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Abstract
Rationale: Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome and concomitant IL-1β responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma.
Objectives: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1β in severe, steroid-resistant asthma.
Methods: We developed mouse models of Chlamydia and Haemophilus respiratory infection–mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1β responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1β responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti–IL-1β antibody. Roles for IL-1β–induced neutrophilic inflammation were examined using IL-1β and anti-Ly6G.
Measurements and Main Results: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1β responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1β expression. Treatment with anti–IL-1β, Ac-YVAD-cho, and MCC950 suppressed IL-1β responses and the important steroid-resistant features of disease in mice, whereas IL-1β administration recapitulated these features. Neutrophil depletion suppressed IL-1β–induced steroid-resistant airway hyperresponsiveness.
Conclusions: NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.
Severe, steroid-resistant asthma is the major unmet need in asthma management, and improved therapies are urgently required. The development of effective therapies has been hampered by a poor understanding of the mechanisms that drive severe, steroid-resistant forms of asthma, the heterogeneity of disease in humans, and lack of representative preclinical models to increase understanding and test drugs.
We show that nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3), caspase-1, and IL-1β responses are increased in novel experimental models of infection-induced severe, steroid-resistant asthma that are representative of neutrophilic, severe, steroid-resistant asthma in humans. Increased NLRP3 and IL-1β responses correlate with increased neutrophil numbers and severity of airflow obstruction and reduced disease control in patients with asthma taking inhaled steroids. We also show that therapeutically targeting NLRP3, caspase-1, and IL-1β responses reduces IL-1β production, steroid-resistant neutrophilic inflammation, and airway hyperresponsiveness in experimental disease. Administration of IL-1β induces the cardinal features of severe, steroid-resistant neutrophilic asthma. These findings demonstrate, for the first time, novel roles for NLRP3-dependent, caspase-1–mediated, IL-1β responses in the pathogenesis of severe, steroid-resistant asthma and highlight that these responses may be targeted therapeutically for the treatment of this disease.
Mild-to-moderate asthma is typically T-helper cell type 2 (Th2) lymphocyte-associated with eosinophilic airway inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR) (1–4). These patients with asthma respond well to corticosteroid-based therapies that control their symptoms. However, asthma is a heterogeneous disease with moderate-to-severe forms more associated with Th1/Th17–mediated responses and monocytic or neutrophilic airway inflammation (2, 5–8). Between 5% and 20% of people with asthma, mostly those with more severe asthma, respond poorly to corticosteroids even at high doses (2, 7, 9, 10). There are currently no effective treatments for severe, steroid-resistant (SSR) asthma and these patients account for 50–80% of health care costs (2, 7). The development of therapies is hampered by the lack of understanding of the mechanisms of pathogenesis (2, 7).
Inflammasomes are multiprotein signaling complexes and the nucleotide-binding oligomerization domain–like receptor (NLR) family, pyrin domain–containing 3 (NLRP3) inflammasome is the most widely characterized and implicated in inflammatory diseases (11). It is comprised of NLRP3, apoptosis-associated speck–like protein containing a pyrin domain and CARD and procaspase-1 domains (12). The assembly of inflammasome complexes is induced by pathogen-associated molecular patterns, including LPS and double-stranded DNA (12). Inflammasomes are then activated by damage-associated molecular patterns, such as ATP and monosodium urate crystals (11, 12). This results in the cleavage and activation of procaspase-1, which in turn cleaves and activates pro–IL-1β and pro–IL-18 into the mature and active proinflammatory cytokines IL-1β and IL-18 (12).
Increasing clinical and experimental evidence specifically implicates NLRP3 inflammasome activation and IL-1β responses in the pathogenesis of severe neutrophilic, compared with mild–moderate, steroid-sensitive, asthma (11, 13–15). NLRP3 and caspase-1 levels are increased in the airways in neutrophilic asthma (15). Furthermore, IL-1β–related genes are overexpressed in these patients and increased IL-1β signaling is a marker of more severe disease (13). IL-1β promotes Th17 differentiation and IL-17 production that induces steroid-resistant neutrophilic inflammation and AHR (14, 16, 17).
