Function And Mechanism Of Deubiquitinase OTUD1 In Antifungal Immunity Response

Innate immunity provides the first line of defense against pathogen infection.The activation of innate immunity depends on the recognition of pathogen associated molecular patterns by the pattern recognition receptors.C-type lectin receptors(CLRs)can recognize the fungi pathogen associated molecular patterns,activate the corresponding signal pathways,induce the production of inflammatory cytokines,and finally eliminate the fungi.CARD9 is an essential adaptor protein in antifungal innate immunity mediated by CLRs.CARD9 can recruit BCL10 and MALT 1 to form a CARD9-BCL10-MALT1(CBM)complex,which activates the canonical NF-?B and MAPKs,leading to the release of pro-inflammatory cytokines,ultimately leading to fungi clearance.The activity of CARD9 is critically regulated by ubiquitination,however,the deubiquitinases involved in CARD9 regulation remain incompletely understood.Here,we identified OTU deubiquitinase 1(OTUD1)as an essential regulator of CARD9.OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9,leading to the formation of the CBM complex and induced the activation of the canonical nuclear factor ?B(NF-?B)and MAPK pathway.OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the pro-inflammatory cytokine production following fungal stimulation.Importantly,Otud1-/-mice were more susceptible to fungal infection than WT mice in vivo.Collectively,our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.The study enriched the activity regulation mechanism of CARD9,and provided a new molecular mechanism for the activation of antifungal immunity response,which will be conducive to the development of new treatment strategies for fungal diseases.Objectives1.To identify the deubiquitinating enzymes(DUBs)that potentially regulate CARD9 deubiquitination;2.To determine the role and mechanism of the DUB acting on CARD9 in the antifungal immunity response;3.To determine the physiological significance of the DUB in the regulation of antifungal innate immunity through the knockout mouse model.Methods1.To screen and identify the DUBs which can deubiquitinate CARD91.1.To screen the DUBs which can deubiquitinate CARD9HEK293T cells were transfected with the indicated combination of V5-CARD9,HA-ubiquitin(HA-Ub)and Flag-tagged or Myc-tagged DUBs.The cell lysates were subjected to IP and western blot.1.2.To identify OTUD1 catalyzes deubiquitination of CARD9 through overexpression experimentHEK293T cells were transfected with HA-Ub,Myc-CARD9 and various doses of Flag-OTUD1 or Flag-OTUD1 mutant C320A.The cell lysates were subjected to IP and western blot.1.3.To confirm OTUD1 catalyzes deubiquitination of CARD9 in vitroThe deubiquitination reaction mixture contains ubiquitinated Flag-CARD9 and recombinant Flag-OTUD1 or OTUD1 mutant C320A.After incubation for 60min,the mixture was detected by western blot analysis with anti-HA Ab.1.4.To determine whether OTUD1 specifically deubiquitinates CARD9 in physiological conditionsOtud1+/+and Otud1-/-BMDMs were treated with HKCK-Y for 20min.Cell lysates were immunoprecipitated with anti-CARD9 or BCL10.The ubiquitinated CARD9 or BCL10 was detected with anti-ubiquitin(Ub)Ab.2.To identify the interaction between OTUD1 and CARD9 and search for the interaction domain2.1.To identify the interaction between OTUD1 and CARD9 through overexpression experimentHEK293T cells were transfected with the indicated plasmids.The cell lysates were IP and then immunoblotted with the indicated antibodies.2.2.To confirm the interaction between OTUD1 and CARD9 in vitroRecombinant Flag-OTUD1 and Flag-CARD9 proteins were incubated in 37?for 4h,then the mixture was subjected to IP and western blot.2.3.To determine the association between endogenous CARD9 and OTUD1BMDMs were stimulated with HKCK-Y for indicated time points,the cell lysates were subjected to IP and western blot.2.4.To search for the interaction domainHEK293T cells were transfected with Flag-OTUD1 and Myc-CARD9,Myc-CARD9(6-98aa),Myc-CARD9(117-420aa),Myc-CARD9(420-536aa).The cell lysates were IP and western blot analysis with indicated antibodies;HEK293T cells were transfected with Myc-CARD9 and Flag-OTUD1,Flag-OTUD1(1-185aa),Flag-OTUD1(1-308aa),Flag-OTUD1(1-450aa),Flag-OTUD1(156-481aa).The cell lysates were IP and western blot analysis with indicated antibodies.3.To investigate the role of OTUD1 on antifungal immunity response3.1.To investigate the role of OTUD1 on the CLRs-induced production of cytokines in cellsWT and Otud1-KO RAW264.7 cells were stimulated with Zymd or ?