Critical role of IL-1 receptor-associated kinase-M in regulating chemokine-dependent deleterious inflammation in murine influenza pneumonia

Abstract

Influenza virus is a common cause of respiratory infection and morbidity, which is often due to deleterious host immune responses directed against the pathogen. We investigated the role of IL-1 receptor-associated kinase-M (IRAK-M), an inhibitor of MyD88-dependent TLR signaling, in modulating the innate inflammatory response during influenza pneumonia using a murine model. The intranasal administration of influenza resulted in the upregulation of IRAK-M mRNA and protein levels in the lungs within 2 d after infectious challenge. Pulmonary influenza infection in mice deficient in IRAK-M (IRAK-M(-/-)) resulted in substantially increased mortality compared with similarly treated wild-type animals. Increased mortality in IRAK-M(-/-) mice was associated with enhanced early influx of neutrophils, high permeability edema, apoptosis of lung epithelial cells, markedly increased expression of inflammatory cytokines/chemokines, and release of neutrophil-derived enzymes, including myeloperoxidase and neutrophil elastase. Early viral clearance was not different in mutant mice, whereas viral titers in lungs and blood were significantly higher in IRAK-M(-/-) mice compared with wild-type animals. Increased lethality observed in IRAK-M(-/-) mice after influenza challenge was abrogated by Ab-mediated blockade of CXCR2. Collectively, our findings indicate that IRAK-M is critical to preventing deleterious neutrophil-dependent lung injury during influenza infection of the respiratory tract.

FIGURE 1

Expression of IRAK-M in lungs of WT mice infected with influenza virus. A, Analysis by real-time quantitative PCR (n = 4). *p < 0.01, significant differences among groups. B, Western blots show two representative samples. Upper panel: IRAK-M (68 kDa). Lower panel: β-actin (42 kDa). Days 2 and 7: Samples collected 2 and 7 d, respectively, postinfection with influenza virus. CTR, control (untreated).

FIGURE 2

Survival rates and viral accumulation in IRAK-M^−/− or WT mice infected with influenza virus. A, Survival rates. Each group consisted of 10–15 mice. ■, mock (saline)-infected IRAK-M^−/− mice; □, mock-infected WT mice; ●, IRAK-M^−/− mice infected with influenza virus; ○, WT mice infected with influenza virus. Survival was significantly shorter in infected IRAK-M^−/− than in infected WT mice (**p < 0.001). B, Viral titers (n = 8). Blood and lung homogenates were collected on days 2 and 7 after influenza virus infection. *p < 0.01; **p < 0.001. NS, no significant differences between groups. WT mice (open circles); IRAK-M^−/− KO mice (closed circles). KO, knockout.

FIGURE 3

Lung injury in infected IRAK-M^−/− mice. A, Histopathological analysis of mouse lungs collected 2 d (a, b, e, f, i, j) or 6 d (c, d, g, h, k, l) after inoculation with influenza virus. IRAK-M^−/− KO mice (a, c, e, g, i, k) and WT mice (b, d, f, h, k, l). H&E-stained tissue sections at magnifications of ×40 (c–h) and ×100 (i–l). Results are representative of four repeated experiments. B, Lung permeability of IRAK-M^−/− mice infected with influenza virus (n = 4) assessed by quantitation of albumin concentrations in BALF from each mouse 2 and 6 d postinfection. Data are expressed as means ±SD. **p <0.001 among groups. WT mice (open bars); IRAK-M^−/− KO mice (closed bars).

FIGURE 4

BAL cells and differential cell types and numbers in lungs of IRAK-M^−/− mice infected with influenza virus. A, BAL cell counts (n = 4). Samples were collected at 2, 4, and 6 d postinfection and total cells, neutrophils, and mononuclear cells were counted. Open circles, WT mice; closed circles, IRAK-M^−/− mice. B, Flow cytometry (n = 4). Lung digests were collected at 2 and 6 d post-infection. Mock, saline-infected control; Flu, influenza virus-infected mice. Open bars, WT mice; closed bars, IRAK-M^−/− mice. *p < 0.01; **p < 0.001. NS, no significant differences among groups.

FIGURE 5

Cytokines/chemokines and neutrophil-derived enzymes in lungs of IRAK-M^−/− mice infected with influenza virus. A, Analysis of lung homogenates by real-time PCR (n = 4). Lung homogenates were collected at 2 and 6 d postinfection. B, Analysis of BALF by ELISA (n = 4). C, Type 1 INF mRNA expression in lung homogenates analyzed by real-time PCR (n = 4). D, Release of MPO and NE (n = 4). Samples were collected at 2 and 6 d postinfection. *p < 0.01; **p < 0.001; significant differences among groups. WT mice (open bars); IRAK-M^−/− KO mice (closed bars). CTR, control (saline-infected).

FIGURE 6

Influenza A viral infection induces secretion of inflammatory cytokines ex vivo. Primary PMs (A) and type II AECs (B) isolated from either WT or IRAK-M^−/− KO mice were exposed to influenza A virus (Flu: closed bars) at a multiplicity of infection of 1.0, PBS (mock: open bars) or LPS at 100 ng/ml (positive control: striped bars), then culture supernatants were collected after 18 h for measurement of selected cytokines by ELISA. *p < 0.01; **p < 0.001. Flu, influenza; NS, no significant differences between the groups (n = 4).

FIGURE 7

Apoptosis in lungs of IRAK-M^−/− mice infected with influenza virus. A, TUNEL stains of lung tissue sections. Lungs were collected 2 d (a, b, e, f ) or 6 d (c, d, g, h) after viral inoculation. IRAK-M^−/− KO mice (a, c, e, g) and WT mice (b, d, f, h). H&E-stained tissue sections at magnifications of ×80 (a–d) and ×200 (e–h). B, Caspase-3 induction in lung homogenates from infected WT and IRAK-M^−/− mice analyzed by real-time PCR (n = 4). *p < 0.01 among groups. WT mice (open bars); IRAK-M^−/− KO mice (closed bars). CTR, control (saline-infected).

FIGURE 8

Beneficial effects of anti-CXCR2 Ab administration in IRAK-M^−/− mice infected with influenza virus. A, Survival rates of infected IRAK-M^−/− mice (15 mice per group). ●, CTR IRAK-M^−/− mice; □, anti-CXCR2 Ab-treated IRAK-M^−/− mice; ○, CTR WT mice; ■, anti-CXCR2 Ab-treated WT mice. Survival was significantly longer after treatment with anti-CXCR2 Ab, than with control IgG in IRAK-M^−/− mice (*p < 0.01), but not significantly different among WT mice (NS). B, Lung permeability of infected IRAK-M^−/− mice (n = 4) shown as albumin concentration in BALF from each mouse. Samples were collected at 2 and 6 d postinfection. CTR IRAK-M^−/− mice (closed bars); +CXCR2 Ab, anti-CXCR2 Ab-treated IRAK-M^−/− mice (open bars). C, BAL cell counts in lungs of infected IRAK-M^−/− mice (n = 4). Samples were collected at 2 and 6 d after influenza virus infection and total cells, neutrophils, and mononuclear cells were counted. D, Viral titers (n = 4). Closed circles and open squares, IRAK-M^−/− mice treated with control IgG or anti-CXCR2 Ab, respectively. Lung homogenates were collected on days 2 and 6 after influenza virus infection. Data are expressed as means ± SD. **p < 0.001; *p < 0.01. CTR, control (IgG treated); NS, no significant differences between groups.