Clinical studies link Chlamydia and Haemophilus respiratory infections with SSR asthma. Chlamydia-associated asthma is less responsive to steroids and people with asthma with elevated antibodies to Chlamydia have increased sputum neutrophils (18–20). Haemophilus influenzae is commonly isolated from the airways of patients with severe asthma, 60% of patients with stable asthma with high airway bacterial load were culture-positive for this bacterium, and these patients were more likely to be taking high doses of inhaled steroids (21). Both infections induce NLRP3 inflammasome–mediated caspase-1 and IL-1β responses (22, 23).
We hypothesized that infection-induced, NLRP3 inflammasome–mediated IL-1β responses are important in the pathogenesis of SSR asthma. To test this we used experimental models of Chlamydia and Haemophilus infection–induced SSR allergic airway disease (AAD) (24, 25) and clinical studies to define previously unrecognized roles for NLRP3 inflammasome–mediated IL-1β responses in the pathogenic mechanisms of SSR asthma. We identify the NLRP3 inflammasome as a therapeutic target.
All procedures were performed with approval from the University of Newcastle Human/Animal Ethics Committees.
Murine models of ovalbumin (Ova)-induced AAD and Chlamydia and Haemophilus respiratory infections were combined to induce SSRAAD as previously described (24, 25). NLRP3, caspase-1, and IL-1β responses were assessed in lung tissues by immunoblot, ELISA, real-time quantitative polymerase chain reaction, or immunofluorescence staining and the effects of steroid (dexamethasone [DEX]), and NLRP3, caspase-1, IL-1β, and Ly6G inhibitor and recombinant mouse (rm) IL-1β treatments on inflammatory cell influx into bronchoalveolar lavage fluid (BALF) and lung function in SSRAAD were determined as previously described (24–33) and/or as in the online supplement.
Statistical analyses of mRNA expression of NLRP3 and IL-1β in sputum with clinical parameters in asthma were performed on existing data (13). Gene expression was normalized to the transcript levels of the housekeeping gene β-actin and the mean of all samples (2−ΔΔCt). Correlations with the proportions and absolute numbers of neutrophils in sputum, Asthma Control Questionnaire score, inhaled corticosteroid (ICS) dose, and FEV1 % predicted were investigated. A comprehensive description of patient details and methods used has been given by Baines and coworkers (13) and appears in the online supplement.
Comparisons between two groups used unpaired Student’s t tests or a nonparametric equivalent where appropriate. Comparisons between multiple groups used a one-way analysis of variance and an appropriate post-test or a nonparametric equivalent where appropriate. Lung function data were assessed using a two-way analysis of variance and an appropriate post-test or a nonparametric equivalent. Correlation analyses of sputum data were made using Spearman rank correlation. Analyses were performed using GraphPad Prism Software (San Diego, CA). All data are representative of individual mice. No data have been pooled.
Acute Ova-induced AAD was established in wild-type BALB/c mice, which were then infected with Chlamydia muridarum or nontypeable H. influenzae (see Figure E1 in the online supplement) (24, 25). The infections peak between 5 and 10 days, and both clear before 20 days (27, 29). The phenotype of AAD wanes over time (unpublished observations); therefore, we recapitulated the phenotype with two additional Ova challenges (Days 33–34). This represents the human scenario of repeated allergen exposures in an individual with asthma and allows the examination of the effect of a resolved respiratory infection on established AAD. Key disease features were assessed on Day 35 with or without treatment with the corticosteroid, DEX (Days 32–34) (24, 25, 27–29, 31, 32, 34–36).
The induction of AAD (Ova) resulted in eosinophilic airway inflammation and AHR compared with nonallergic (Sal) control subjects (Figures 1A–1G). Infection in AAD suppressed eosinophilic, and increased neutrophilic, airway inflammation but did not affect the magnitude of AHR (Ova/Cmu) compared with AAD. This recapitulates a phenotype of severe neutrophilic asthma. Nonallergic, infected (Sal/Cmu) groups had similar airway inflammation and AHR to sham-infected (Sal) control groups at this time point (i.e., immediately after OVA rechallenge in other groups, Day 35), suggesting that Chlamydia-induced neutrophilic AAD results from an altered phenotype of AAD rather than the additive effects of infection and AAD. DEX treatment suppressed airway inflammation and AHR in AAD (Ova/DEX) compared with untreated (Ova) control subjects to baseline levels observed in nonallergic, sham-infected (Sal) groups (Figures 1A–1G). Significantly, in infected groups with AAD (Ova/Cmu/DEX), airway inflammation and AHR were completely steroid resistant. We observed similar effects when mice were infected with nontypeable H. influenzae during AAD (see Figure E2). These data demonstrate that Chlamydia and Haemophilus respiratory infections in AAD induce SSRAAD, recapitulating the phenotype of SSR asthma.
