p40 phox -Deficient Mice Exhibit Impaired Bacterial Clearance and Enhanced Pro-inflammatory Responses during Salmonella enterica serovar Typhimurium Infection

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of acute gastroenteritis in humans. During infection, reactive oxygen species (ROS), generated from NADPH oxidase (a multisubunit enzyme complex), are required for pathogen killing upon phagocytosis and for regulating pro-inflammatory signaling in phagocytic cells. Mutations in subunits forming the NADPH complex may lead to enhanced susceptibility to infection and inflammatory disease. Compared to other NADPH oxidase subunits, the function of p40 ^phox is relatively understudied, particularly in the context of intestinal bacterial infection. In this study, we utilized genetically engineered mice to determine the role of p40 ^phox in the response to S. Typhimurium infection. We show that mice lacking p40 ^phox are more susceptible to oral infection with S. Typhimurium, as demonstrated by significantly enhanced bacterial dissemination to spleen and liver, and development of exacerbated bacterial colitis. Moreover, we demonstrate that the increased infection and disease severity are correlated with markedly increased F4/80⁺ macrophage and Ly6G⁺ neutrophil infiltration in the infected tissues, coincident with significantly elevated pro-inflammatory cytokines (IL-1β and TNF-α) and chemoattractant molecules in the infected tissues. Functional analysis of macrophages and neutrophils further shows that p40 ^phox deficiency impairs bacteria- or PMA-induced intracellular ROS production as well as intracellular killing of Salmonella. These observations indicate that the p40 ^phox subunit of NADPH oxidase plays an essential role in suppressing intracellular multiplication of Salmonella in macrophages and in the regulation of both systemic and mucosal inflammatory responses to bacterial infection.

Keywords: NADPH oxidase; Salmonella infection; bacterial killing; inflammation; mucosa.

Figure 1

p40^phox deficiency exacerbates bacterial translocation and mortality in Salmonella-infected mice. Wild-type (WT) mice and mice lacking p40^phox were infected orally with 3 × 10⁸ CFU of Salmonella enterica serovar Typhimurium (SL1344 strain). (A) Mice were monitored daily for survival. (B) Body weight changes were measured 72 h post infection. Significance was determined using a paired t-test for body weight (n = 5/group; *p < 0.05; **p < 0.01; ***p < 0.001). Bacterial translocation to spleens (C) and livers (D) was determined 72 h after infection using a Student’s t-test. The number of CFUs per gram of tissue is shown (n = 9–12/group; ***p < 0.001). (E) Immunofluorescence microscopy data show the distribution of Salmonella (in Green) in spleen sections (magnification 40×). Neutrophils were identified by positive staining with anti-Ly6G. Data shown are generated from three independent experiments.

Figure 2

p40^phox−/− mice develop more severe spleen and liver inflammation after Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Spleens and livers were harvested 72 h after S. Typhimurium infection. Representative spleen (A) and liver (B) sections stained by hematoxylin and eosin [(A) magnification 20×; (B) magnification 40×]. Arrows indicate inflammatory infiltrates. (C–E) The gene expression of IL-1β and TNF-α was assessed via quantitative PCR. Values represent the fold increase compared to baseline obtained from uninfected wild-type (WT) mice (n = 5/group; *p < 0.05). Significance was determined by one-way ANOVA with Tukey’s post hoc test. (F–I) Cytokine levels of IFN-γ, IL-17A, TNF-α, and IL-1β were measured by ELISA. The data shown are mean ± SEM from one of three experiments performed showing similar results (n = 5/group; *p < 0.05). Significance was determined by a Student’s t-test.

Figure 3

p40^phox deficiency in Salmonella enterica serovar Typhimurium (S. Typhimurium)-infected mice enhances the recruitment of macrophages. Mice were infected with S. Typhimurium (3 × 10⁸ CFU) and sacrificed 24 h post infection. Single-cell suspensions from spleens were prepared and stained for macrophages with anti-F4/80 and anti-CD11b (A) or neutrophils with anti-Ly6G and anti-CD11b antibodies (B). The percentages of F4/80⁺CD11b⁺ macrophages (C) and Ly6G⁺CD11b⁺neutrophils (D) are shown as mean ± SEM (n = 5/group; NS = Not Significant; *p < 0.05; **p < 0.01). FACS plots are representative of three independent experiments. Spleen from uninfected B6 and p40^phox KO mice (left) and infected B6 and p40^phox KO mice (right) were stained with anti-F4/80 for macrophages and anti-Ly6G (Red) for neutrophils (magnification × 100) (E). (F–H) Analysis of splenic chemoattractants (KC, MCP1, and MIP2) gene expression was performed by quantitative PCR (n = 5/group; *p < 0.05). Significance was determined by one-way ANOVA with Tukey’s post hoc test.

