Uric acid pathway activation during respiratory virus infection promotes Th2 immune response via innate cytokine production and ILC2 accumulation

Abstract

Respiratory syncytial virus (RSV) infects a majority of infants and can cause severe disease leading to increased risk to develop asthma later in life. In the present studies we detected high levels of uric acid pathway components during RSV infection and examined whether they altered the pathogenesis of RSV infection. Inhibition of uric acid (UA) pathway activation during RSV infection in airway epithelial cells using XOI decreased the expression of IL-33, thymic stromal lymphopoietin (TSLP), and CCL2. In addition, treatment of RSV infected bone marrow-derived macrophages with XOI decreased production of IL-1β. Thus, UA activation of different cell populations contributes different innate immune mediators that promote immunopathogenesis. When mice were treated with XOI or interleukin-1 receptor antagonist (IL1-ra) during RSV infection decreased pulmonary mucus was observed along with significantly reduced numbers of ILC2 and macrophages, accompanied by decreased IL-33 in bronchoalveolar lavage of the treated mice. These findings provide mechanistic insight into the development of RSV immunopathology and indicate that xanthine metabolites and UA are key immunoregulator molecules during RSV infection. Moreover, these findings suggest uric acid and IL-1β as possible therapeutic targets to attenuate severe RSV disease.

Figure 1.. Animal model:

Female Balb/c mice 6–7 weeks old were intratracheally infected with RSV line 19 (1×10^5 pfu). The mice were treated with xanthine oxidase inhibitor (XOI) (20mg/kg/mouse) intraperitoneally or recombinant murine IL-1ra (100ug/kg/mouse) intranasally every day during the time of infection.

Figure 2.. RSV infection increases Uric acid production in the airways.

A) qPCR of Xanthine Oxidase (XO) in the lung of RSV infected mice shows significantly increased expression of XO during infection. B) The uric acid levels in bronchoalveolar lavages (BAL) of RSV infected mice were significantly increased in the BAL of infected mice compared with the uric acid level in naive mice. Data represents the Mean ± SE from 4–5 mice (experimental repeats 3–4). * P≤0.05, ^** P≤.01, ^*** P≤0.001. C) Uric acid levels from samples of lung aspirates from RSV+ and normal infants, with significant upregulation in RSV+ infants. * P≤0.05, ^** P≤.01.

Figure 3.. Human Airway epithelial cells increase the expression of XO during RSV infection and induce inflammatory innate cytokines.

A) Expression of XO in A549 infected with RSV compared with uninfected cells. B-D) IL-33, TSLP and CCL2 expression in A549 cells treated with XOI with or without RSV infection. E) Expression of IFNβ in A549 cells treated with XOI with or without RSV infection. Data represents the Mean ± SE from 4 replicates per group (experimental repeats 3). * P≤0.05, ^** P≤0.01, ^*** P≤0.001, ^****P≤0.0001.

Figure 4.. Primary mouse airway epithelial cells increase the expression of XO during RSV infection and induce inflammatory innate cytokines.

Cultures of primary airway epithelial cells (AECs) from naïve Balb/c mice were infected with RSV for 24 h and underwent different treatments A) Xanthine oxidase expression in RSV infected cells compared with uninfected cells. B and C) TSLP, IL-33 and IL-25 and IFNβ expression in AECs infected with RSV treated with or without XOI. D and E) CCL2 expression and production in in AECs infected with RSV treated with or without XOI. F and G) IL-33 and CCL2 expression in AECs that were treated with uric acid, RSV or RSV+Uric acid. H and I) IL-33 and CCL2 expression in AECs that were treated with murine recombinant IL-1β (IL-1β), RSV or RSV+IL1β. J) IL-33 expression in AECs that were treated with murine recombinant IFNβ (IFNβ), RSV or RSV+ IFNβ. K) IFNβ expression in AECs that were treated with murine recombinant IL-33 (IL-33), RSV or RSV+ IL-33. Data represents the Mean ± SE from 4 replicates per group (experimental repeats 3–4). * P≤0.05, ^** P≤.01, ^*** P≤0.001.

Figure 5.. Bone marrow-derived macrophages (BMDMs) treated with XOI during RSV infection show decreased expression of IL-1β and CCL2.

A) qPCR XO expression in BMDMs that were RSV infected. B and C) Expression and production of IL-β by BMDMs treated with or without XOI during RSV infection. D and E) CCL2 expression and production in BMDMs treated with or without XOI during RSV infection. F and G) Expression of IL-1β and CCL2 in BMDMs treated with uric acid during RSV infection. Data represent the Mean ± SE from 4 replicates per group (experimental repeats 3). * P≤0.05, ^** P≤.01, ^*** P≤0.001.

Figure 6.. Targeting the xanthine/uric acid pathway during RSV infection attenuates the immunopathology response.

A) Uric acid detection in the BAL of infected mice was significantly decreased in mice treated with XOI compared with control and RSV infected mice. B) Lung histopathology in Hematoxylin and Eosin stain (H&E) showed inflammatory infiltrates and Periodic acid-Schiff stain (PAS) to detected mucus that was reduced in the lungs of mice treated with XOI, as well as C) decreased Gob 5 mRNA expression. D and E) IL-13 lung mRNA expression and production in lung extracts. F) Lung draining lymph nodes from mice at 8 were re-stimulated with RSV (MOI of 1, 48 hrs.), and T-cell cytokine levels were measured by Bio-Plex. G) qPCR of total lung RNA IFNβ mRNA expression. H and I) ELISA of IL-33 and IL-1β from BAL samples collected at 6dpi. J-M) Flow cytometry of lung leukocytes, ILC2, neutrophils, dendritic cells, and interstitial macrophages from naïve and infected mice at 8dpi. Data represents the Mean ± SE from 4–5 mice (experimental repeats 4–5). * P≤0.05, ^** P≤.01, ^*** P≤0.001

Figure 7.. Targeting IL-1 pathway during RSV infection attenuates the immunopathology response.

A) Lung histopathology (H&E) showed inflammatory infiltrated and mucus (PAS staining) were reduced in lungs of mice with IL-1ra as well as B) decreased Gob 5 and Muc5 mRNA expression, and C) IL-13 and IL-5 mRNA expression. D) and E) IL-13 and IL-5 production in lung extracts. F) qPCR of total lung RNA IFNβ expression. G) Lung draining lymph nodes from mice at 8dpi were re-stimulated with RSV (MOI of 1, 48 hrs.), and T-cell cytokine levels were measured by Bio-Plex. H) ELISA of IL-33 from BAL samples collected at 6dpi. I-M) Flow cytometry of lung leukocytes, ILC2 cells, neutrophils, interstitial macrophages, dendritic cells, and T cells from naïve and infected mice at 8dpi. Data represents the Mean ± SE from 4–5 mice (experimental repeats 3–4). * P≤0.05, ^** P≤.01, ^*** P≤0.001.

Figure 8.. RSV induces Uric acid and leads to Th2 response.

RSV infected airway epithelial cells (AECs) trigger the production of uric acid, IL-33, TSLP, and CCL2. AECs and macrophages increase the expression of innate cytokines when uric acid is upregulated. CCL2 can recruit monocytes, APC, and T cells to the lung, while IL-33 along with TSLP and IL-1β will recruit and activate ILC2.