Figure 1. Chlamydia infection induces severe, steroid-resistant, neutrophilic allergic airway disease (AAD) that is associated with increased IL-1β, caspase 1 (CASP1), and nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) responses. (A–E) Total leukocytes (A), eosinophils (B), neutrophils (C), macrophages (D), and lymphocytes (E) were enumerated in bronchoalveolar lavage fluid (BALF) on Day 35 of the study protocol (see Figure E1) in Chlamydia (Cmu)- and sham (SPG)-infected groups with ovalbumin (Ova)-induced AAD with or without steroid (dexamethasone) treatment compared with nonallergic control subjects (Sal). (F and G) Airway hyperresponsiveness in terms of airway resistance in response to increasing doses of methacholine (Mch) (F), and 10 mg/ml of Mch (G; shows statistics at maximal dose from airway hyperresponsiveness curves [F]) was also determined in all groups on Day 35 (≥2 experiments; n = 5–8). (H) Lung mRNA expression of Il-1b and Casp1 on Day 30 and Day 35 of the study protocol in Chlamydia (Cmu)- and sham (SPG)-infected, allergic groups. (I) Lung cytoplasmic protein levels of pro-CASP1 (45 kD), CASP1 (10 kD), and CASP1/pro-CASP1 ratio normalized to β-actin (ACTB; 42 kD) were determined on Day 35 in Cmu- and sham-infected groups after Ova-induced AAD by quantification of immunoblot (see Figure E4) by densitometry (one experiment; n = 6). (J and K) Representative photomicrographs of NLRP3 immunofluorescence (Alexa Fluor 488 with Hoechst 33342 nuclear counterstain) in lung histologic sections (J) and BALF leukocytes (K) on Day 35. Arrows in histologic photomicrographs indicate NLRP3+ epithelial cells in the airway lumen. Scale bars = 50 μm. (L and M) NLRP3+ leukocytes in BALF were enumerated (L) and graded (M) (formula in Figure E5) to determine mean expression index (n = 3). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DEX = dexamethasone; Rn = airway resistance; SPG = sucrose phosphate glutamate.
[More] [Minimize]To investigate the role of IL-1β in SSR asthma, we assessed the levels of IL-1β and caspase-1 mRNA expression in whole lungs in SSRAAD (Figure 1H) (26). Groups with SSRAAD (Ova/Cmu) had increased Il-1b and Casp1 expression on Day 30 (before Ova rechallenge), but only Il-1b on Day 35 (after rechallenge), compared with those with AAD (Ova). Thus, Chlamydia infection increases Il-1b and Casp1 expression in SSRAAD.
We next examined whether SSRAAD was associated with increased levels of caspase-1 (10 kD) that results from proteolytic cleavage and activation of procaspase-1 (45 kD). Cytoplasmic fractions from lung lysates had increased caspase-1 and decreased procaspase-1 levels in SSRAAD compared with AAD (Figure 1I; see Figure E4).
We then assessed the levels of NLRP3 in lung sections and BALF. We found that infection induced NLRP3 expression in airway epithelial and infiltrating immune cells in SSRAAD (Figure 1J). There were corresponding increases in the numbers of NLRP3+ leukocytes in BALF and levels of NLRP3 in these cells (Figures 1K–1M; see Figure E5). These data show that factors associated with NLRP3 inflammasome responses are increased in SSRAAD.
