Analysis of NLRP3 in the development of allergic airway disease in mice

Abstract

The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat-containing (NLR) family, to the development of allergic airway disease is currently controversial. In this study, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathologic condition, mucus production, and airway hyperresponsiveness between wild-type and Nlrp3(-/-) mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite Ag exposure. Although we did not observe significant phenotypic differences in any of the models tested, we did note a significant reduction of IL-13 and IL-33 in Nlrp3(-/-) mice compared with wild-type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However, this difference did not greatly alter the clinical outcome of the disease. This finding suggests that the role of NLRP3 in allergic asthma must be re-evaluated.

Figure 1. NLRP3 is not required for the development of OVA-alum driven airway inflammation

A. BALF cellularity from wild type and Nlrp3^-/- mice was evaluated following OVA-alum immunization and i.n. OVA challenge. B. The leukocyte composition of the BALF was evaluated via cellular morphology following differential staining. C. Lung histopathology was evaluated and scored utilizing a semi-quantitative scoring system. D. H&E stained sections were evaluated and found to have significant levels of inflammation, concentrated around the airways and vasculature. E. Mucus levels were quantified along the main bronchiole (represented as Vs). F. AB/PAS staining was used to quantify mucus production. Data shown are representative images of AB/PAS+ cells. G. Muc5B gene expression in whole lung homogenates was evaluated. Saline, n=7; Wild Type, n=15; Nlrp3^-/-, n=9. Data are representative of 5 individual experiments.

Figure 2. NLRP3 does not attenuate Th2 associated cytokine production in the OVA-alum model

A) The expression of select Th2 associated cytokines in whole lungs following OVA mediated allergic airway inflammation was determined. B) IL-13 levels in the BALF were assessed by ELISA. A-B. Saline, n=7; Wild Type, n=15; Nlrp3^-/-, n=7. Data are representative of 5 individual experiments. C) IL-1β production in whole lung homogenates was assessed by Western blot. IL-1β and IL-18 were not detected by either Western blot or ELISA (data not shown). Saline, n=2; Wild Type, n=5; Nlrp3^-/-, n=3. Data are representative of 3 individual experiments.

Figure 3. NLRP3 does not contribute to OVA-alum mediated airway hyperreactivity

A) Airway resistance (Raw) and B) tissue damping (G) in the airways, in response to methacholine (MCh), was evaluated in wild type and Nlrp3^-/- mice. Wild Type, n=9; Nlrp3^-/-, n=4. Data are representative of 3 individual experiments.

Figure 4. NLRP3 is not required for the development of OVA mediated airway inflammation in a chronic, alum free, model of allergic airway disease

A. BALF cellularity from wild type and Nlrp3^-/- mice following 6 weeks of i.n. OVA challenge was evaluated. B. The leukocyte composition of the BALF was evaluated via morphology following differential staining. C. Lung histopathology was evaluated and scored utilizing a semi-quantitative scoring system. D. H&E stained sections were evaluated and found to have significant levels of inflammation, which was more diffuse and interstitial in nature. E. Mucus levels along the main bronchiole were quantified (represented as Vs). F. AB/PAS staining was used to assess mucus production. Data shown are representative images of AB/PAS+ cells in the large conducting airway. Vehicle, n=6; Wild Type, n=6; Nlrp3^-/-, n=6. Data are representative of 4 individual experiments.

Figure 5. IL-33 was significantly attenuated in Nlrp3^-/- mice in the chronic alum-free OVA model

A) Total serum IgE was evaluated by ELISA. B-C) IL-13 and IL-33 levels in the BALF were assessed by ELISA. IL-1β and IL-18 were not detected by either Western blot or ELISA (data not shown). Vehicle, n=6; Wild Type, n=6; Nlrp3^-/-, n=6. *p < 0.05. Data are representative of 4 individual experiments.

Figure 6. NLRP3 is not required for the development of HDM induced allergic airway disease

A. BALF cellularity from wild type and Nlrp3^-/- mice was evaluated following either acute (2 weeks) or chronic (10 weeks) HDM challenges. B. The leukocyte composition of the BALF was evaluated via morphology following differential staining. C-D. H&E stained sections were evaluated and found to have low levels of inflammation following either C) 2 weeks or D) 10 weeks of HDM exposure. E. Lung histopathology was evaluated and scored utilizing a semi-quantitative scoring system. Vehicle, n=6; Wild Type 2 weeks, n=7; Nlrp3^-/- 2 weeks, n=7; Wild Type 10 weeks, n=7; Nlrp3^-/-10 weeks, n=7. Data are representative of 8 individual experiments.

Figure 7. NLRP3 does not attenuate Th2 associated cytokine production following acute HDM administration

A) The expression of select Th2 associated cytokines from whole lungs following 2 weeks of i.n. HDM challenges was determined. B) IL-13 levels in the BALF were assessed by ELISA. C) Total IgE levels in the serum were assessed. D) Nlrp3 expression from whole lungs was evaluated by RT-PCR. IL-1β, IL-18 and IL-33 were not detected by either Western blot or ELISA from BALF or whole lung homogenates (data not shown). Vehicle, n=6; Wild Type 2 weeks, n=7; Nlrp3^-/- 2 weeks, n=7; Wild Type 10 weeks, n=7; Nlrp3^-/-10 weeks, n=7. Data are representative of 8 individual experiments.

Figure 8. NLRP3 does not contribute to HDM mediated airway hyperreactivity

A) Airway Resistance (Raw) and B) tissue damping (G), in response to methacholine (MCh), was evaluated in wild type and Nlrp3^-/- mice that were subjected to sub-chronic (2 weeks) of HDM administration. Wild Type, n=37; Nlrp3^-/-, n=30. Data shown was combined from 3 individual experiments.

Figure 9. NLRP3 is not required for the development of OVA-alum driven airway inflammation in 129SvEv mice

A. BALF cellularity from wild type and Nlrp3^-/- mice was evaluated following OVA-alum immunization and i.n. OVA challenge. B. BALF cellularity was evaluated via cellular morphology following differential staining. C. Lung histopathology was evaluated and scored. D. H&E stained sections were evaluated and found to have significant levels of inflammation. E. Mucus levels were quantified along the main bronchiole (represented as Vs). F. AB/PAS staining was used to quantify mucus production. Data shown are representative images of AB/PAS+ cells. G-H. BALF levels of IL-13 and serum levels of OVA specific IgE were assessed by ELISA. Wild Type Saline, n=4; Nlrp3^-/- Saline, n=3; Wild Type OVA-alum, n=8; Nlrp3^-/- OVA-alum, n=4. Data are representative of 2 individual experiments.