-mannan for indicated time points,qPCR analyzed the expression of IL-6,TNF-? and IL-1?.Otud1+/+and Otud1-/-BMDMs were stimulated with Zymd or ?-mannan for indicated time points.The expression of IL-6,TNF-?,IL-1?,IL12p35,IL12p40 and IL23p19 and secretion of pro-inflammatory cytokines(IL-6,TNF-?,IL-1? and IL-12p40)and chemokines(CXCL1 and CXCL2)were measured by quantitative RT-PCR and ELISA,respectively.We also stimulated Otud1+/+and Otud1-/-BMDMs with HKCA-Y,HKCA-H or TDB and measured the above mentioned pro-inflammatory cytokines and chemokines expression and secretion.Otud1+/+and Otud1-/-BMDCs were stimulated with Zymd or ?-mannan for indicated time points.The similar methods assessed pro-inflammatory cytokines and chemokines expression and secretion.Otud1+/+and Otud1-/-BMDMs were reconstituted with WT mOTUD1 or mOTUD1 mutant C293A,then stimulated with Zymd for 24h,ELISA analyzed the production of IL-6 and TNF-?.3.2.To investigate the role of OTUD1 on the activation of CLRs signaling pathwayOtud1+/+ and Otud1-/-BMDMs were stimulated with Zymd or ?-mannan for indicated time points,the phosphorylation levels of Syk,PLC-y2,PKC-?,I?B?,p65,Jnk,Erk and p38 were detected by western blot analysis with indicated antibodies;Otud1+/+and Otud1-/-BMDMs were stimulated with HKCA-Y or C.albicans for indicated time points,the phosphorylation levels of I?B?,p65,Jnk,Erk and p38 were detected by western blot analysis with indicated antibodies.3.3.To investigate the role of OTUD1 on the phagocytosisOtud1+/+and Otud1-/-BMDMs and BMDCs were stimulated with HKCA-Y for indicated time points,the rate of phagocytosis was measured by flow cytometry analysis.4.To clarify the mechanism of OTUD1 in antifungal innate immunity4.1.To investigate the role of OTUD1 on the CBM complex formationHEK293T cells were transfected with Myc-CARD9,Flag-BCL10,HA-Ub and with Flag-OTUD1 or Flag-OTUD1 mutant C320A.The cell lysates were subjected to IP and western blot.Otud1+/+and Otud1-/-BMDMs were stimulated with HKCK-Y for indicated time points,the cell lysates were subjected to IP and western blot;4.2.To explore the ubiquination form of CARD9 regulated by OTUD1HEK293T cells were transfected with the indicated combination of Myc-CARD9,Flag-OTUD1 and HA-Ub or its mutants.The cell lysates were subjected to IP,followed by western blot.4.3.To detect which polyubiquitin chain of CARD9 cleaved by OTUD1 regulates the CBM complexHEK293T cells were transfected with Myc-CARD9,Flag-BCL10,HA-Ub lysine residue mutants and with Flag-OTUD1 or Flag-OTUD1 mutant C320A.The cell lysates were subjected to IP,followed by western blot.4.4.To investigate the function of OTUD1 in the CARD9 stability and CARD9 K27-linked polyubiquitinationProteins in BMDMs from Otud1+/+and Otud1-/-mice stimulated with Zymd or?-mannan for indicated times,western blot analysis the CARD9 expression.Otud1+/+and Otud1-/-BMDMs were treated with HKCK-Y for 20min.Cell lysates were immunoprecipitated with anti-CARD9 Ab.The K27 ubiquitinated CARD9 was detected with anti-Ubiquitin(linkage-specific K27)Ab.5.To investigate the role of OTUD1 in antifungal immune responses in vivoOtud1+/+(n=9)and Otud1-/-(n=9)mice(age-matched littermates)were infected with C.albicans strain SC5314(2×105 fungal cells per mouse)by intravenous injection,the survival and weight loss of the mice were examined;Kidney sections from C.albicans-infected Otud1+/+and Otud1-/-mice were stained with haematoxylin and eosin(H&E),periodic