We next assessed the links between increasing asthma severity, steroid responsiveness, neutrophilic airway inflammation, and NLRP3 activity. We measured the levels of mRNA expression of NLRP3 and IL-1β in the sputum of subjects with asthma with mild, steroid-sensitive through to SSR disease and with diverse sputum inflammatory profiles (cohort described in online supplement and Reference 13). Both the proportion (r = 0.40 [P < 0.0001] and r = 0.51 [P < 0.0001], respectively) (Figures 2A and 2B) and absolute numbers (r = 0.48 [P = 0.0001] and r = 0.51 [P < 0.0001], respectively) (Figures 2C and 2D) of neutrophils strongly and positively correlated with increased expression of NLRP3 and IL-1β mRNA. Furthermore, worsened Asthma Control Questionnaire score (r = 0.35 [P = 0.0003] and r = 0.32 [P = 0.0010], respectively) (Figures 2E and 2F) positively correlated, and lower FEV1 % predicted (r = −0.27 [P = 0.0059] and r = −0.22 [P = 0.0252]) (Figures 2G and 2H) negatively correlated with NLRP3 and IL-1β mRNA expression. This was despite ongoing treatment with ICS. These data show that increased lung expression of NLRP3 and IL-1β mRNA are linked with key clinical parameters of SSR asthma, specifically increased neutrophilic airway inflammation, and decreased asthma control and lung function.
Figure 2. Neutrophilic airway inflammation and disease severity correlate with nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) and IL-1β expression in human asthma despite ongoing treatment with inhaled corticosteroids. (A–H) Neutrophil proportion (%) (A and B) and absolute number (per ml) (C and D), Asthma Control Questionnaire score (E and F), and FEV1 % predicted (G and H) were correlated with sputum (mRNA) expression of NLRP3 and IL-1β in a population of stable subjects with asthma (n = 104, described previously [13]). Associations for each comparison are expressed as Spearman rank correlation coefficient (Spearman rho; r). (I and J) mRNA expression of NLRP3 and IL-1β in patients with severe asthma compared with mild asthma. Data are represented as the median with error bars as interquartile range. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ACQ = Asthma Control Questionnaire.
[More] [Minimize]We also show that both NLRP3 (P = 0.004) and IL-1β (P < 0.001) mRNA expression is higher in patients with severe asthma compared with mild asthma (Figures 2I and 2J). NLRP3 gene expression is higher in patients taking ICS, whether the dose is low (<1,000 μg daily beclomethasone dipropionate equivalent) or high (≥1,000 μg daily beclomethasone dipropionate equivalent) (P = 0.0009) (no ICS, n = 24, 0.42 [0.19–0.84]; low ICS, n = 26, 1.34 [0.51–2.63]; high ICS, n = 52, 1.35 [0.64–3.33]). IL-1β gene expression is higher in patients taking high doses of ICS (P = 0.002) (no ICS, n = 24, 0.40 [0.17–1.15]; low ICS, n = 26, 0.90 [0.41–3.32]; high ICS, n = 52, 1.39 [0.55–5.38]). Although we show that NLRP3 (P = 0.0001) and IL-1β (P = 0.0002) gene expression is higher in patients taking ICS (n = 80) compared with those not taking ICS (n = 24), neither NLRP3 (r = 0.08; P = 0.499), IL-1β (r = 0.09; P = 0.433), nor neutrophils (neutrophil % r = −0.01, P = 0.907; neutrophil number r = 0.005, P = 0.968) correlate with ICS dose. Together, these data show that, NLRP3, IL-1β, and neutrophil responses are increased in patients with severe asthma who are taking high-dose ICS treatment. Although these markers are higher in patients taking ICS, there is no correlation between ICS dose and NLRP3, IL-1β expression, or neutrophils, suggesting that asthma severity is driving the association rather than ICS treatment. Notably, our murine models of SSR asthma (Figures 1, 3–6; see Figures E1–E3) accurately replicate these associations. Thus, we next systematically interrogated the temporal association between excessive NLRP3 inflammasome–mediated IL-1β responses, neutrophilic airway inflammation, and SSR asthma using our models.
Figure 3. Inhibition of IL-1β suppresses cardinal features of Chlamydia-induced severe, steroid-resistant allergic airway disease. (A) Lung IL-1β protein levels were assessed by ELISA on Day 35 of the study protocol (see Figure E1) in Chlamydia- and sham (SPG)-infected groups with ovalbumin-induced allergic airway disease, with or without steroid (dexamethasone) or anti(α)-IL-1β or isotype antibody treatment (two experiments; n = 4–5). (B–F) Total leukocytes (B), eosinophils (C), neutrophils (D), macrophages (E), and lymphocytes (F) were enumerated in bronchoalveolar lavage fluid on Day 35. (G and H) Airway hyperresponsiveness in terms of airway resistance (Rn) in response to increasing doses of methacholine (Mch) (G) and 10 mg/ml of Mch (H; shows statistics at maximal dose from airway hyperresponsiveness curves [G]) was also determined in all allergic groups on Day 35 (two experiments; n = 5–12). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. BALF = bronchoalveolar lavage fluid; Cmu = Chlamydia; DEX = dexamethasone; Ova = ovalbumin; SPG = sucrose phosphate glutamate.
