Function And Mechanism Of TRIM32 On The Regulation Of Antibacterial Innate Immunity

Tripartite motif protein 32(TRIM32)is an RBCC-NHL-type TRIM member containing of a RING domain,two B-boxes,a coiled-coil region,and the C-terminal NHL domain composed of 6 NHL repeats.TRIM32 is ubiquitously expressed in mammalian tissues and has been reported to be involved in different biological processes,including innate immunity,development,differentiation,signal transduction,tumorigenesis,et al.TRIM32 has been reported to be involved in the regulation of several innate immune signaling pathways by ubiquitinating XIAP,PIASy and STING.TRIM32 also plays a key role on the pathogen-host interaction by ubiquitinating polymerase basic protein 1 of the influenza A virus or binding Salmonella effector Sse K3.However,the role and mechanism of TRIM32 in intrinsic immunity against Gram positive bacteria have still not reported.We investigated the function and mechanism of TRIM32 on the regulation of innate immunity against extracellular and intracellular bacteria using Streptococcus suis and Listeria monocytogenes as model bacteria respectively.S.suis is an important emerging worldwide pig pathogen and zoonotic agent causing severe meningitis and streptococci toxic-shock-like syndrome in humans.Listeria monocytogenes is a foodborne pathogen causing bacteremia and organ failure in susceptible immunocompromised people and fetuses,and even fetal death with improper treatment.Previous studies showed that S.suis invades the host immune system and results in an early burst of proinflammatory cytokines,leading shock and sudden death.If the patient survives,the pathogen might traverse across the blood-brain barrier via paracellular manner and cause meningitis.L.monocytogenes activates innate immune response by releasing its double DNA and c-diGMP.These pathogen-associated molecular patterns then activate NF-k B,MAPK and STAT signaling to produce interferon and inflammatory cytokines,and aggravate the host's inflammatory infection.In the first part of this study,we constructed TRIM32 gene knockout mouse and RAW264.7 cells by TALEN and CRISPR/Cas9 gene editing technology,respectively,which were important materials to study the function and mechanism of TRIM32 on defense against Gram positive bacterial infection.In the second part,we focused on the role of TRIM32 on streptococcal toxic-shocklike syndrome and meningitis.Wild-type and TRIM32 knockout mice were intraperitoneally or intravenously injected high-dose of S.suis 05ZYH33,and we then investigated the survival rate,bacteremia and the level of cytokines and chemokines in blood.Our results demonstrated that compared to wild-type mice,the survival rate of TRIM32 knockout mice was significantly increased,the bacterial level in blood,the level of proinflammatory cytokines(IL-6,TNF?,IL-18,IL-1?,IFN-?)and chemokines(MCP-1,MCP-3,GRO?,MIP-1?,MIP-2,IP-10,RANTES)were significantly decreased.These results indicated that TRIM32 positively regulated S.suis-induced innate immune signaling.Besides,in order to study the function of TRIM32 on the development of S.suis meningitis,wild-type and TRIM32 knockout mice were intraperitoneally injected with low-dose of S.suis 05ZYH33 to allow for the development of S.suis meningitis.We measured the bacterial loads and pathological injury in brain,and permeability changes of blood-brain barrier,as well as the component and number changes of innate immune cells in blood and brain.Our results demonstrated that bacterial loads as well as the pathological injury in brain of TRIM32 knockout mice were significantly decreased than those of wild-type mice,indicating deficiency of TRIM32 relieves the development of S.suis meningitis.Further studies showed that compared to wild type mice,TRIM32 knockout mice revealed the pathogenic characters of increasing permeability of blood-brain barrier and infiltration of inflammatory monocytes and polymorphonuclear neutrophils to brain,which contributed to clear the bacteria,decrease the bacterial loads in brain,prevent and relieve the development of S.suis meningitis.In the third part,we focused on the function of TRIM32 on defense against L.monocytogenes.We first found that the expression of TRIM32 was increased during the course of L.monocytogenes,indicating that TRIM32 might involve in the host defense against L.monocytogenes infection.Our further results demonstrated that compared to the wild-type mice,the survival rate of TRIM32 knockout mice was significantly increased,the bacterial loads and the pathological injury in spleen and liver of TRIM32 knockout mice were significantly decreased,the levels of inflammatory cytokines such as IL-12,IL-18,IL-6,TNF?,IFN-?,IFN-? in sera were also significantly decreased which were in accord with the decreased levels of p44/p42,JNK,STAT and TBK1 phosphorylation.The above results suggested that TRIM32 positively regulated the innate immune signaling pathway.Next,we explored the possible mechanisms of decreased susceptibility of TRIM32 knockout mice to L.monocytogenes infection.We first investigated the dynamic changes of population of innate immune cells.Our experimental results demonstrated that there were higher rates of macrophages in spleen of TRIM32 knockout mice at day 1 post infection and neutrophils in liver of TRIM32 knockout mice at day 3 post infection than those of wild type mice,indicating TRIM32 deficiency contributed to bacterial clearance of the phagocytes.Then,we further investigated the viability and bactericidal ability of innate immune cells.Our results demonstrated that compared to wild-type mice,there were higher rate of viable neutrophils secreting more perforin and granzyme B in spleen of TRIM32 knockout mice.In addition,TRIM32 deficiency significantly increased the production of IFN-? and TNF? of NK cells as well as the secretion of IL-12 and i NOS of macrophages in spleen.Finally,we found that the survival rate of L.monocytogenes in macrophages of TRIM32 knockout mice was significantly decreased than that of wild-type mice.The above results indicated that TRIM32 played important roles to decrease the defense ability of innate immune cells against L.monocytogenes infection via multiple mechanisms.In summary,our study demonstrated that TRIM32 positively regulated the innate immune signaling pathways during the course of Gram-positive bacterial infection and decreased the bactericidal ability via multiple mechanisms.Based on the findings that TRIM32 plays important roles of host defense against bacterial infection,TRIM32 might be a potential target to treat bacterial infection.