Figure 4

p40^phox−/− mice show more severe Salmonella-induced mucosal injury and enterocolitis. Mice lacking p40^phox and wild-type (WT) mice were treated with streptomycin prior to infection with Salmonella enterica serovar Typhimurium (10⁸ CFU) and sacrificed 24 h post infection. (A) Representative hematoxylin and eosin-stained cecum sections from each group of mice (original magnification 40×). (B) Histopathological score of cecal inflammation in mice with or without infection of Salmonella. The scores were assessed by determination of infiltration of inflammatory cells (score range, 0–4), together with the evaluation of cecal tissue damage (score range, 0–4). (C,D) Representative immunofluorescence-stained cecum sections from each group of mice showing increased intestinal bacterial loads in the cecum of p40^phox deficient mice compared to WT mice. Numbers of bacteria recovered from liver (E) and spleen (F) homogenates of Salmonella-infected mice were determined using a Student’s t-test. The number of CFUs per gram of tissue is shown (n = 5–6/group; *p < 0.05). Cecal levels of IL-1β (G) and TNF-α (H) were determined by quantitative PCR (n = 5/group; *p < 0.05; **p < 0.01). Data are representative of two independent experiments.

Figure 5

p40^phox deficiency enhances the recruitment of macrophages and neutrophils in cecal lamina propria. Mice deficient in p40^phox and normal mice were treated with streptomycin prior to Salmonella enterica serovar Typhimurium infection and sacrificed 24 h post infection. Ceca from uninfected B6 (A) and p40^phox KO (B) mice and infected B6 (C) and p40^phox KO (D) mice were stained with anti-F4/80 for macrophages and anti-Ly6G (Red) for neutrophils (magnification 100×). The mean number of F4/80⁺ cells (E) and Ly6G⁺ cells (F) detected in each high power field was determined by counting four fields from each sample (samples from three mice per group were counted) (*p < 0.05; **p < 0.01). Cecal levels of KC (G) and MCP1 (H) were determined by quantitative PCR (n = 5/group; *p < 0.05). Data are representative of two independent experiments.

Figure 6

p40^phox−/− macrophages exhibit normal bacterial phagocytosis and reduced bactericidal activity. Peritoneal macrophages were isolated from wild-type (WT) and p40^phox−/−mice. (A) Peritoneal macrophages were exposed to Salmonella enterica serovar Typhimurium (10 bacteria/cell) for 1 h. At different time points after infection (2, 6 h), the number of intracellular bacteria was determined by plating cell lysates onto LB plates supplemented with streptomycin. Results are expressed as mean ± SEM (n = 5/group; NS = Not Significant; *p < 0.05). The data shown are representative of three experiments with similar results. (B) Immunofluorescence microscopy data show the number of internalized Salmonella in peritoneal macrophages (stained with Cy3 anti-F4/80) at 2 and 6 h after gentamicin treatment. Salmonella bacteria were detected with a rabbit antibody against Salmonella, followed by fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG antibody. (C) Real-time PCR analysis reveals that Salmonella LPS treatment (100 ng/ml) significantly upregulated the gene expression of IL-1β and TNF-α in both WT and p40^phox−/− cells (n = 3–5/group; *p < 0.05; **p < 0.01; ***p < 0.001). The results are displayed as mean ± SEM and are representative of three independent experiments.

Figure 7

p40^phox−/− macrophages have impaired reactive oxygen species (ROS) production. Intestinal and peritoneal macrophages were isolated from wild-type and p40^phox−/−mice. (A,B) Time course of lucigenin-elicited chemiluminescence in peritoneal macrophages after addition of 200 nM PMA or the same volume of HBSS. ROS release was monitored every 5 min for a period of 1 h at 37°C. (C–F) Bacterial and PMA-induced intestinal (C,D) and peritoneal macrophage (E,F) intracellular ROS production was measured using the 2′,7′-dichlorofluorescin diacetate (DCFDA) Cellular ROS Detection Assay Kit. Results are expressed as fluorescent intensity of samples minus blank (DCFDA-unstained cells) (n = 3–5 mice/group; *p < 0.05). Each experiment was performed in triplicate.