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Figure 4. Treatment with the pan-caspase inhibitor z-VAD-fmk (ZVAD) suppresses airway hyperresponsiveness (AHR) in Chlamydia-induced severe, steroid-resistant allergic airway disease. (A) Lung IL-1β protein levels were assessed by ELISA on Day 35 of the study protocol (see Figure E1) in Chlamydia (Cmu)- and sham (SPG)-infected groups with ovalbumin (Ova)-induced allergic airway disease, with or without steroid (dexamethasone [DEX]) or z-VAD-fmk or dimethyl sulfoxide (vehicle) treatment (one experiment; n = 5–6). (B) Total leukocytes, (C) eosinophils, (D) neutrophils, (E) macrophages, and (F) lymphocytes were enumerated in bronchoalveolar lavage fluid on Day 35. (G and H) AHR in terms of airway resistance in response to increasing doses of methacholine (Mch) (G) and 10 mg/ml of Mch (H; shows statistics at maximal dose from AHR curves [G]) was also determined in all allergic groups on Day 35. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The data for the four control groups, Ova ± DEX and Ova/Cmu ± DEX, are repeated in Figures 4–6 and Figure E6 (all experiments were performed concurrently, ≥2 experiments; n = 5–15). BALF = bronchoalveolar lavage fluid; Rn = airway resistance; SPG = sucrose phosphate glutamate.
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Figure 5. Inhibition of caspase 1 suppresses IL-1β levels and cardinal features of Chlamydia-induced severe, steroid-resistant allergic airway disease. (A) Lung IL-1β protein levels were assessed by ELISA on Day 35 of the study protocol (see Figure E1) in Chlamydia (Cmu)- and sham (SPG)-infected groups with ovalbumin (Ova)-induced allergic airway disease, with or without steroid (dexamethasone [DEX]) or Ac-YVAD-cho or dimethyl sulfoxide (vehicle) treatment (two experiments; n = 6). (B) Total leukocytes, (C) eosinophils, (D) neutrophils, (E) macrophages, and (F) lymphocytes were enumerated in bronchoalveolar lavage fluid on Day 35. (G and H) Airway hyperresponsiveness in terms of airway resistance in response to increasing doses of methacholine (Mch) (G) and 10 mg/ml of Mch (H; shows statistics at maximal dose from airway hyperresponsiveness curves [G]) was also determined in all allergic groups on Day 35. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The data for the four control groups, Ova ± DEX and Ova/Cmu ± DEX, are repeated in Figures 4–6 and Figure E6 (all experiments were performed concurrently, ≥2 experiments; n = 5–15). BALF = bronchoalveolar lavage fluid; Rn = airway resistance; SPG = sucrose phosphate glutamate.
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Figure 6. Treatment with a highly selective nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 inhibitor MCC950 suppresses cardinal features of Chlamydia-induced severe, steroid-resistant allergic airway disease. (A) Lung IL-1β protein levels were assessed by ELISA on Day 35 of the study protocol (see Figure E1) in Chlamydia (Cmu)- and sham (SPG)-infected groups with ovalbumin (Ova)-induced allergic airway disease, with or without steroid (dexamethasone [DEX]) or MCC950 or dimethyl sulfoxide (vehicle) treatment (two experiments; n = 5–6). (B) Total leukocytes, (C) eosinophils, (D) neutrophils, (E) macrophages, and (F) lymphocytes were enumerated in bronchoalveolar lavage fluid (BALF) on Day 35. (G and H) Airway hyperresponsiveness in terms of airway resistance in response to increasing doses of methacholine (Mch) (G) and 10 mg/ml of Mch (H; shows statistics at maximal dose from airway hyperresponsiveness curves [G]) was also determined in all allergic groups on Day 35. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The data for the four control groups, Ova ± DEX and Ova/Cmu ± DEX, are repeated in Figures 4–6 and Figure E6 (all experiments were performed concurrently, ≥2 experiments; n = 5–15). Rn = airway resistance; SPG = sucrose phosphate glutamate.