acid-Schiff(PAS)or Ly-6G(neutrophil marker)staining to show the fungal inflammation,fungal growth and neutrophil infiltration,respectively;C.albicans colony-forming units(CFU)in the kidney,liver and spleen of Otud1+/+and Otud1-/-mice 5 d after infection were detected;IL-6 and TNF-? production in serum obtained from Otud1+/+and Otud1-/-mice after infection with C.albicans for 24 h were detected by ELISA.Results and conclusion1.Identification of OTUD1 as a CARD9 DUB1.1.OTUD1 reduces CARD9 ubiquitination levelsWe overexpressed V5-CARD9,HA-ubiquitin(HA-Ub)together with DUBs into HEK293T cells,then the Co-IP and western blot were used to analyze the ubiquitination of CARD9.The result showed that OTUD1 can significantly reduce CARD9 ubiquitination.1.2.OTUD1 regulates CARD9 deubiquitination via its deubiquitinase activityWe transfected increased amount of OTUD1 WT or mutant C320A together with Myc-CARD9 and HA-ubiquitin into HEK293T cells,then the IP and western blot was used to analyze the ubiquitination of CARD9.The result showed that OTUD1 WT but not the mutant C320A attenuated CARD9 ubiquitination in a dose dependent manner,indicating that OTUD1-mediated CARD9 deubiquitination requires its deubiquitinase activity1.3.OTUD1 directly deubiquitinates CARD9We performed in vitro ubiquitination assays to confirm that OTUD1 directly catalyzes deubiquitination of CARD9.The recombinant OTUD1 WT or OTUD1 C320A was co-incubated together with the enriched ubiquitinanted CARD9,western blot was used to analyze the ubiquitination of CARD9.Recombinant OTUD1 WT but not its mutant C320A was found to remove the polyubiquitination chain from CARD9,indicating that OTUD1 can directly deubiquitinate CARD9 depending on its deubiquitinating enzyme activity.1.4.OTUD1 specifically deubiquitinates CARD9 in physiological conditionsTo determine whether OTUD1-mediated CARD9 deubiquitination occurs in physiological conditions,we isolated Otud1+/+and Otud1-/-mice BMDMs and stimulated with HKCA-Y,then analyzed the endogenous CARD9 ubiquitination status through Co-IP and western blotting.The results showed that HKCA-Y stimulation increased endogenous CARD9 ubiquitination,importantly,the level of endogenous CARD9 ubiquitination was further increased in Otud1-/-BMDMs compared with that in Otud1+/+BMDMs upon HKCA-Y stimulation.To determine whether OTUD1 specifically deubiquitinates CARD9,we also assessed the ubiquitination level of BCL10,and observed that BCL10 ubiquitination were comparable between Otud1+/+and Otud1-/-BMDMs before and after being stimulated with HKCA-Y.All together,these data demonstrated that OTUD1 removes ubiquitin chains from CARD9 through its DUB enzymatic activity.2.OTUD1 regulates CARD9 by interacting with CARD92.1.OTUD1 interacts with CARD9In order to investigate whether OTUD1 deubiquitinates CARD9 through the association between OTUD1 and CARD9,we transfected OTUD1 WT or mutant C320A together with Myc-CARD9 into HEK293T cells,then the IP and western blot was used to analyze the intaeraction between OTUD1 and CARD9.The result showed that both OTUD1 and OTUD1 C320A can associate with CARD9,indicating that the catalytic activity of OTUD1 is dispensable for its association with CARD9.2.2.OTUD1 directly interacts with CARD9We performed in vitro binding assays to confirm the association between OTUD1 and CARD9.Recombinant OTUD1 was found to interact with CARD9,confirming direct protein-protein interactions.2.3.Endogenous OTUD1 interacts with CARD9In order to determine whether the association between OTUD1 and CARD9 occurs in physiological conditions,we performed endogenous binding experiments.We also noticed the interaction between endogenous CARD9 and OTUD1 in BMDMs.Notably,the association between endogenous CARD9 and OTUD1 was substantially enhanced upon HKCA-Y stimulation in BMDMs2.4.OTUD1 interacts with CARD9 through OTU and UIM region of OTUD1 and the coiled-coil domain of CARD9In order to further determine the binding domains for CARD9 and OTUD1,the truncation mutants of CARD9 and OTUD1 were overexpressed in HEK293T cells,and Co-IP results showed that OTUD1 combines with CCD region of CARD9 through OTU and UIM region.3.OTUD1 positively regulates antifungal immune response3.1.OTUD1 positively regulates CLRs-induced production of cytokinesTo explore the potential role of OTUD1 in antifungal immune response,we first generated Otud1-KO RAW264.7 cells with a CRISPR/Cas9 technology.These cells were stimulated with Zymd and ?