Both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary Streptococcus pneumoniae infection

Abstract

Since secondary Streptococcus pneumoniae infections greatly increase the mortality of influenza infections, we determined the relative roles of neutrophil-dependent and -independent mechanisms in increased susceptibility to S. pneumoniae during influenza infection. Mice infected with influenza for 6 days, but not 3 days, showed a significant increase in susceptibility to S. pneumoniae infection compared to mice not infected with influenza. There was significant neutrophil accumulation in the lungs of S. pneumoniae-infected mice regardless of whether or not they were infected with influenza for 3 or 6 days. Depletion of neutrophils in these mice resulted in increased susceptibility to S. pneumoniae in both the non-influenza-infected mice and mice infected with influenza for 3 days but not in the mice infected with influenza for 6 days, indicating that a prior influenza infection of 6 days may compromise neutrophil function, resulting in increased susceptibility to a S. pneumoniae infection. Neutrophils from the lungs of mice infected with influenza for 3 or 6 days exhibited functional impairment in the form of decreased phagocytosis and intracellular reactive oxygen species generation in response to S. pneumoniae. In addition, neutrophil-depleted mice infected with influenza for 6 days were more susceptible to S. pneumoniae than neutrophil-depleted mice not infected with influenza, indicating that neutrophil-independent mechanisms also contribute to influenza-induced increased susceptibility to S. pneumoniae. Pulmonary interleukin-10 levels were increased in coinfected mice infected with influenza for 6 days but not 3 days. Thus, an influenza infection of 6 days increases susceptibility to S. pneumoniae by both suppression of neutrophil function and by neutrophil-independent mechanisms such as enhanced cytokine production.

FIG. 1.

S. pneumoniae CFU (A) and influenza PFU (B) in lungs after 24 h of S. pneumoniae infection for mice infected with S. pneumoniae only or infected with S. pneumoniae after 3 or 6 days of influenza infection. Data were pooled from three independent experiments with 4 to 5 mice per group per experiment. Significant differences between mice infected with S. pneumoniae only and mice coinfected with influenza and S. pneumoniae are indicated as follows: ***, P < 0.001; NS, P > 0.05. Horizontal lines represent median values for each infection. Data were analyzed using Mann-Whitney U test.

FIG. 2.

Percentage of neutrophils in blood of total blood leukocytes (A), total number of neutrophils in BALF (B), and lung S. pneumoniae CFU (C) of nondepleted and RB6-depleted mice at 24 h after S. pneumoniae infection. Mice were infected with S. pneumoniae for 24 h after 3 or 6 days of influenza infection or were only infected with S. pneumoniae for 24 h. Data represent results from two independent experiments with 4 to 5 mice per group per experiment. Significant differences between neutrophil-depleted mice and nondepleted mice are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, P > 0.05. Significant differences between neutrophil-depleted mice infected with S. pneumoniae only and neutrophil-depleted mice coinfected with influenza for 6 days followed by S. pneumoniae as well as differences between neutrophil-depleted mice coinfected with influenza for 3 days followed by S. pneumoniae and neutrophil-depleted mice coinfected with influenza for 6 days followed by S. pneumoniae are indicated as follows: #, P < 0.05; ##, P < 0.01. Horizontal lines represent median values for each infection. Data were analyzed using Mann-Whitney U test.

FIG. 3.

Neutrophil counts for BALF (A and B) and bone marrow (C and D) samples from noninfected mice, LPS-stimulated mice, or mice infected with influenza for 3 or 6 days with (B and D) or without (A and C) LPS stimulation. Data represent results from two independent experiments with 3 sets of 2 mice per group. Significant differences between LPS-stimulated mice and mice with both influenza infection and LPS stimulation are indicated as follows: , P < 0.001. Data are expressed as means ± standard deviations. Data were analyzed using unpaired t test.

FIG. 4.

S. pneumoniae association after 0 or 60 min incubation with lung (A) or bone marrow (B) neutrophils from noninfected mice, LPS-stimulated mice, or mice infected with influenza for 3 or 6 days with (A) or without (B) subsequent LPS stimulation. Data represent results from one (A) or two (B) independent experiments with 3 sets of 2 mice per group. Significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to LPS-stimulated mice; +, P < 0.05 compared to noninfected (none) mice; ##, P < 0.01 compared to mice infected with influenza for 3 days. Data are expressed as means ± standard deviations. Data were analyzed using unpaired t test.

FIG. 5.

ROS generation by lung (A and B) and bone marrow (C and D) neutrophils from noninfected mice, LPS-stimulated mice, or mice infected with influenza for 3 or 6 days with (A and B) or without (C and D) subsequent LPS stimulation. Neutrophils were incubated with S. pneumoniae (A and C) or PMA (B and D) for 0 or 60 min. Data represent results from one (A and B) or two (C and D) independent experiments with 3 sets of 2 mice per group. Significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to LPS-stimulated mice; ##, P < 0.01 compared to mice infected with influenza for 3 days. Data are expressed as means ± standard deviations. Data were analyzed using unpaired t test.

FIG. 6.

Lung cytokine levels in neutrophil-depleted and nondepleted mice with or without S. pneumoniae for 24 h and/or influenza virus for 3 or 6 days. TNF (A), IFN-γ (B), MCP-1, IL-10, and IL-6 levels were measured in nondepleted and neutrophil-depleted mice which were not infected, infected with S. pneumoniae only for 24 h, infected with influenza only for 3 or 6 days, or infected with influenza for 3 or 6 days followed by S. pneumoniae for 24 h. Significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 for mice infected with influenza virus for 6 days followed by S. pneumoniae compared to noninfected mice, mice infected with S. pneumoniae only, mice infected with influenza virus only, and mice infected with influenza virus for 3 days followed by S. pneumoniae with the same depletion status; ¶, P < 0.05 for mice with the same influenza infection and depletion status to determine the effects of a S. pneumoniae infection; #, P < 0.05 for mice with the same S. pneumoniae infection and depletion status to determine the effects of an influenza infection; †, P < 0.05; ††, P < 0.01; †††, P < 0.001 for mice with the same influenza and S. pneumoniae infections to determine the effects of neutrophil depletion; §, P < 0.05 for mice with the same S. pneumoniae infection and depletion status to determine the effects of 3 days versus 6 days of influenza infection; ¤, P < 0.05; ¤¤, P < 0.01; ¤¤¤, P < 0.001 for mice with the same depletion status to determine the effects of a coinfection or S. pneumoniae infection versus no infection. Data are expressed as means ± standard deviations. Data were analyzed using one-way analysis of variance, followed by the Bonferroni post test.