[More] [Minimize]We first examined the role of IL-1β in Chlamydia-induced SSRAAD. Groups with SSRAAD (Ova/Cmu) had increased lung IL-1β protein levels compared with AAD (Ova) control groups (Figure 3A). DEX treatment had no effect on IL-1β levels or key features of SSRAAD (Ova/Cmu/DEX). Treatment with neutralizing anti–IL-1β antibody (α-IL-1β) (see Figure E1A) inhibited the Chlamydia-induced production of excess IL-1β in the lungs in SSRAAD (Ova/Cmu/α-IL-1β vs. Ova/Cmu) to baseline levels observed in AAD (Ova) control subjects (Figure 3A). Neutralization of IL-1β suppressed total airway inflammation and inhibited the excessive influx of neutrophils and macrophages in SSRAAD with numbers returned to those in AAD (Ova; Figures 3B–3F). α-IL-1β also suppressed AHR to levels in steroid-treated AAD (Ova/DEX; Figures 3G and 3H). Similar effects were observed with α-IL-1β treatment in Haemophilus-induced SSRAAD (see Figure E2). These data demonstrate that steroid-resistant airway inflammation and AHR in Chlamydia- and Haemophilus-induced SSRAAD are IL-1β–dependent and that targeting IL-1β may have therapeutic benefit in SSR asthma.
Caspase-1 cleaves and activates pro–IL-1β into mature IL-1β (12). Caspase-1 is increased in severe asthma (15) and we show similar increases in SSRAAD (Figure 1I; see Figure E4). Again, lung IL-1β levels were elevated and steroid treatment was not effective in SSRAAD (Figures 4A–4H). Treatment with the pan-caspase inhibitor, z-VAD-fmk (see Figure E1B), induced a close to statistically significant decrease in Chlamydia-induced lung IL-1β levels, had no effects on airway inflammation, but suppressed AHR in SSRAAD (Ova/Cmu/ZVAD; Figure 4).
We then more specifically inhibited caspase-1 using Ac-YVAD-cho (see Figure E1B). Specific targeting inhibited the Chlamydia-induced production of excess IL-1β in the lungs in SSRAAD (Ova/Cmu/YVAD vs. Ova/Cmu) with levels returned to those in AAD (Ova; Figure 5A). Treatment also suppressed steroid-resistant neutrophilic airway inflammation but had no effect on the numbers of total leukocytes or other infiltrating immune cells (Figures 5B–5F). Treatment also suppressed AHR in SSRAAD to levels comparable with steroid-treated AAD (Ova/DEX; Figure 5G and 5H). Similar effects were observed with treatment in Haemophilus-induced SSRAAD (see Figure E3). These data suggest that caspase-1–mediated activation of IL-1β in the lungs drives key features of Chlamydia- and Haemophilus-induced SSRAAD.
We next assessed the therapeutic potential of direct targeting of the NLRP3 inflammasome with MCC950, a potent (inhibition of NLRP3-driven production of IL-1β IC50 = 7 nM) and highly selective NLRP3 inhibitor we recently developed (37). In optimization studies we showed that low-dose treatment with MCC950 (1 mg/kg) (see Figure E1B) potently suppressed neutrophilic airway inflammation and induced a close to statistically significant decrease in the numbers of total leukocytes and AHR in SSRAAD (see Figure E6). However, low-dose treatment in the presence of DEX (Ova/Cmu/MCC950/DEX) had no additional effects on airway inflammation and DEX reversed the suppressive effect of MCC950 on AHR. These data suggest that treatment with MCC950 is not steroid sparing and that higher doses in the absence of steroids may be a more effective therapeutic strategy.