-mannan,which are recognized by Dectin-1 and Dectin-2/3 respectively.We found the mRNA levels of IL-6,TNF-? and IL-1? were decreased in Otud1-KO RAW264.7 cells compared with that in WT cells after Zymd and ?-mannan stimulation.In addition,we prepared BMDMs from Otud1+/+and Otud1-/-mice and stimulated these cells with Zymd and ?-mannan.We found that the secretion and mRNA levels of pro-inflammatory cytokines(IL-6,TNF-?,IL-1? and IL-12)and chemokines(CXCL1 and CXCL2)were lower in Otud1-/-BMDMs compared to that in Otud1+/+ BMDMs after Zymd-and ?-mannan stimulation.We also repeated this experiment in BMDCs and observed the similar results;Further,we stimulated Otud1+/+and Otudl-/-BMDMs with HKCA-Y,HKCA-H and TDB and found that the secretion and mRNA levels of IL-6 and TNF-? were attenuated in Otud1-/-BMDMs compared to that in Otud1+/+BMDMs after HKCA-Y-,HKCA-H-and TDB stimulation.In order to confirm the decreased expression of cytokines in CLRs signaling pathway was due to OTUD1 deficiency,we restored the expression of OTUD1 in BMDMs through retroviral infection.Reconstitution of mouse WT OTUD1,rather than the OTUD1 mutant C293A,rescued Zymd-induced the production of IL-6 and TNF-?.Taken together,these data suggested that OTUD1 positively regulates CLRs-mediated cytokine production.3.2.OTUD1 positively regulates CLRs-induced activation of NF-?B and MAP kinasesNext,we assessed the role of OTUD1 in the regulation of CLRs-induced signaling.We prepared BMDMs from Otud1+/+and Otud1-/-mice,followed by stimulation with Zymd and ?-mannan.We analyzed the phosphorylation status of the main kinases in the CLR s-induced signaling.We found that phosphorylation of Syk,PLC-y2 and PKC-? was comparable in Otud1-/-and Otud1+/+BMDMs upon stimulation with Zymd and ?-mannan,indicating that OTUD1 was not involved in receptor-proximal signaling.However,Otud1-/-BMDMs exhibited reduced levels of phosphorylated p65,I?B,Jnk,Erk and p38 upon stimulation with Zymd and?-mannan.Moreover,we also evaluated the NF-?B and MAPKs activation in absence of OTUD1 upon HKCA-Y and C.albicans stimulation.We found that the phosphorylation levels of I?B,p65 and MAPKs were also decreased in Otud1-/-BMDMs after HKCA-Y and C.albicans stimulation.Together,these data demonstrated that OTUD1 positively regulates CLRs-induced antifungal innate signaling and production of pro-inflammatory cytokines.3.3.OTUD1 has no effect on the phagocytosis in innate immune cellsPhagocytosis is the first line of defense against invading fungal pathogens,we also assessed whether OTUD1 affected the phagocytosis ability of BMDMs and BMDCs and found that the cells phagocytosis abilities of innate immune were similar between Otud1-/-and Otud1+/+ cells.These data suggested that OTUD1 has no effect on the phagocytosis ability of innate immune cells.4.OTUD 1-m ediated CARD9 deubiquitination potentiates CBM complex formation4.1.OTUD1-mediated CARD9 deubiquitination promotes the assembly of CBM complexesCBM signalosomes play a vital role in the antifungal immune response,we reasoned that OTUD1-mediated deubiquitination of CARD9 might affect the assembly of CBM complex.To confirm this hypothesis,we transfected Myc-CARD9,HA-Ub,Flag-BCL10 with Flag-OTUD1 or OTUD1 mutant C320A into HEK293T cells.Overexpression of HA-Ub(which increased CARD9 ubiquitination level)impaired CARD9-BCL10 interaction,notably,OTUD1,but not OTUD1 mutant,restored CARD9-BCL10 interaction.We also stimulated BMDMs from Otudl-/-and Otud1+/+mice with HKCA-Y.The result showed that HKCA-Y stimulation promoted the interaction between CARD9 