analysis of target genes; Observe the expression of mi R-146 a, mi R-30 b in THP-1 cells group, healthy human primary mononuclear cells group, the expression of "immunologically active normal" group of patients with cryptococcal meningitis; Probeexpression pattern of mi R-146 a in THP-1 cells induced by C. neoformans, and detect the effect in MAPK ∕ NF-κB pathway observed exerted by mi R-146a; Analysis immune regulation and possible mechanisms of mi R-146 a targeting IRAK1 and TRAF6 in THP-1 cells induced by C. neoformans. So as to provide a theoretical basis of the body’s immune system for further research the pathogenicmechanisms of C. neoformans.Part I Differences expression of Micro RNA in THP-1 cell induced by Cryptococcus neoformans and related bioinformatics analysis of target genesWe screened the differential expression of mi RNA in a single nuclear macrophage THP-1 cells induced by C. neoformans(WN148), and predicted the target genes of these mi RNA, and analyzed the biological information. mi RNA expression profiling of 0 h and 6 h C. neoformans-induced THP-1cells(n= 4 each group). mi RNA were isolated by separating total RNAs on denaturing polyacrylamide gel electrophoresis and cutting a portion of the gel corresponding to the size 18-30 nucleotides. Total RNA was extracted using Trizol reagent(Invitrogen, CA, USA) according to the manufacturer’s protocol, and the RNA concentration and purity were determined by the Agilent Technologies 2100 Bioanalyzer. Small RNA library construction and sequencing,analysis of sequencing data, stem-loop q RT-PCR for mi RNA,bioinformatics analysis were finished by the Amplicon Gene Biotechnology Companies(Shanghai, China). Validation of mi RNA: Cells in each group were collected, extracted total RNA, used Takara Prime ScriptTMRT reagent Kit specific mi RNA stem loop primers reversal,used SYBR? Premix Ex Taq TM II detection reagent box and ABI7900 amplification and detection. U6 is selected as the reference, the relative expression of mi RNA calculated by 2-△△Ct. mi RNA target gene were obtained by the validated targets way in mi RWalk, A list of the predictions target genes will be carried on GO(gene ontology, GO) enrichment analysis through DAVID and biological pathway enrichment analysisthrough KEGG(Kyoto Encyclopedia of genes and genomes, KEGG).We identified mi R-4792, mi R-30b-5p(mi R-30b), mi R-30c-5p, mi R-223-3p, mi R-15b-3p, mi R-146a-5p(mi R-146a), and mi R-155-5p among the altered cases were significantly upregulated relative to the control group. When compared with the expression profiles established by Illumina sequencing, the q RT-PCR validation results demonstrate great congruence between the expression patterns determined using these two techniques for these mi RNAs.The target genes related to immunity were enriched in cell proliferation, transcription regulation, metabolic organelles, intracellular signal transduction, membrane transport, all types of gene expression regulation, nucleic acids and other biological processes.The biological pathway mainly enriched in cancer signaling pathway, MAPK signaling pathway, factor interaction, T cell signaling pathways, Toll-like receptor regulation of adhesion molecules and other signaling pathway.Part II Observation of the expression ofmi R-146 aand mi R-30 b in different groupsThe expression of mi R-146 a, mi R-30 b were evaluatedin THP-1 cells, human primary monocytes of healthy controls and “immunocompetent” patients respectively. For all experiments, cells were induced with WN148 at a MOI of 1:5. The supernatant and the cell pellet was collected separately after incubation. Peripheral blood mononuclear cells(PBMCs) were obtained from peripheral venous blood samples using Ficoll-Hypaque density gradient centrifugation. Human peripheral blood samples for purification of PBMCs were drawn at 7 to 8 am from patients with Cryptococcal meningitis and healthy controls from Department of Dermatology and Key Laboratory of Medical Mycology, Changzheng Hospital, Second Military Medical University. Human monocytes were purified from PBMCs by anti-CD14 microbeads(BD Bioscience) according to the manufacturer’s directions. Monocytes were harvested for related experiments. Cells were collected from each group, and the total RNA was extracted, and mi RNA were reversed through the stem loop primers specific. Amplificated and detected on ABI7900 quantitative PCR instrument, and observed the expression of mi R-146 a and mi R-30 b in each group of cells.We found, the basal levels of mi R-146 a was more low than mi R-30 b. Due to this reason possibly, mi R-146 a expression immediately increased in the early phase of incubation and reached the peak at 3h, then gradually declined from 3h to 12 h. However the expression levels of mi R-30 b increased progressively from 0h to 12 h in THP-1 cells following incubation. The dysregulation of mi R-146 a and mi R-30 b expression has been observed in primary human macrophages from patients relative to