Influenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances susceptibility to secondary methicillin-resistant Staphylococcus aureus infection

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. In this study, we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, even though MRSA superinfection had no significant effect on viral burdens. Compared with mice infected with bacteria alone, postinfluenza MRSA-infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for reactive oxygen species production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91(phox)-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to wild-type animals, gp91(phox-/-) mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91(phox+/-) mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Taken together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection.

Figure 1. Influenza enhances susceptibility to MRSA infection during the recovery stage of viral infection

(A) Bacterial burdens, (B) influenza-induced bacterial outgrowth, and (C) viral burdens in the lungs at various days after 3×10³ PFU H1N1 CA04 influenza virus infection of B6 WT mice and 24 h after infection with 10⁷ CFU/mouse of MRSA BAA-1692 (5–7 mice/group). In (B), the relative increases of bacterial burdens in influenza-infected mice 24 h after MRSA infection are represented as fold increases relative to mean lung bacterial CFUs in mice infected with MRSA alone (A) included at each time point. In (A&C) P<0.001, ANOVA, in (B), P<0.01, ANOVA; ***P< 0.001, Tukey’s multiple comparisons test.

Figure 2. Influenza infection impairs innate immunity against respiratory MRSA challenge

(A) Bacterial and (B) viral burdens in the lungs 24 h after inoculation of naïve (MRSA) or day 7 postinfluenza infected (Flu+MRSA) C57BL/6 mice with 10⁷ CFU MRSA BAA-1695 (Flu+MRSA) or PBS (Flu). ***P< 0.001, compared to mice not infected with influenza virus, unpaired t test. (C) Survival of C57BL/6 mice after infection with 2×10⁸ CFU of MRSA BAA-1695 alone (MRSA) or on day 7 after PR8 infection (Flu + MRSA). Also shown, survival of mice infected with 50 PFU PR8 and inoculated with PBS control, instead of MRSA, 7 days later (Flu).

Figure 3. Influenza infection regulates the antibacterial ability of airway phagocytes

(A) Airway phagocyte profiles (3 mice/group), and (B) intracellular ROS levels (3 mice/group) at 24 h after infection with 10⁷ CFU of MRSA BAA-1695 alone (MRSA) or on day 7 after PR8 infection (Flu + MRSA). In (A&B), P<0.001, ANOVA; *P< 0.05, ***P< 0.001, Tukey’s multiple comparisons test. Control mice were inoculated with only PBS (PBS) or infected with 50 PFU PR8 and inoculated with PBS (Flu) 7 days after PR8 infection. The data are representative of two independent experiments.

Figure 4. Macrophage oxidative burst is essential for lung S. aureus clearance

Numbers of bacteria remaining in the lungs 24 h after MRSA BAA-1695 infection of C57BL/6 (A) WT, NOS2^−/−, gp91^phox−/−, and p47^phox−/− mice, (B) α-PMN antibody-treated WT and gp91^phox−/− mice, and (C) Mafia mice. In (B), mice were treated with RB6-8C5 mAb for neutrophil depletion. In (C), Mafia mice were depleted of macrophages using AP20187 dissolved in ethanol. Control WT mice were also treated with AP20187. (D) Survival of α-PMN antibody-treated WT mice after i.n. infection with 10⁸ CFU of MRSA. Control WT mice were treated with Rat IgG. (E) Survival of α-PMN antibody-treated WT and gp91^phox−/− mice after i.n. infection with 10⁷ CFU of MRSA. In (A, B&C), P<0.001, ANOVA; ***P< 0.001, Tukey’s multiple comparisons test.

Figure 5. Influenza infection inhibits NADPH oxidase-dependent bacterial killing

(A) Numbers of bacteria (4–8 mice/group) in the lungs, and (B) airway inflammatory cell accumulation at 24 h after infection WT and gp91^phox−/− mice (n=4) with 10⁷ CFU of MRSA BAA-1695 alone (MRSA) or on day 7 after PR8 infection (Flu + MRSA). In (A&B), P<0.001, ANOVA; *P< 0.05, ***P< 0.001, Tukey’s multiple comparisons test.

Figure 6. Influenza infection inhibits NADPH oxidase-dependent immune defense against S. aureus

(A) Survival of WT and gp91^phox−/− mice after infection with 10³ PFU of PR8 virus alone. (B) Survival of WT and gp91^phox−/− mice after infection with 1×10⁸ CFU of MRSA BAA-1695 alone. (C&D) Survival of WT and gp91^phox−/− mice after infection with (C) 2×10⁸ CFU and (D) 1×10⁸ CFU of MRSA BAA-1695 on day 7 after PR8 infection.

Figure 7. Influenza infection inhibits cell-intrinsic contribution of NADPH oxidase to phagocytic bacterial killing

(A) Flow cytometry analysis of gp91^phox expression in blood neutrophils (CD11b⁺Ly6G⁺) from female gp91^phox WT, knockout (KO) and mosaic mice (n=5). (B) Composition of gp91^phox WT and deficient neutrophils in blood and airways of gp91^phox+/− mosaic mice (n=5) at day 7 after influenza infection. (C) Numbers of bacteria (5 mice/group) in the lungs 24 h after challenge of naïve (MRSA) or day 7 postinfluenza infected (Flu+MRSA) female WT and gp91^phox+/− mosaic mice with 10⁷ CFU MRSA BAA-1695. In (C), P<0.01, ANOVA; **P< 0.01, ***P< 0.001, Tukey’s multiple comparisons test.