and BCL10,however,OTUD1 deficiency profoundly decreased CARD9-BCL10 interaction.Taken together,these results demonstrated that OTUD1 cleaves polyubiquitin chains from CARD9 and potentiates CBM complex formation.4.2.OTUD1 mainly removes K29-,K33-and K63-linked polyubiquitin chains from CARD9In order to explore which ubiquination form of CARD9 is regulated by OTUD1,we transfected Myc-CARD9,Flag-OTUD1 and HA-Ub or its mutants into HEK293T cells.The Co-IP result showed OTUD1 mainly removes K29-,K33-and K63-linked polyubiquitin chains from CARD9.4.3.K33-linked polyubiquitin chain removed by OTUD1 from CARD9 promotes the assembly of CBM complexesThe previous results clarified that OTUD1 promotes the assembly of the CBM complex by regulating the deubiquitination of CARD9 and found that OTUD1 mainly cleaved K29-,K33-and K63-linked polyubiquitin chains from CARD9,so we next explored which polyubiquitin chain of CARD9 cleaved by OTUD1 promoted the CBM complex.We found that only K3 3-linked polyubiquitin chain removed by OTUD1 from CARD9 enhanced the CARD9-BCL10 interaction,K29-and K63-linked polyubiquitin chains removed by OTUD1 had no effect on the CARD9-BCL10 interaction.Because OTUD1 did not cleave K6-,K11-,K27-and K48-linked polyubiquitin chains from CARD9,the CARD9-BCL10 interaction was not affected by OTUD1 in HA-Ub K6,K11,K27 and K48 transfected cells.4.4.OTUD1 has no effect on the CARD9 stability and can not catalyze K27-linked polyubiquitinationIn order to explore whether OTUD1 is involved in the degradation of CARD9,we assessed the stability of CARD9 in Otudl+/+and Otud1-/-BMDMs after stimulated with Zymd and ?-mannan.We observed that CARD9 protein level was comparable between Otud1+/+and Otudl-/-BMDMs upon Zymd and ?-mannan stimulation,indicating OTUD1 does not affect CARD9 expression.TRIM62 mediates K27-linked ubiquitination of CARD9 at K125,which promotes CARD9 activation.However,we found that OTUD1 did not affect the K27-linked ubiquitination levels of CARD9 in BMDMs after stimulated with HKCA-Y.5.OTUD1 deficiency impairs antifungal immune responses in vivoIn the C.albicans induced fungal model,we observed that Otud1+/+mice showed moderate weight loss,while,Otud1-/-mice exhibited substantial weight loss after infection with C.albicans and had much lower survival rates than Otud1+/+mice.Otud1-/-mice showed more renal inflammation and increased numbers of C.albicans hyphae in the kidney.Additionally,Otud1-/-mice had more numbers of infiltrated neutrophils in the kidneys than in the kidneys of Otud1+/+mice.Consistently,Otud1-/-mice exhibited more C.albicans colony-forming units(CFU)in the kidney,liver and spleen compared to that in Otud1+/+mice.Moreover,the pro-inflammatory cytokines IL-6 and TNF-? in the serum of Otud1-/-mice were significantly lower than that in Otud1+/+mice.These data demonstrated OTUD1 is an effective regulator in the regulation of antifungal immune responses in vivo.Innovation1.At present,there are few studies on the deubiquitination of CARD9,and it has not been found that deubiquitination of CARD9 regulates the assembly of CBM complex.We found for the first time that the deubiquitination of CARD9 by OTUD1 could promote the recruitment for BCL10,which provides a new direction for the functional exploration of CARD9.2.It has been reported that OTUD1 is related to tumor development and RNA virus escape,but its function in antifungal innate immune response has not been explored.Our research confirmed that OTUD1 played a positive role in the antifungal pathway and could enhance the antifungal ability of the body.3.Fungal infection with high incidence and mortality rate has become a major public health problem in the world,especially for the immunocompromised population,seriously endangers human health.In this study,we identified OTUD1 cleaved polyubiquitination chains from CARD9,thus enhancing the activation of NF-?B pathway.This discovery can provide a new idea and treatment bootstrap for clinical treatment of fungal infectious diseases.