| ObjectiveProgrammed cell death(PCD)is a kind of active,controlled cell death,which is essential for normal cells development and homeostasis maintenance in multicellular organism.Apoptosis is a caspase-dependent manner of cell death.It could induce cellular components to form “apoptotic bodies” orderly to be engulfed by macrophages,which is a rapid manner for clearance of dead cells.Necroptosis(or programmed necrosis),on the other hand,is a newly recognized,highly controlled form of cell death.It is activated by certain ligands and leads to loss of plasma membrane integrity and inflammatory response.Several molecules,such as receptor interacting serine/threonine kinase(RIPK)1,RIPK3,mixed lineage kinase like protein(MLKL),PGAM5,DAI and cylindromatosis(CYLD),have been recognized to be participated in regulation of necroptosis.RIPK3 is a key molecule of necroptosis pathway.RIPK3 forms complex IIb(necrosome)with RIPK1 and MLKL.Fas associated death domain(FADD)is an important regulator of apoptosis and component of complex IIa,which could bind Fas to promote apoptosis.Tumor suppressor CYLD is a deubiquitinating enzyme specific to remove K63 ubiquitin chains.It could deubiquitinate RIPK1 to promote necroptosis.Caspse-8 cleaves CYLD on its D215 site and inhibits its deubiquitination of RIPK1.However,whether other proteins in necroptosis pathway could also be regulated by CYLD and its relevant mechanism is unknown.While FADD and RIPK3 have been extensively studied in viral infection,bacterial infection,cardiovascular disease,ischemia-reperfusion injury,chronic colitis,pancreatitis and liver injury,little is known about their roles during fungal infections.Among the major fungal pathogens,Cryptococcus neoformans is the cause of life-threatening invasive infections in both immunocompromised and immunocompetent hosts.While insufficiency of immune response is the major trigger of cryptococcal infection,excessive inflammation that occurs during therapy-induced immune reconstitution in cryptococcosis patients importantly contributes to worsening of the symptoms and patients’ mortality.It is reported that cryptococcal infection could induce death of macrophages and T cells,however,whether this kind of cell death influences the immune response to Cryptococcus neoformans.Therefore,we carry out two aspects of experiments.Firstly,we investigated the influence of D215 site mutation on apoptosis and necroptosis and its molecular mechanism.Secondly,we use intranasally inhalation model of cryptococcal infection to study the regulation of RIPK3 and FADD on lung cryptococcal infection.Methods1.The influence of CYLD and its D215 A mutation on PCDWe generated D215 A site mutation mice using CRISPR-Cas9 technology and isolated MDF and BMDM for in vitro cell viability assay.Western blot was used to detect key molecules expression in necroptosis and apoptosis pathways.Besides,we measured NF-κB,MAPKs and inflammasome activation in MDF and BMDM.In vivo animal models involved cerulein induced acute pancreatitis,LPS+z VAD induced necroptosis model and anti-Fas induced apoptosis model.2.The role of RIPK3&FADD on immune response in lung post cryptococcal infectionWe explored role of RIPK3 and FADD in mouse model of cryptococcal infection and identified yet unknown,critical contribution of apoptosis signaling in pulmonary immune responses to cryptococcal infection.We use Ripk3-/-and Ripk3-/-Fadd-/-mice to examine the survival course and fungal burdens in lung,brain and spleen in mice with infection.Lung sections and pulmonary leukocytes number were also assessed at 7 and 10 day post infection(dpi).We also determined pulmonary and systemic cytokine production,dendritic cells differentiation and activation in lung-associated lymph nodes(LALN),M1/M2 polarization patterns and fungicidal ability of bone marrow-derived macrophages after cryptococcal infection to explore the molecular mechanism.Results1.Deletion of CYLD inhibited apoptosis and necroptosis,while D215 A mutation of CYLD promoted apoptosis and necroptosis in MDF and BMDM.When treated with TNFα+Smac,Cyld D215A/D215 A MDF could induced necroptosis,which was confirmed by immunofluorescence.Immunoprecipitation showed that D215 A mutation promoted RIPK1 and RIPK3 interaction.D215 A mutation did not influence NF-κB,MAPKs and inflammasome activation.CyldD215A/D215 A mice showed similar phenotype with WT mice in cerulein induced acute pancreatitis,LPS+zVAD induced necroptosis model and anti-Fas induced apoptosis model.2.In mouse model of cryptococcal infection,we observed that deletion of RIPK3 alone or combined with FADD deletion lead to progressively robust Th1/Th17-biased responses with M1-biased macrophage activation in C.neoformans-infected lungs.These responses,rather than being protective,lead to paradoxical expansion of C.neoformans and rapid clinical deterioration in Ripk3-/-and Ripk3-/-Fadd-/-mice.Increased mortality in Ripk3-/-even more profoundly accelerated in Ripk3-/-Fadd-/-mice,was attributed to profound pulmonary damage due to neutrophil-dominant infiltration with prominent up-regulation of proinflammatory cytokines TNFα,IFNγ and IL-12.This was associated with selectively decreased frequencies of apoptotic neutrophils and CD4+T cells found in the infected Ripk3-/-Fadd-/-mice.Conclusions1.It is first proved that D215 A mutation of CYLD promotes apoptosis and necroptosis in primary cells.D215 A mutation does not influence NF-κB,MAPKs and inflammasome activation.Although D215 A mutation promotes PCD in intro,it does not show significant difference in animal model of apoptosis and necroptosis.CYLD is not an essential factor in necroptosis and its function might be tissue-specific.2.It is showed that RIPK3 and FADD,serving as physiological breaks,preventing the development of excessive Th1/Th17 bias and “unchecked” inflammation,which in turn leads to pulmonary damage and defective fungal clearance.Our study highlights the crucial importance of FADD and RIPK3 in preventing detrimental “side effects” of otherwise protective Th1 response in anti-fungal host defenses. |
PDF Full Text Request