Inhibition of uric acid or IL-1β ameliorates respiratory syncytial virus immunopathology and development of asthma

Abstract

Background: Respiratory syncytial virus (RSV) affects most infants early in life and is associated with increased asthma risk. The specific mechanism remains unknown.

Objective: To investigate the role of uric acid (UA) and IL-1β in RSV immunopathology and asthma predisposition.

Methods: Tracheal aspirates from human infants with and without RSV were collected and analyzed for pro-IL-1β mRNA and protein to establish a correlation in human disease. Neonatal mouse models of RSV were employed, wherein mice infected at 6-7 days of life were analyzed at 8 days postinfection, 5 weeks postinfection, or after a chronic cockroach allergen asthma model. A xanthine oxidase inhibitor or IL-1 receptor antagonist was administered during RSV infection.

Results: Human tracheal aspirates from RSV-infected infants showed elevated pro-IL-1β mRNA and protein. Inhibition of UA or IL-1β during neonatal murine RSV infection decreased mucus production, reduced cellular infiltrates to the lung (especially ILC2s), and decreased type 2 immune responses. Inhibition of either UA or IL-1β during RSV infection led to chronic reductions in pulmonary immune cell composition and reduced type 2 immune responses and reduced similar responses after challenge with cockroach antigen.

Conclusions: Inhibiting UA and IL-1β during RSV infection ameliorates RSV immunopathology, reduces the consequences of allergen-induced asthma, and presents new therapeutic targets to reduce early-life viral-induced asthma development.

Keywords: allopurinol; asthma; interleukin-1 beta; respiratory syncytial virus; uric acid.

Figure 1.. RSV induces pulmonary IL-1β expression in humans and mice neonates.

A) cDNA from tracheal aspirates from human infants with RSV and from control patients intubated for non-infectious reasons underwent qPCR to determine pro-IL-1β mRNA (N ≥ 9). B) Tracheal aspirates from human infants underwent Bioplex to determine IL-1β protein concentration (N ≥ 6). C) Tracheal aspirates from human infants were measured via Bioplex to determine CCL5 protein concentration (N ≥ 7). D) Mice infected with RSV were sacrificed at 2, 4, 8, and 14 days post-infection (dpi) and compared with age-matched controls. Lungs were homogenized and mRNA extracted to determine CCL5, pro-IL-1β, and xanthine oxidase mRNA expression (N ≥ 5). E) BAL fluid from mice with RSV and age-matched controls was taken 8 days post-infection and assayed for uric acid (N ≥ 6). Data represent mean +/− SEM. *p < 0.05, **p < 0.01.

Figure 2.. Administration of the XOI or IL-1RA ameliorates RSV infection.

Mice were infected with RSV and treated with the XOI or IL-1RA and compared with infected mice at 6 or 8 days post infection. A) Lungs were embedded in paraffin and Periodic acid-Schiff (PAS) stained to visualize mucous (pink/purple staining) or hematoxylin/eosin (H/E) stained to evaluate inflammatory cell infiltrates. Representative photos shown (N ≥ 5). B) Mucus scoring was performed on blinded histological slides on a scale of 1–4 for mucus production (N ≥ 4). C) Lungs were homogenized and mRNA extracted to determine Gob5 mRNA expression (N ≥ 5) and CCL5 mRNA expression (N ≥ 6). D) BAL fluid at 6 days post-infection was collected as described and assayed for uric acid content (N ≥ 4). Data represent mean +/− SEM. *p < 0.05. **p < 0.01, ***p < 0.001.

Figure 3.. Administration of the XOI or IL-1RA reduces pulmonary immune infiltrates and lymphocyte responsiveness with RSV infection.

Mice were infected with RSV and treated with the XOI or IL-1RA and compared with infected mice at 8 days post-infection. A and B) Lungs were processed into single-cell suspension, then stained and analyzed via flow cytometry for type 2 innate lymphoid cells (N ≥ 6) and neutrophils (N ≥ 5). C, D, E, and F) Lung draining lymph nodes were processed into single-cell suspension and re-stimulated with RSV in vitro for 48 hours to determine cytokine protein levels in the supernatant including IL-4, IL-5, IL-13, and IFN-γ (N ≥ 3). Data represent mean +/− SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.. Administration of the XOI or IL-1RA during RSV infection leads to durable immune changes.

Mice were infected with RSV and treated with the XOI or IL-1RA and compared with infected mice at 5 weeks’ post infection. A, B, C, D) Lungs were processed into single-cell suspensions, then stained and analyzed via flow cytometry for type 2 innate lymphoid cells, macrophages, CD103+ dendritic cells, and eosinophils (N ≥ 4). E, F, G, and H) Lung draining lymph nodes were processed into single-cell suspension and re-stimulated with RSV in vitro for 48 hours to determine cytokine protein levels in the supernatant including IL-4, IL-5, IL-13, and IFN-γ (N ≥ 6). Data represent mean +/− SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.. Administration of the XOI or IL-1 receptor antagonist during RSV infection ameliorates lung immunopathology during subsequent cockroach antigen sensitization.

Mice were infected with RSV and treated with the XOI or IL-1RA. 5 weeks after the start of infection, mice underwent CRA sensitization and challenge. Control mice were uninfected, CRA-treated mice (CRA only), and RSV-infected/CRA-treated mice (RSV/CRA). A and B) Lungs were embedded in paraffin and Periodic acid-Schiff (PAS) stained to visualize mucous (pink/purple staining) or hematoxylin/eosin (H/E) stained to evaluate inflammatory cell infiltrates. Representative photos shown (N ≥ 9). C) Mucus scoring was performed on blinded histological slides on a scale of 1–4 for mucus production (N ≥ 9). D and E) Lungs were homogenized and mRNA extracted to determine Gob5 (N ≥ 8) and IL-13 mRNA expression (N ≥ 3). Data represent mean +/− SEM. *p < 0.05, **p < 0.01.

Figure 6.. Administration of the XOI or IL-1 receptor antagonist during RSV infection ameliorates type 2 immune responses during subsequent cockroach antigen sensitization.

Mice were infected with RSV and treated with the XOI or IL-1RA. 5 weeks after the start of infection, mice underwent CRA sensitization and challenge. Control mice were uninfected, CRA-treated mice (CRA only) as well as RSV-infected/CRA-treated mice (RSV/CRA). A, B, C, D, E, F) Lungs were processed into single-cell suspensions, then stained and analyzed via flow cytometry for type 2 innate lymphoid cells (N ≥ 7), macrophages (N ≥ 8), CD4+ T cells (N ≥ 4), eosinophils (N ≥ 8), CD11b+ dendritic cells (N ≥ 8), and CD103+ dendritic cells (N ≥ 8). G, H, I, and J) Lung draining lymph nodes were processed into single-cell suspension and re-stimulated with RSV in vitro for 48 hours to determine cytokine protein levels in the supernatant including IL-4, IL-5, IL-13, and IFN-γ (N ≥ 8). Data represent mean +/− SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.