Higher dose treatment with MCC950 (10 mg/kg) (see Figure E1B) completely inhibited Chlamydia-induced production of excess IL-1β in the lungs in SSRAAD (Ova/Cmu/MCC950 vs. Ova/Cmu) with levels returned to those in AAD (Ova; Figure 6A). Treatment also potently suppressed steroid-resistant airway inflammation and AHR in SSRAAD to levels comparable with steroid-treated AAD (Ova/DEX; Figures 6B–6H). Similar effects were observed with MCC950 treatment in Haemophilus-induced SSRAAD (see Figure E3). These data demonstrate that the key features of Chlamydia- and Haemophilus-induced SSRAAD are induced by an NLRP3 inflammasome–dependent, caspase-1–mediated signaling axis that drives the production of excess IL-1β in the lungs. Treatment with the highly selective NLRP3 inhibitor MCC950 potently suppresses the hallmark features of SSRAAD, and potentially SSR asthma.
To further examine the roles of NLRP3 inflammasome–dependent, IL-1β responses in SSRAAD, we intranasally administered rmIL-1β to naive mice and mice with eosinophil-dominated, steroid-sensitive AAD in the absence and presence of DEX treatment (see Figures E1C and E1D). rmIL-1β administration to the extracellular compartment of the airways and lungs of naive mice resulted in increased numbers of leukocytes, predominantly neutrophils and macrophages, in the airways (Figures 7A–7E) that corresponded with increased AHR (Figures 7F and 7G). Furthermore, rmIL-1β administration during AAD suppressed eosinophils and increased neutrophils in the airways (see Figures E7A–E7E). rmIL-1β–induced inflammation and AHR was completely steroid-resistant in naive mice and during AAD (Figures 7F and 7G; see Figures E7F and E7G). Thus, intranasal administration of rmIL-1β induces the cardinal features of SSRAAD in the absence of infection in naive mice and mice with AAD.
Figure 7. Administration of recombinant IL-1β results in neutrophilic airway inflammation and airway hyperresponsiveness (AHR) that are steroid resistant. (A–E) Total leukocytes (A), eosinophils (B), neutrophils (C), macrophages (D), and lymphocytes (E) were enumerated in bronchoalveolar lavage fluid on Day 4 of the study protocol (see Figure E1C) in naive mice treated with recombinant mouse IL-1β (rmIL-1β) with or without steroid (dexamethasone) and neutrophil-depleting anti-Ly6G (α-Ly6G) treatments compared with vehicle-treated control subjects (phosphate-buffered saline). (F and G) AHR in terms of airway resistance in response to increasing doses of methacholine (Mch) (F) and 10 mg/ml of Mch (G; shows statistics at maximal dose from AHR curves [F]) was also determined in all groups on Day 4. Data are presented as means ± SEM. ***P < 0.001; ****P < 0.0001 (one experiment; n = 4–6). BALF = bronchoalveolar lavage fluid; DEX = dexamethasone; PBS = phosphate-buffered saline; Rn = airway resistance.
[More] [Minimize]To examine the potential role of neutrophil responses in IL-1β–induced SSRAAD, we intraperitoneally administered anti-Ly6G to naive mice treated with rmIL-1β (see Figure E1C). We found that neutrophil depletion suppressed steroid-resistant inflammation and AHR (Figures 7A–7G). These data demonstrate that the key features of SSRAAD are induced by IL-1β in the absence of infection and that IL-1β–induced neutrophil responses play key roles in the induction of disease features. Finally, we treated mice intranasally with α-IL-1β during AAD (see Figure E1) to show that targeting IL-1β is also capable of suppressing the cardinal features of steroid-sensitive, eosinophilic inflammation and AHR (see Figure E7).
Here, we demonstrate that Chlamydia and Haemophilus respiratory infections both induced steroid-resistant, neutrophil-dominated airway inflammation and AHR and SSRAAD (Figure 1; see Figures E1 and E2) that are associated with increased NLRP3 inflammasome (Figure 6; see Figure E3), caspase-1 (Figure 5; see Figure E3), and IL-1β (Figure 3; see Figure E2) responses in the lungs. We also show that NLRP3 and IL-1β responses correlated with increased neutrophilic inflammatory responses, steroid resistance, and asthma severity in humans (Figure 2). Hence, responses associated with the NLRP3 inflammasome correlate with disease severity, and the disease features of our murine models of SSRAAD replicate those of human SSR asthma. We then used a combination of neutralizing α-IL-1β antibodies, the caspase-1 inhibitor, Ac-YVAD-cho, and a highly specific and potent NLRP3 inflammasome inhibitor, MCC950, to define previously unrecognized roles for an NLRP3/caspase-1/IL-1β signaling axis, and the possibilities for therapeutic use, in infection-induced SSRAAD and potentially SSR asthma (Figure 8). Finally, we show that IL-1β responses can induce the cardinal features of SSRAAD without infection in the absence and presence of AAD, and that IL-1β–induced, steroid-resistant inflammation and AHR are dependent on neutrophil responses.
