The Role Of P40^phox Subunit Of NADPH Oxidase In Host Defense Against Bacterial Infection

NADPH oxidase is a membrane-bound enzyme complex that generates reactive oxygen species (ROS), critical for pathogen killing upon phagocytosis and for regulating pro-inflammatory signaling in phagocytic cells. Deficiency of NADPH oxidase may lead to enhanced susceptibility to infection and inflammatory disease. Salmonella enterica serovar Typhimurium(S. Typhimurium) is a facultative intracellular pathogen, capable of causing disease in a variety of hosts. This pathogen can penetrate the intestinal epithelium and spread into the circulatory system, inducing enteric and systemic disease. Unlike the entero-invasive Salmonella, Citrobacter rodentium (C. rodentium) is a classically noninvasive pathogen, which belongs to the attaching and effacing family of enteric bacteria. In this study, genetically engineered p40-/- mice were used to determine the role of p40phox, one subunit of NADPH oxidase, in host response to S. Typhimurium and C. rodentium infection.1 The Role of p40phox in Host Defense against Systemic Salmonella InfectionTo test whether mice lacking p40phox are more susceptible to S. Typhimurium infection, p40phox-/- mice and congenic C57BL/6 wild-type mice were infected orally with 3 x 108 CFU of S. Typhimurium. Results showed that p40phox deficiency significantly increased mortality and bodyweight loss of mice during oral Salmonella infection. Compared with C57BL/6 controls, significantly greater bacterial numbers were present in spleens and livers of p40phox-/- mice. This enhanced bacterial dissemination was accompanied by exacerbated inflammation and tissue damage in p40phox-/- mice.In contrast to wild-type mice, the gene expression of inflammatory cytokine interleukin (IL)-1β and tumor necrosis factor (TNF)-a in infected p40phox-/-mice was significantly enhanced. ELISA analysis further confirmed that Salmonella-infection induced a marked increase in TNF-a production in mice lacking p40phox. Consistent with more severe inflammatory changes, it was observed that p40phox deficiency led to markedly increased recruitment of CDllb+F4/80+ macrophages and Ly6G+CD11b+ neutrophils in the infected tissues. Furthermore, the increased cell infiltration present in p40phox deficient mice was correlated with the up-regulated gene expression of keratinocyte chemoattractant (KC), monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2. CD4+T cell recruitment was also increased in p40phox-/- mice after oral infection. To further study the impact of p40phox deficiency on phagocytes recruitment, mice were injected intra-peritoneally (i.p.) with 5,000 CFU of S. Typhimurium. It was found that p40phox-/- mice exhibited normal ability to recruit phagocytes to the peritoneal cavity after infection with Salmonella. In accordance with the results observed from oral administration study, p40phox deficiency also exacerbated tissue inflammation and bacterial translocation in Salmonella-infected mice. These results suggest that p40phox is necessary for the innate immune defense against systemic Salmonellosis after infection.2 The Role of p40phox in Regulating Intestinal Inflammation Caused by Salmonella InfectionTo decipher whether and how p40phox deficiency affects mucosal inflammatory responses, a streptomycin-pretreated mouse model was utilized to analyze Salmonella-induced intestinal inflammation. Histological examination showed more extensive pathological changes in p40phox-/- mice, including pronounced submucosal edema, dramatic disruption of epithelial architecture, and marked inflammatory cell infiltrates. Compared with wild-type mice, p40phox-/- mice developed severe barrier disruption, as evidenced by the clear alteration in E-cadherin and zonula occludens (ZO)-1 distribution and significantly higher pathogen burdens. Moreover, p40phox deficiency in S. Typhimurium-infected mice could regulate the expression of mucins (MUC) and enzymes involved in glycan modifications, as demonstrated by lower expression levels of MUC2 and sialyltransferase St3gal4 in p40phox-/- mice. The induction of fucosyltransferase FUT9 expression was also impaired in p4hphox-/- mice after Salmonella infection. Further, a significantly higher frequency of both F4/80+ macrophages and Ly6G+ neutrophils was noted in the cecal lamina propria of p40phox knockout mice with Salmonella infection compared to wild-type mice, consistent with higher levels of chemokine KC in infected p40phox-/- mice. Interestingly, it was observed that the macrophages and neutrophils appeared to be distributed differently between two mouse strains. In wild-type mice, the macrophages and neutrophils were dispersed, while in the p40phox-/- tissues, they were lined up at the base of the epithelium. These findings indicate that p40phox is required for host defense during Salmonella-induced colitis.3 The Role of p40phox in Regulating Antimicrobial Activity of MacrophagesTo evaluate the bacterial killing capacity of p40phox-/- macrophages, peritoneal macrophages were isolated and the microbicidal function was measured using gentamicin protection assay. Results showed that p40phox-/- macrophages exhibited normal bacterial phagocytosis and reduced bactericidal activity. Using a lucigenin-dependent chemiluminescence assay, it was demonstrated that the production of ROS was markedly reduced in p40phox-/- macrophages and p40phox-/-neutrophils, which may contribute to the observed impaired bacterial killing activity. Functional analysis of macrophages further showed that p40phox deficiency impaired autophagy-mediated intracellular killing of Salmonella, as supported by decreased expression and processing of microtubule-associated protein light chain (LC)-3, low expression levels of autophagy-related protein (Atg)-3 and Atg12-Atg5 conjugate, and infrequent of co-localization of LC3 and Salmonella in p40phox-/- macrophages. Yet these macrophages produced significantly more IL-1β and had higher level of caspase-1 activity in response to bacterial stimulation. These observations imply that p40phox plays an essential role in suppressing intracellular multiplication of Salmonella in macrophages, and in the regulation of autophagy and inflammasome activity during bacterial infection.4 The Role of p40phox in Host Defense against Citrobacter rodentium InfectionTo investigate whether defects in p40phox impair host immune response in C. rodentium-induced colitis, p40phox-/- mice and wild-type mice were infected orally with 5×108 CFU of C. rodentium. Results showed that p40phox deficiency did not increase susceptibility of mice to C. rodentium infection, since no differences were found in body weight loss, bacterial clearance or colonic pathology between p40phox-/- and wild-type mice. However, it was observed that p40phox-/- mice exhibited an elevated level of TNF-a and IL-1β at baseline, further confirmed by the high frequency of macrophages present in uninfected p40phox-/- mice. Moreover, impaired Thl/Thl7 responses and higher IL-10 levels were observed in the supernatants of MLN cells and splenocytes isolated from infected p40phox-deficient mice. Further, higher expression level of inducible nitric oxide synthase (iNOS) was also noted in mice lacking p40phox. In contrast to wild-type mice, p40phox-knockout mice exhibited greater nitric oxide (NO) production after lipopolysaccharide (LPS) or bacterial antigen re-stimulation. These results suggest that p40phox-/- mice do not develop worsened colitis, and the underlying mechanisms may involve an impaired Thl/Th17 response and enhanced IL-10 and iNOS levels.In summary, these observations indicate that the p40phox subunit of NADPH oxidase is critical for host resistance and survival during Salmonella infection and Salmonella-mediated intestinal inflammation, but not for C. rodentium infection.