Interferon-β deficiency at asthma exacerbation promotes MLKL mediated necroptosis

Abstract

Defective production of antiviral interferon (IFN)-β is thought to contribute to rhinovirus-induced asthma exacerbations. These exacerbations are associated with elevated lung levels of lactate dehydrogenase (LDH), indicating occurrence of cell necrosis. We thus hypothesized that reduced lung IFN-β could contribute to necrotic cell death in a model of asthma exacerbations. Wild-type and IFN-β^-/- mice were given saline or house dust mite (HDM) intranasally for 3 weeks to induce inflammation. Double-stranded RNA (dsRNA) was then given for additional 3 days to induce exacerbation. HDM induced an eosinophilic inflammation, which was not associated with increased expression of cleaved caspase-3, cleaved PARP or elevated bronchoalveolar lavage fluid (BALF) LDH levels in wild-type. However, exacerbation evoked by HDM + dsRNA challenges increased BALF levels of LDH, apoptotic markers and the necroptotic markers receptor-interacting protein (RIP)-3 and phosphorylation of mixed linage kinase domain-like protein (pMLKL), compared to HDM + saline. Absence of IFN-β at exacerbation further increased BALF LDH and protein expression of pMLKL compared to wild-type. We demonstrate that cell death markers are increased at viral stimulus-induced exacerbation in mouse lungs, and that absence of IFN-β augments markers of necroptotic cell death at exacerbation. Our data thus suggest a novel role of deficient IFN-β production at viral-induced exacerbation.

Figure 1

HDM induces lung inflammation. (A) Differential cell count in BALF (B) Total protein (C) Release of the pan-necrotic marker LDH in BALF (D) H&E staining of mice lung sections (E) Inflammation scored in lung tissue. (F) Representative immunoblots of cleaved caspase-3 and cleaved PARP. (G) Quantification of protein expression of cleaved caspase-3 and (H) cleaved PARP from immunoblots. Optical density was measured and bands related to housekeeping protein GAPDH and normalized towards saline. The data are presented as mean ± SEM (n = 5–6). *p < 0.05 vs saline, **p < 0.01 vs saline.

Figure 2

Exacerbating mice have increased expression of apoptotic and necroptotic markers. Representative immunoblots and quantification of (A) cleaved caspase-3, (B) cleaved PARP (C) full-length caspase-8 (D) RIP3 (E) pMLKL from homogenized lungs. Optical density was measured and bands were related to housekeeping protein GAPDH and normalized towards HDM:dsRNA 50. The data are presented as mean ± SEM (n = 5–6). *p < 0.05, **p < 0.01.

Figure 3

Interferon-β is protective against cell BALF LDH release at exacerbation. (A) Total cell count in BALF (B) Total protein in BALF (C) Release of the pan-necrotic marker LDH in BALF (D) Gene expression of IL-1β from lung homogenate. (E) Inflammation scored in lung tissue. (F,G) H&E staining of mice lung sections. The data are presented as mean ± SEM (n = 4–10) (n = 4–5 in control groups). *p < 0.05, **p < 0.01.

Figure 4

Interferon-β is protective against necroptosis. Representative immunoblots and quantification of (A) cleaved caspase-3 (B) cleaved PARP (C) full-length caspase-8 (D) RIP3 (E) pMLKL from homogenized lungs. Optical density was determined and bands were related to housekeeping protein GAPDH and normalized towards HDM:dsRNA. (F) Representative TUNEL-staining. Nucleus is stained blue. TUNEL-postive cells are stained green. The data are presented as mean ± SEM (n = 5–10). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.