healthy volunteers.Part III Study on the expression of mi R-146 a in the monocytes induced by Cryptococcus neoformansFirst, we establish the C. neoformans induced cell model and observed the mi R-146 a expressionin THP-1 cellsinducedby C. neoformans; According to the front of the bioinformatics analysis results, observed the MAPK signal transduction pathway being closely related to mi R-146 a expression. THP-1 cells were incubated with the heat-killed C. neoformans WN148 for 0h, 3h, 6h, 9h, 12 h. Collected supernatant and cell pellet by centrifugation, tested the effect of mi R-146 a expression on NF-κB gene of MAPK signal pathway and the expression of inflammatory cytokines TNF-a, IL-1β. THP-1 cells were intervented with PDTC(NF-κB inhibitor) for 12 h, and then induced by the heat-killed C. neoformans WN148, and the further observations of mi R-146 a effect on expression of NF-κB and inflammatory cytokines TNF-a, IL-1β were conducted. To provide additional evidence of the mechanism of mi R-146 a in the regulation of the mi R-146a-NF-κB axis, we examined the effect of mi R-146 a on NF-κB expression in THP-1 cells transfected with mi R-146 a mimics and mi R-146 a inhibitors. The efficiency of transfection is shown obviously, when THP-1 cells were transfected with mi R-146 a mimics and inhibitors for 6 h, followed by C. neoformans induction for 3h.The results showed that the expression of mi R-146 a in the of the 3h reached the peak, and then gradually decreased from 3h to 12 h. At the same time, the expression of NF-κB protein level was increased continuously. The expression of TNF-a and IL-1β. PDTC treatment suppressed the expression levels of IL-1β and TNF-a. The results showed that the mi R-146 a level was significantly increased by C. neoformans induction in the early phase, and be associated with the expression of inflammatory cytokines. These data indicated that C. neoformansinduction increases mi R-146 a expression via a NF-κB-dependent mechanism.THP-1 cells were transfected with mi R-146 a mimics and inhibitors, mi R-146 a mimics significantly attenuated the the expression of p65, mi R-146 a inhibitorselevated the expression of p65, which means that mi R-146 a can significantly regulate the activationof NF-κB caused by C. neoformans. The changes in the activation of NF-κB affect the release of IL-1β and TNF-a. The results showed that NF-κB activation can be regulated by mi R-146 a via transfection with mi R-146 a mimics and inhibitors. These data indicated that mi R-146 a could exert negative regulation on the NF-κB pathway.Part IV mi R-146 a played a negative regulate role via directly targeting IRAK1 and TRAF 6 in THP-1 cellsmicro RNAs(mi RNAs) bind to the 3’UTRs of the target m RNAs and leading degradation or transcriptional repression of target m RNA at post-transcriptional level. We analysis and predict target genes of mi R-146 a by Target Scan and Miranda targetscan database, and chosed IRAK1 and TRAF6 as target genes of mi R-146 a for the further research.Bioinformatics prediction show that IRAK1 and TRAF6 3’UTR contains the complementary to the target sequence of mi R-146 a. We construct the expression vector containing IRAK1 and TRAF6 3’ UTR binding sequence of firefly luciferase, Renilla luciferase as a reference. We detected the influence of luciferase activity on overexpression or silencing of mi R-146 a by dual luciferase reporter assay system. In addition, the expression levels of IRAK1 and TRAF6 protein and gene in THP-1 cells transfected with overexpression or inhibited of mi R-146 a, were detected using quantitative PCR and Western blotting.We found that mi R-146 a could significantly inhibit the luciferase activity of the reporter gene plasmid of IRAK1 and TRAF6 3’UTR binding sequences. Transfection of mi R-146 a mimics group, expression of IRAK1 and TRAF6 m RNA were not obvious change; However, IRAK1 and TRAF6 protein were significantly lower than the control group of mimics mi R-146 a.In mi R-146 a inhibitor group, the expression of IRAK1 and TRAF6 m RNA was not significant change too, IRAK1 and TRAF6 protein were significantly higher than that of mi R-146 a inhibitor control group. These results suggest that the effect of mi R-146 a on the expression of irak1 and TRAF6 is post transcriptional regulation, inhibite the expression of IRAK1 and TRAF6 protein chiefly, not affect IRAK1 and TRAF6 m RNA.Based on the above study, we found that mi RNA was closely associated with inflammatory reaction in monocytes induced by C. neoformans. mi R-146 a is upregulated by NF-κB passageway, whereas mi R-146 a negatively regulates NF-κB activation through targeting IRAK1 and TRAF6 and then inhibiting expression of the NF-κB, release of inflammatory cytokines in the monocytes, which help to fine-tune the immune response. Taken together, our results suggest mi R-146 a may represent a future therapeutic target for regulation the inflammatory response in the host innate immune response to C. neoformans infection.