NOX2 protects against progressive lung injury and multiple organ dysfunction syndrome

Abstract

Systemic inflammatory response syndrome (SIRS) is a common clinical condition in patients in intensive care units that can lead to complications, including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate, and it is unclear why some patients resolve SIRS, whereas others develop MODS. Although oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91(phox-/y) (NOX2-deficient) mice compared with wild-type mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histological examination showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation.

Keywords: MODS; SIRS; chronic granulomatous disease; gp91phox; inflammation.

Fig. 1.

NADPH oxidase 2 (NOX2)-deficient mice exhibit significant mortality following low-dose zymosan injection. Kaplan-Meier curve displaying survival of wild-type (WT) and NOX2-deficient mice following intraperitoneal (ip) injection of 0.4 mg/g zymosan (n ≥ 29 per genotype from a total of 6 separate experiments; ***P < 0.001).

Fig. 2.

NOX2-deficient mice display ongoing weight loss, hypothermia, hypotension, and symptoms of inflammation following zymosan challenge. Weight (A), core body temperature (B), mean arterial pressure (C), and inflammatory symptoms (D) were recorded at several time points following low-dose ip zymosan injection. Mean ± SE (n ≥ 9 mice per genotype per time point). Data were analyzed by 2-way ANOVA with Bonferroni posttests; ***P < 0.001.

Fig. 3.

Enhanced neutrophil recruitment to the peritoneum of NOX2-deficient mice. Peritoneal cells were recovered by lavage, counted, and neutrophils (Ly6G+) and macrophages (F4/80+) were differentiated by flow cytometry. A: number of cells recovered from the peritoneum, and percentage of neutrophils and macrophages at 14 (B) and 21 days (C) postinjection. Mean + SE (n ≥ 8 mice per genotype per time point). **P < 0.01; ***P < 0.001.

Fig. 4.

NOX2-deficient neutrophils and macrophages have altered activation states. The geometric mean index (GMI) of receptor for advanced glycation end products (RAGE) (A, B), CXCR2 (C, D) and macrophage mannose receptor (MMR) (E, F) was measured by flow cytometry for gated neutrophil (Ly6G+) and macrophage (F4/80+) populations isolated from the peritoneum. Mean + SE (n ≥ 8 mice per genotype per time point). *P < 0.05; **P < 0.01.

Fig. 5.

Increased polymorphonuclear leukocyte (PMN) generation by NOX2-deficient bone marrow. The percentage of neutrophils (Ly6G+) in the bone marrow determined by flow cytometry at 14 (A) and 21 (B) days postinjection. Mean + SE (n ≥ 8 mice per genotype per time point). **P < 0.01; ***P < 0.001.

Fig. 6.

Histological images of pyogranulomatous inflammation in NOX2-deficient mice encapsulating the spleen (A) and adhered to the liver and lining of the stomach (B). Both images are from NOX2-deficient mice killed 21 days postinjection.

Fig. 7.

NOX2-deficient mice have ongoing recruitment of inflammatory neutrophils to the lungs. A: number of cells isolated from the bronchoalveolar lavage fluid (BALf) of WT and NOX2-deficient mice. Percentages of neutrophils and macrophages in BALf at 14 (B) and 21 days (C) postinjection were determined by flow cytometry. The GMI of CD11b (D, E) and RAGE (F, G) surface expression on neutrophils and macrophages from the BALf. Mean + SE (n ≥ 7 mice per genotype per time point). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 8.

NOX2-deficient mice have significant multiple organ pathology. Histological sections of lungs (A), spleens (B), livers (C), and kidneys (D) were scored for pathology. Mean + SE (n ≥ 8 per genotype at 14 days and n ≥ 5 per genotype at 21 days). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 9.

NOX2-deficient mice have more inflammatory lesions in organs by histology. A: lungs from WT and NOX2^−/y mice had multifocal inflammation (arrows, top). In NOX2^−/y mice, inflammation was more extensive with more neutrophils and macrophages often associated with eosinophilic crystals (arrowheads, bottom right). Thrombi (arrow, bottom right) were detected in inflamed lung with vascular congestion/hemorrhage. B: spleens from WT and NOX2^−/y mice. Splenic red pulp from NOX2^−/y mice had increased cellularity (asterisks, top right). This increased cellularity was composed primarily of granulocytic to myeloid predominant extramedullary hematopoiesis. Note the granulocytic lineage cells with donut-shaped nuclei (arrows, bottom right and inset). C: livers from WT and NOX2^−/y mice had multifocal inflammation (arrows, top) but NOX2^−/y livers had granulocytic/myeloid predominant extramedullary hematopoiesis (arrowheads, top right) detected frequently. Thrombi (arrow, bottom right) were more frequent in NOX2^−/y livers and were associated with nearby hepatocyte cell death (arrowhead, bottom right). D: kidneys from WT and NOX2^−/y mice. Kidneys from NOX2^−/y mice had multifocal tubular inflammation and remodeling changes (arrows, top right). Ectatic tubules (arrows, bottom right) were filled with neutrophilic cellular debris and affected tubules had basophilic cuboidal epithelium with mitotic figure indicative of tubular hyperplasia. Inset: note the neutrophils in tubular lumen and in peritubular interstitium (arrows, inset box).

Fig. 10.

Inflammatory chemokines and cytokines are elevated in the lungs of NOX2-deficient mice. MIP-1α (A), MIP-1β (B), KC (C), IL-1α (D), G-CSF (E), and MCP-1 (F) levels in murine BALf obtained at 14 and 21 days after zymosan injection. Mean + SE (n ≥ 8 mice per genotype per time point). **P < 0.01; ***P < 0.001.

Fig. 11.

NOX2-deficient and WT mice have differential circulating cytokine profiles. G-CSF (A), IL-6 (B), IL-17 (C), IL-12p40 (D), KC (E), and IL-1α (F) plasma levels in mice at 14 and 21 days after zymosan injection. Mean + SE (n = 6 mice per genotype per time point). *P < 0.05; **P < 0.01; ***P < 0.001.