Immune Protection Of NLRC4-mediated Macrophage Activation In The Pulmonary Infection Caused By Cryptococcus Gattii

Background:Cryptococcus is an opportunistic pathogen that exists widely in nature,which mainly infects the respiratory tract and causes cryptococcal meningitis and cryptococcal pneumonia.There will be a very high mortality rate,if patients are not diagnosed and treated in time.Despite the serious threat of cryptococcal infection to human health,current treatment options are limited.Therefore,it is very important to study the targeted virulence characteristics of Cryptococcus,the response pathway that causes damage,and the mechanism of regulating the host’s immunity for the effective treatment of infection caused by Cryptococcus.Nucleotide-binding and oligomerization domain-like receptors("NLR")are cytoplasmic pattern-recognition receptors("PRRs")that play key roles in innate immune responses.Recent studies showed that NLR was associated with the recognition of fungal invasion by the host.The inflammasome is key component in downstream immune recognition of the PRRs.and the activation of the inflammasome complex would activate caspase 1 and promote the release of Interleukin-1β(IL-1β)and Interleukin-18(IL-18),and trigger cell pyroptosis.Currently,increasing evidence suggests that the inflammasome plays a crucial role in the development of a variety of inflammatory diseases.Until now,the role of the inflammasome family has notbeen clarified thoroughly.At present,among the NLR family,more studies focued on the NLRP3 inflammasome,while the study of NLRC4 was relatively rare.Several studies had showed that the flagellin or bacterial secretion system of pathogens such as Salmonella typhimurium,Streptococcus mutans,and Legionella pneumophila can lead to the activation of the NLRC4 inflammasome in macrophages,and affect the expression of downstream pro-inflammatory cytokines,ultimately trigger proptosis and restriction of bacterial replication inside the host’s body.Studies had also confirmed that the NLRC4 inflammasome can be activated in fungal infections such as dermatophytes(Malassezia)and oropharyngeal Candida,and played a protective role.Some studies found that the expression of inflammatory factors in oral mucosal cells of NLRC4-/-mice were lower than that of normal mice when both were infected with Candida albicans orally,indicating that NLRC4 played a role in the early resistance of host’s mucosal tissue to Candida albicans infection.In vulvovaginal candidiasis,NLRC4 regulated the production of IL-18,and IL-18 helped to increase the activity of Interleukin 22(IL-22),so NLRC4 played an important role in regulating the network interaction of IL-18.In view of the partial similarity between the above pathogens infection and cryptococcal infection,the latter may also be directly or indirectly recognized by the host through NLRC4,and then triggers changes of downstream inflammatory factors cell behavior.There are few researches and no literature report at home and abroad on whether NLRC4 inflammasome inhibits Cryptococcus by inducing pyroptosis.Therefore,a study is designed and conducted through related experiments to identify whether NLRC4 inflammasome can be activated by Cryptococcus gattii infection and play a role.Objective:This study is aimed to investigate,through in vivo and in vitro experiments,whether inflammasome NLRC4 can be activated by Cryptococcus gattii and release inflammatory factors IL-1β and IL-18,thereby cause pyroptosis,and help to kill Cryptococcus and inhibit its intracellular parasitism,so as to provide an evidencen for identification of the interaction between Cryptococcus and the host’s immune response.Material and Methods:1.qRT-PCR technology was used to detect the changes of gene transcription levels of inflammatory factors such as NLRC4 and IL-1β in human monocytic-leukemia cells(THP-1)and mice Bone Marrow-Derived Dendritic Cells(BMDC)infected with Cryptococcus gattii.2.Western blot was used to detect the expression level of NLRC4 protein and the maturation of caspase-1 in THP-1 cells infected with Cryptococcus gattii;the secretion of downstream inflammatory response-related proteins was examined after knockdown of NLRC4 by Small interfering RNA(siRNA)transfection.3.Models of NLRC4-/-mice and wild-type mice with pulmonary infection caused by Cryptococcus gattii were established.Survival analysis was made:the survival status of mice was continuous observed,recorded and summarized using statistical method,and assessed in the way of Kaplan-Meier plotter curve.The fungal burden in the lungs was detected by in vivo fluorescence imaging for small animals,living body and in vitro tissue Micro-CT scans were conducted to compare differences in nodules and histopathological lesions due to cryptococcal infection.PAS staining was utilized to identify the distribution of Cryptococcus gattii in lungs.The fungal burden of extrapulmonary organs was determined by tissue isolation and colony counting.4.The downstream inflammatory response-related proteins of NLRC4 in whole-lung protein samples were quantitatively analyzed by ELISA.5.The peritoneal macrophages were extracted from the mice for plating and infection.The effect of NLRC4 on the activation of caspase-1 caused by Cryptococcus gattii was detected by Caspase-1 detection kit.6.The release of ROS from macrophages stimulated by Cryptococcus gattii was detected in the way of immunofluorescence and flow cytometry.Lactate dehydrogenase(LDH)in the supernatant of cell lysate was quantified as a measure of the pyroptosis of the macrophages.The number of leukocytes in the bronchoalveolar lavage fluid(BALF)of the two groups of mice were compared.7.Flow cytometry technology was used to examine the phagocytosis of macrophage in the NLRC4-/-group.Results:1.Cellular level experiments confirmed that the Cryptococcus gattii could induce increased expression of IL-1β and IL-18 in BMDC,and could activate caspase-1 and NLRC4 inflammasomes in THP-1-derived macrophages,and affect the release of downstream IL-1βand IL-18.2.In experiments on animal models,compared with wild-type mice,the survival rate of NLRC4-/-mice with Cryptococcus gattii infection was significantly lower(p=0.0208),the lung fungal burden was increased,and the histopathological damage was more significant.In the quantitative analysis of whole-lung protein samples,it was found that the expression of TNF-α and IL-1β in the whole lung of the NLRC4-/-mice with cryptococcal infection was lower than that of the wild group.3.When exploring the mechanism,we found that NLRC4 significantly affected the activation of caspase-1 caused by Cryptococcus gattii,and also affected the release of ROS and LDH,the number of macrophages and the occurrence of phagocytosis due to Cryptococcus gattii.Conclusions:Based on clinical problems,this study has verified the following scientific problems from the level of gene transcription,protein translation,cell experiments and animal in vivo experiments:1.Cryptococcal infection can cause the activation of the NLRC4 inflammasome,and the activation of the caspase-1 pathway and the increased expression of IL-1β and IL-18 in the downstream.2.The experiments have found that the NLRC4 inflammasome plays an important role in the host’s resistance to Cryptococcus infection:the expression level of NLRC4 significantly affects the fungal load in the lungs,the histopathological damage and survival of the mice with Cryptococcus gattii infection.3.As to the mechanism,we have found that NLRC4 affects the release of ROS and LDH caused by Cryptococcus gattii and the phagocytosis of macrophages,indicating that NLRC4 can help the body to clear Cryptococcus.In conclusion,the activation of NLRC4 inflammasome is of great significance to the prognosis of Cryptococcus gattii infection,and may play an important auxiliary role in anti-Cryptococcus gattii therapy by regulating the expression of NLRC4.Limitation:this study is subject to certain limitations.First,our research on the mechanism of NLRC4 regulating the activation of casepase-1 is not in-depth enough,and the specific signaling pathway and action site have not been identified.Second,issues like whether the activation of the NLRC4 inflammasome would affect the efficacy of anti-cryptococcal infection drugs and whether the inflammasome can be used as a drug targeting site still require further research and investigation.