Figure 8. Mechanisms of pathogenesis and potential treatment of severe, steroid-resistant asthma by inhibition of the nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome. Infection in allergic airway disease/asthma induces NLRP3 inflammasome activity that cleaves pro–caspase 1 (pro-CASP1) into CASP1, which in turn cleaves pro–IL-1β into active IL-1β, causing steroid resistance. This pathway may be targeted therapeutically by the specific inhibition of the NLRP3 inflammasome. AHR = airway hyperresponsiveness; ASC = adaptor protein apoptosis-associated speck-like containing a caspase recruitment domain; CARD = caspase recruitment domain; DAMP = damage-associated molecular patterns; PAMP = pathogen-associated molecular patterns; PYD = pyrin domain; SSR = severe, steroid-resistant.
[More] [Minimize]Our murine models of neutrophil-enriched, SSR asthma bear the hallmark features of neutrophilic asthma, and are associated with increased NLRP3, caspase-1, and IL-1β responses (13, 15). Importantly, we also extend previous clinical observations by showing that increased expression of NLRP3 and IL-1β in induced sputum from a cohort of adults with stable asthma correlate with increasing neutrophilic inflammation and asthma severity (worsened Asthma Control Questionnaire score and decreased lung function) (Figure 2), despite steroid treatment. Thus, our experimental and clinical data indicate for the first time that excessive NLRP3 inflammasome–mediated responses correlate with neutrophilic airway inflammation and SSR asthma.
We then examined the functional contribution and potential for therapeutic targeting of IL-1β in SSRAAD by in vivo inhibition with α-IL-1β. Unlike steroid administration, α-IL-1β treatment inhibited infection-induced IL-1β to levels in AAD (Figure 3A; see Figure E2). IL-1β inhibition also suppressed airway inflammation and AHR in SSRAAD (Figures 3B–3H; see Figure E2). Thus, the key features of infection-induced SSRAAD are IL-1β dependent, and targeting elevated IL-1β in the lungs instead of using steroids may be an effective treatment. These outcomes are consistent with other studies showing that treatment with IL-1R antagonist reduced inflammation and AHR in a murine asthma model (38), and suppressed acute ozone-induced BALF neutrophilia and AHR (39). Anakinra, a recombinant human IL-1R antagonist (12, 40), could be used to inhibit the biologic activity of IL-1β in SSR asthma. However, it is strictly licensed for systemic delivery and its use may increase the frequency of upper respiratory tract infections (40), thereby limiting its therapeutic potential in SSR asthma. In contrast, the specific targeting of NLRP3 to reduce the excessive activation of NLRP3 and NLRP3-specific IL-1β production in SSR asthma will suppress disease while still allowing IL-1β production through other inflammasomes/pathways that can still protect against infection.
Infection also increased Casp1 mRNA expression on Day 30 and protein levels (10 kD) in SSRAAD (Figures 1H and 1I; see Figure E4). Mature caspase-1 proteolytically cleaves and activates pro–IL-1β (41). Here, we showed that treatment with the pan-caspase inhibitor, z-VAD-fmk, suppressed AHR but not airway inflammation in Chlamydia-induced SSRAAD and induced a close to statistically significant decrease in lung IL-1β levels (Figures 4A–4H). This was surprising, because z-VAD-fmk is the most commonly used experimental caspase inhibitor that reportedly inhibits caspase-1–mediated activation of pro–IL-1β (42). z-VAD-fmk exhibits varying specificity against different members of the caspase family and has a short half-life in vivo (43). Thus, it is likely that treatment with z-VAD-fmk targeted a range of caspases and so a reduced level was available to specifically suppress caspase-1. Caspase-8 may have been inhibited, which promotes the apoptosis of epithelial and inflammatory cells during Chlamydia infection (44, 45). