DOCK2 Promotes Antifungal Immunity By Regulating The Activation Of Rac GTPase

Fungal infections are major public health threats,especially HIV patients,immunecompromised people with hematopoietic stem cell transplantation(HSCT)and chemotherapy,or patients with primary immune deficiencies are susceptible to fungal infections.It has been reported that lots of fungal species bloom in the gut of COVID-19 patients.Fungal infection causes approximately 1.5 million deaths each year worldwide,Candida albicans are among the most prevalent opportunistic fungal pathogens in humans.Although several anti-fungal drugs have been developed,the mortality rate of fungal infections still exceeds 40%.The major reasons for the high mortality of fungal infections are limited antifungal drugs and the emergence of drug-resistant species.Therefore,a better understanding of the mechanisms of host defense against fungi is crucial for the development of effective preventive and therapeutic strategies and drugs.The CLRs,such as Dectin-1,Dectin-2,Dectin-3,and Mincle are mainly expressed on macrophages,neutrophils and dendritic cells,and they recognize fungal cell wall components such as β-glucan,α-mannan,and glycolipids,respectively.Recognition of fungal cell wall components by various CLRs results in activation of spleen tyrosine kinase(Syk)in macrophages and dendritic cells(DCs),thereby triggering activation of CARD9-BCL10-MALT1(CBM)complex-dependent mitogen-activated protein kinases(MAPKs)and nuclear factor-κB(NF-κB)signaling pathways,inducing downstream cytokines and chemokines(including IL-1β,IL-12,IL-6,IL-23,TNFα,CXCL1,etc).Moreover,these cytokines and chemokines recruit neutrophil infiltration,and macrophage maturation for the distinguishing of fungal infections.DOCK2(dedicator of cytokinesis 2)is a member of the CDM family protein(CED-5in Caenorhabditis elegans and DOCK180 in mammals),which is specifically expressed in hematopoietic cells.It controls actin recombination and regulates lymphocyte activation,migration and morphological changes.It is also found that the double allelic mutation of DOCK2 led to the impairment of Rac1 activation in T cells of clinical patients and migration defects of T cells,B cells and NK cells induced by chemokines.Here,we report that DOCK2 is indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages.DOCK2-deficient macrophages have decreased Rac GTPase activation and ROS(reactive oxygen species)synthesis after fungal stimulation,which in turn attenuates the killing of intracellular fungi and the activation of downstream signalling pathways.Mechanistically,Syk and DOCK2 are interacted with each other when co-expressed in 293 T cell.When truncated Syk and DOCK2 are co-expressed in 293 T cell,we can get that the SH3 domain in DOCK2 interacts with Syk and the kinase domain in Syk interacts with DOCK2.The endogenous interaction becomes gradually enhanced after fungal stimulation.The tyrosine kinase Syk phosphorylates DOCK2,and the endogenous phosphorylation becomes gradually enhanced after fungal stimulation.After treatment with the kinase inhibitor Piceatannol,the phosphorylation of DOCK2 is significantly reduced.When Syk and DOCK2 are co-expressed in 293 T cell,and co-IP DOCK2-Flag by Flag antibody and then eluted by 3×Flag peptide solution,the tyrosine sites of DOCK2 are identified at the122、224、985 and 1405 by mass spectrometer.After the four tyrosine in DOCK2 are mutated respectively,we can get that the phosphorylated DOCK2 was significantly reduced when mutated the DOCK2 at the 985 th and 1405 th tyrosines.When fungal stimulation the recursed vector,DOCK2,DOCK2-Y985+1405F in DOCK2 deficient THP-1 cell,the activation of Rac1,synthesis of ROS and downstream signaling activation are significantly reduced.Based on the molecular and cellular mechanism we show that nanoparticlemediated delivery of in vitro transcribed(IVT)Rac1 m RNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo.Taken together,this study not only identifies a critical role of DOCK2 in antifungal immunity by regulating Rac1 activity,but also provides a proof-of-concept for the treatment of invasive fungal infections by using m RNA.