Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R alpha-, TLR4-, and IFN-beta-dependent

Abstract

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed "Th1" and "Th2" cytokine storm. We hypothesized that differentiation of "alternatively activated" macrophages (AA-M phi) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4R alpha/STAT6-dependent AA-M phi differentiation, and significantly enhanced inflammation in the lungs of IL-4R alpha(-/-) mice. Adoptive transfer of wildtype macrophages to IL-4R alpha(-/-) mice restored RSV-inducible AA-M phi phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4(-/-) and interferon (IFN)-beta(-/-) macrophages and mice also failed to express AA-M phi markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptor gamma expression, a DNA-binding protein that induces AA-M phi genes, whereas IFN-beta regulates IL-4, IL-13, IL-4R alpha, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-M phi. These data suggest new treatment strategies for RSV that promote AA-M phi differentiation.

Fig. 1. RSV infection induces differentiation of AA-Mϕ

(A) Highly purified WT BALB/c BAL macrophage cultures were treated with medium only or infected with RSV. Supernatants were collected at the indicated time points and analyzed for IL-4 and IL-13 by ELISA. (B) WT BALB/c BAL macrophages were treated with medium alone, rIL-4, or RSV. Gene expression was analyzed by real-time PCR. Data are means ± SEM from a single representative experiment (N = 4). (C) WT C57BL/6 mice were mock- or RSV-infected and lungs harvested 4 d p.i. Frozen lung sections were stained for nuclei (DAPI; blue), F4/80 (green), and arginase-1 (red), then viewed by confocal microscopy. Arrows are positioned identically on each panel within a treatment to provide a reference point. The arrowhead in panels f–j illustrates a rare cell that is positive for F4/80, but not for arginase-1.

Fig. 2. Failure to induce AA-Mϕ prolongs CA-Mϕ phenotype

(A) WT BALB/c and IL-4Rα^−/− peritoneal macrophages were treated as in Fig. 1 and mRNA expression measured. Data are derived from a single representative experiment (N = 3). (B) WT and IL-4Rα^−/− mice were mock- or RSV-infected. Mice were sacrificed 4 d p.i., and arginase-1 and IL-12 p40 mRNA measured in lungs by real-time PCR. (C) WT and IL-4Rα^−/− mice were treated as in (B). Lung pathology was scored as described in Methods. Results are compiled from 3 independent experiments. (D) WT and IL-4Rα^−/− mice were mock- or RSV-infected. Lungs were harvested 4 d p.i. Lung pathology was scored (N = 4; 4 mice/treatment). Images shown are at 100x.

Fig. 3. Adoptive transfer of WT macrophages reconstitutes AA-Mϕ phenotype in IL-4Rα^−/− mice

(A) WT BALB/c and IL-4Rα^−/− mice, along with IL-4Rα^−/− mice that received either 1.5 × 10⁷ WT (WT→IL-4Rα^−/−) or IL-4Rα^−/− (IL-4Rα^−/−→IL-4Rα^−/−) macrophages i.p., were mock- or RSV-infected 5 d post-transfer. Lungs were harvested 4 d p.i. and analyzed for gene expression by real-time PCR. (B) Lung sections from mice in (A) were H&E stained and scored for lung pathology. Data represent one of 2 separate experiments with similar outcomes (10 mice/treatment).

Fig. 4. Differentiation of AA-Mϕ by RSV is TLR4-dependent

(A) WT C57BL/6 and TLR4^−/− peritoneal macrophages were treated as indicated in Fig. 1 and analyzed for mRNA gene expression by real-time PCR (N = 3). (B) WT and TLR4^−/− macrophages were treated as in (A) and analyzed for PPARγ mRNA gene expression by real-time PCR. (C) WT and TLR4^−/− mice were mock- or RSV-infected. Lungs were harvested 4 d p.i. Lung pathology was scored (N = 4; 4 mice/treatment). Images shown are at 200x.

Fig. 5. RSV-induced AA-Mϕ and IL-10 production are IFN-β-dependent

(A) WT and IFN-β^−/− peritoneal macrophages were treated as indicated and analyzed for mRNA by real-time PCR (N = 2). (B) WT or IFN-β^−/− mice were mock- or RSV-infected. At 4 d p.i., mice were sacrificed and lungs analyzed for arginase-1, FIZZ1, and MR mRNA. (C) IL-10 and COX-2 mRNA were measured in the lungs of RSV-infected mice (see legend to (B)). (C) Mice were infected as in (B) and lungs fixed and stained with H&E 4 d p.i. (N = 4; 4 mice/treatment). Images shown are at 400x.

Fig. 6. Rapid activation of AA-Mϕ in cotton rats during re-infection and in response to COX-2 inhibition

(A) Cotton rats (4/group) were administered saline or infected with RSV. Animals were then RSV-infected 60 d after initial treatment. Lungs were harvested, total RNA isolated, and analyzed for IL-4, IL-13, and arginase-1 mRNA by RT-PCR and Southern blotting. (B) Cotton rats were mock- or RSV-infected. Infected rats were treated on day 3 p.i. with vehicle (saline) or paracoxib (100 mg/kg) i.p. All animals were sacrificed 4 d p.i. and lung samples evaluated for mRNA expression for the indicated genes. Each lane represents RNA from one cotton rat and results are representative of 3 separate experiments.

Fig. 7. Hypothetical model for the role of AA-Mϕ during RSV infection