Depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections

Abstract

Viruses such as influenza suppress host immune function by a variety of methods. This may result in significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. PKH26-phagocytic cell labeling dye was administered intranasally to label resident alveolar macrophages (AMs) in a well-established murine model before influenza infection to determine turnover kinetics during the course of infection. More than 90% of resident AMs were lost in the first week after influenza, whereas the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with S. pneumoniae. Early AM-mediated bacterial clearance was significantly impaired in influenza-infected mice: ~50% of the initial bacterial inoculum could be harvested from the alveolar airspace 3 h later. In mock-infected mice, by contrast, >95% of inocula up to 50-fold higher was efficiently cleared. Coinfection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of early innate host protection. Local GM-CSF treatment partially restored the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that resident AM depletion occurs during influenza infection. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs, resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial superinfections.

Figure 1. In vivo labeling of lung-resident macrophages confirms validity of flow cytometric gating strategy

(A) Flow cytometry dot plots show the gating strategy of alveolar macrophages (AMs, R4 gate) and interstitial macrophages (IMs, R5 gate) of mock-infected mice in BALF (top plots) and post-lavage lung homogenate (bottom plots). (B) In vivo labeling of lung-resident macrophages using PKH26-PCL dye before influenza infection can distinguish AMs (solid red bars) from IMs (solid black bars) based on MFI of PKH26-PCL dye in both BALF and post-lavage lung homogenate (n = 4). ***P < 0.001 Tukey’s multiple comparison test (ANOVA). The bar graphs show the average ± SD

Figure 2. AMs are depleted during influenza infection

Absolute numbers of AMs in bronchoalveolar lavage fluid (solid bars) and post-lavage lung homogenates (open bars) of PR8-infected (A), or 2009 pandemic H1N1-infected mice (B) 7 days after influenza infection are significantly lower than in mock-infected (naive) mice (n ≥5 each). ***P < 0.001 compared with mock-infected (naïve) mice. The bar graphs show the average ± SEM.

Figure 3. Tracking of AMs and IMs dynamic changes during influenza infection

Absolute numbers of AMs and IMs in BALF (A) and post-lavage lung homogenate (B) of PR8-infected mice harvested 1, 3, 5, 7, 9, 11, and 14 days p.i. Tracking the changes in MFI of PKH26-PCL dye that distinguishes resident macrophages in BALF (C) and post-lavage lungs (D) during influenza infection. *P < 0.05,**P < 0.01,***P < 0.001 compared with mock-infected (naïve) mice (Fig. A, B) or influenza-infected mice 1 day p.i. (Fig. C, D), n ≥4. Dunnett’s multiple comparison test (ANOVA). The bar graphs show the average ± SD

Figure 4. Influenza infection induces significant AM death

Total numbers of dead AMs (A) and IMs (B) are calculated during influenza infection. (C) Cytospin of Diff quick-stained BALF cells from mock-infected and PR8-infected mice 3 days p.i at magnification × 500. *P < 0.05, **P < 0.01 Dunnett’s multiple comparison test (ANOVA) compared with mock-infected naive mice. The bar graphs show the average ± SD.

Figure 5. Murine influenza-pneumococcal co-infection model

(A) Body weight loss of single influenza-infected (influenza and PBS 7 days p.i.), single pneumococcal-infected (PBS and S. pneumoniae), and co-infected (influenza and S. pneumoniae) mice (n > 5 each). (B) Survival rate of single influenza-infected, single pneumococcus-infected, and co-infected mice (n > 5 each). (C) Thorax bioluminescence of luciferase-expressing A66.1 pneumococcus in co-infected mice showing development of pneumococcal pneumonia. The bar graphs show the average ± SD. *** P < 0.001 Student’s t-test at each timpoint compared with single influenza-infected mice group (Fig. A), *** P < 0.001 log-rank test on the Kaplan Meier survival data (Fig. B). (D) Pneumococcal titers harvested 3 h after bacterial inoculation (inoculum of 200 or more CFUs) from alveolar airspace of mock-infected and influenza-infected mice 7 days p.i. are shown as percentage of inoculum. ***P < 0.001 compared with mock-infected (naïve) mice.

Figure 6. Early pneumococcal clearance from alveolar airspace is impaired during the AM depletion phase in influenza-infected mice

(A) Pneumococcal CFUs harvested 3 h after bacterial inoculation (inoculum of 200 CFUs) from the alveolar airspace of mock-infected and influenza-infected mice 1, 3, 5, 7, 9, 11, and 14 days p.i. Secondary pneumococcal pneumonia development (B) and mortality (C) are manifested in influenza-infected mice that are secondarily pneumococcal-infected during the AM depletion phase (n ≥4). *P < 0.05,***P < 0.001 compared with mock-infected mice, Dunnett’s multiple comparison test (ANOVA) (Fig. A). *P < 0.05,** P < 0.01 *** P < 0.001 compared with Day 1 or Day 14 co-infection groups, log-rank test on the Kaplan Meier survival data (Fig. B, C)

Figure 7. Local recombinant GM-CSF treatment improved secondary pneumococcal pneumonia development in influenza-infected mice

(A) Intranasal administration of recombinant GM-CSF into PR8-infected mice on days −1 and +1 before and after infection, followed by 200 CFUs of pneumococcus on day 3 improved early pneumococcal clearance from the alveolar airspace (C) and secondary pneumococcal pneumonia development (n = 9) (D) compared with mock-treated co-infected mice (n = 4). (B) The absolute numbers of AMs and IMs increased in GM-CSF-treated influenza-infected mice analyzed 3 days p.i (n ≥4). *P < 0.05,***P < 0.001 Dunnett’s multiple comparison test (ANOVA), compared with mock-treated mock-infected mice (Fig. B, C). The bar graphs show the average ± SD. *P < 0.05 compared with Day 1 or Day 14 co-infection groups, log-rank test on the Kaplan Meier survival data (Fig. D).