Pulmonary immunostimulation with MALP-2 in influenza virus-infected mice increases survival after pneumococcal superinfection

Abstract

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.

FIG 1

MALP-2 increased leukocyte recruitment and cytokine release in IAV-infected lungs. Mice were treated intratracheally with 0.5 μg MALP-2 or solvent 5 days after infection with 10² PFU IAV PR8 or sham infection with PBS. At 6 days postinfection, BAL was performed, and BALF leukocytes (A) and cytokines (B) were quantified. Dotted lines indicate the lower limit of the cytokine assay working range and are missing if all values were within the working range. b.t., values below the threshold. Values are given as means plus SEM (n = 6 to 8 [A] or 5 to 8 [B] for each group). *, P < 0.05; **, P < 0.01 (for the indicated comparisons). #, P < 0.05; ##, P < 0.01; ###, P < 0.001 (versus the corresponding sham-infected group).

FIG 2

Pulmonary MALP-2 stimulation did not affect the systemic leukocyte response and clinical parameters in IAV-infected mice. Mice were intratracheally treated with 0.5 μg MALP-2 or solvent 5 days after infection with 10² PFU IAV or sham infection with PBS. (A) At 6 days postinfection, blood leukocytes were analyzed by flow cytometry. Values are given as means plus SEM (n = 5 to 7 for each group). ##, P < 0.01 versus the corresponding sham-infected group. (B) Survival and body weight loss were monitored for 10 days after IAV infection. Body weight values are given as means plus SEM (n = 10 for MALP-2 group and 9 for solvent group).

FIG 3

Pulmonary MALP-2 treatment of IAV-infected mice improved survival after pneumococcal superinfection. Mice infected with 10² PFU IAV were treated with 0.5 μg MALP-2 or solvent on day 5. Secondary infection with 10³ CFU S. pneumoniae (S.pn.) was performed on day 6. (A) Survival was monitored every 12 h for 10 days after IAV infection. (B) Body weight loss within the first 24 h of secondary pneumococcal infection and body temperature were measured on day 7. Values are given as means plus SEM (n = 10 for MALP-2 group and 9 for solvent group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus the corresponding solvent-treated group).

FIG 4

MALP-2 treatment of IAV-infected lungs did not increase pulmonary inflammation after subsequent secondary pneumococcal infection. Mice infected with 10² PFU IAV or sham infected with PBS were treated with 0.5 μg MALP-2 or solvent on day 5. Secondary infection with 10³ CFU S. pneumoniae or sham infection with PBS was performed on day 6. Seven days after IAV infection, BAL was performed, and BALF leukocytes (A) and cytokines (B) were quantified. Values are given as means plus SEM (n = 5 to 8 [A] or 3 to 8 [B] for each group). Dotted lines indicate the lower limit of the cytokine assay working range and are missing if all values were within the working range. b.t., values below the threshold. *, P < 0.05; **, P < 0.01 (for the indicated comparisons).

FIG 5

MALP-2 altered neutrophil accumulation in IAV-induced bronchointerstitial pneumonia without aggravating suppurative bronchopneumonia after secondary pneumococcal infection. Mice infected with 10² PFU IAV were treated with 0.5 μg MALP-2 or solvent on day 5 and challenged with 10³ CFU S. pneumoniae or sham infected with PBS on day 6. Lungs were harvested on day 7, and formalin-fixed and paraffin-embedded sections were prepared and stained with hematoxylin and eosin for histopathological analyses. (A to D) IAV infection induced bronchointerstitial pneumonia, accompanied by numerous neutrophils within bronchi and alveolar spaces after MALP-2 stimulation (D; black arrowheads). (E and F) Secondary pneumococcal infection resulted in suppurative bronchopneumonia in lungs of both solvent-treated and MALP-2-treated IAV-infected mice. (G) Lung sections from sham-infected and solvent-treated mice served as a negative control. Representative images are shown (n = 3 or 4 for each group). (H) Total lung areas affected by inflammation. Values are given as means plus SEM (n = 3 or 4 for each group).

FIG 6

The pulmonary IAV load remained unaltered by MALP-2 stimulation. Mice infected with 10² PFU IAV were treated with 0.5 μg MALP-2 or solvent on day 5 and challenged with 10³ CFU S. pneumoniae or PBS on day 6. Seven days after IAV infection, lung sections were prepared, and immunohistochemistry for IAV (red staining) was performed. IAV was observed mainly within bronchi and in alveolar macrophages (black arrowheads) of lungs from both solvent (A and C)- and MALP-2 (B and D)-treated mice. (E) Lung sections from sham-infected and solvent-treated mice served as a negative control. Representative images are shown (n = 3 or 4). (F) Lung viral loads after secondary bacterial infection were determined on day 7. Values are given as individual data and means (n = 11). The dotted line indicates the lower detection limit.

FIG 7

MALP-2 reduced the pulmonary bacterial load after secondary pneumococcal infection. Mice infected with 10² PFU IAV were treated with 0.5 μg MALP-2 or solvent on day 5 and challenged with 10³ CFU S. pneumoniae on day 6. (A to E) Bacteria were detected by immunohistochemistry with an anti-S. pneumoniae antibody (red staining) on day 7. Pneumococci were observed within bronchi and alveolar spaces (black arrowheads) in 50% of lungs from solvent-treated infected mice (A and C), but not in lung tissue from MALP-2-stimulated infected mice (B and D). (E) Lung sections from sham-infected and solvent-treated mice served as a negative control. Representative images are shown (n = 4). (F) Lung bacterial loads after secondary pneumococcal infection were determined on day 7. Values are given as individual data and means (n = 10). The dotted line indicates the lower detection limit. *, P < 0.05 versus solvent-treated group.