Potential role for alternatively activated macrophages in the secondary bacterial infection during recovery from influenza

Abstract

Purpose: Secondary bacterial infections are a common complication of influenza. Innate immune host defenses appear to be impaired following influenza, leading to susceptibility to subsequent bacterial infections. Alternatively activated macrophages (AAM) in the lungs may play a critical role in eliciting the hypersusceptibility to secondary bacterial pneumonia.

Methods: C57BL6 mice were challenged with sublethal doses of the mouse-adapted A/PR/8/34 (PR8) influenza virus or saline and allowed to recover. At complete recovery (day 14), mice were re-challenged with sublethal doses of Streptococcus pneumoniae serotype 3 (Sp3).

Results: PR8-recovered mice developed a rapidly fatal pulmonary infection to a 100-fold sublethal pneumococcal challenge, whereas PR8-naive mice demonstrated no mortality or illness. The cytokines which induce AAM (IL-4 and IL-13) and the expression of genes associated with AAM (Arginase-1, FIZZ1, and YM1) were elevated after PR8 infection. Flow cytometry suggests that alveolar macrophages demonstrate the AAM-phenotype, as indicated by MGL-1 and MHCII expression, in response to PR8 infection. Recovery from PR8 was associated with blunted cytokine responses to TLR ligands.

Conclusions: The mechanisms of immune regulation during recovery from influenza are being elucidated. We provide evidence that pulmonary AAM are induced during influenza infection and may contribute to the elicitation of hypersusceptibility to a secondary bacterial infection.

Figure 1. Recovery from primary PR8 influenza strain infection

(Panel A) The changes in weight after challenge with saline (n=10), PR8 2×10³ TCID₅₀ (n=20), and PR8 2×10⁴ TCID₅₀ (n=17) were recorded and show recovery of weight by day 14 among mice receiving sublethal (PR8 2×10³ TCID₅₀) infection. * indicates that at day 9, there is incomplete data due to 65% mortality. The viral counts from lungs and BAL fluid (Panel B) and H&E stains on lungs (Panel C, 40X magnification) were performed on the days indicated after challenge with sublethal (PR8 2×10³ TCID₅₀) infection and show clearance of virus by day 10 and normalization of lung histology at day 14. ND = not detectable.

Figure 2. Lethality of secondary Sp3 infection during recovery (14 days) from a primary influenza infection

(Panel A) Kaplan-Meier curves were created for mice that recovered from primary saline (n=11) or PR8 2×10³ TCID₅₀ (n=25) followed by challenge with sublethal 5×10³ CFU of Sp3 and show significant hypersusceptibility during recovery from influenza; Logrank p-values are indicated. Mice challenged with sublethal Sp3 (5×10³ CFU) show significant inflammatory cell infiltration (Panel B) on lung H&E staining (40X magnification) and pneumococcal dissemination on organ bacterial counts (Panel C) at the times indicated among mice in recovery (day 14) from sublethal PR8 (2×10³ TCID₅₀) infection. *p=0.002 **p=0.0002

Figure 3. Gene expression associated with AAM

Gene expression was measured from the lungs of mice at the indicated times after sublethal PR8 ± Sp3 infection for the direct inducers (Panel A) and prototype signature markers (Panel B) of AAM and for anti-inflammatory (Panel C) and pro-inflammatory (Panel D) cytokines. Gene expression by real-time PCR is reported as relative gene expression compared with lungs of mice exposed to saline only. Experiments were performed in triplicate and data are presented as mean ± SEM. *p <0.05 **p<0.005

Figure 4. Flow cytometric identification of the AAM phenotype

(Panel A) Primary murine macrophages that were treated with IL-4 (AAM, solid line) stained positive for MGL-1, whereas un-treated macrophages (CM, dashed line) lacked the expression of this marker. (Panel B) Lung homogenates were stained with a multichromatic flow cytometry panel (Naïve mouse shown). The plot depicts CD11b and CD11c following exclusion of dead cells (ViviD) and granulocytes (Gr1⁺). Populations identified were interrogated for the expression of MGL-1, MHCII, F4/80, and autofluorescence. Only alveolar macrophages (AM, Gr1 ⁻ CD11b⁻ CD11c^high) stained positive for MGL-1; they also were MHCII^dim and highly autofluorescent. DC (dendritic cell) and CM (classical macrophage) do not express MGL-1.

Figure 5. Kinetics of lung AAM

AAM were defined as alveolar macrophages (autofluorescent Gr1⁻ CD11b⁻ CD11c^high F4/80^dim) which express both MGL-1 and MHCII. Panel A depicts the MGL-1 and MHCII positive alveolar macrophage population after sublethal PR8 ± Sp3 infection. (Panel B) The percentage of lung AAM (percentage relative to alveolar macrophages) after sublethal PR8 ± Sp3 are summarized in 5–95 percentile box-whisker plots (line indicates mean values). (Panel C) The numbers of AAM per gram of lung tissue after sublethal PR8 ± Sp3 are summarized in minimum-maximum box-whisker plots (line indicates mean values) with dots that indicate individual animal data. Panel D summarizes the number of CM (Gr1⁻ MGL-1⁻ CD11b⁺ CD11c- F4/80⁺) per gram of lung tissue after sublethal PR8 ± Sp3, by minimum-maximum box-whisker plots (line indicates mean values) with dots that indicate individual animal data. The data are from 3 separate experiments. *p <0.05.

Figure 6. Blunted splenocyte responses to TLR stimuli

Splenocytes from mice at the indicated times after sublethal PR8 infection were stimulated with: medium alone (non-stimulated), 100 ng/mL LPS, 10 μg/mL CpG, or 100 ng/mL Pam3Cys. After 18 h incubation, supernatants were harvested and cytokine production was measured by ELISA. Data are expressed as the mean ± SEM of cytokine production and are representative of three independent experiments for Day 0 and 14, two experiments for Day 3. *p <0.05 **p<0.005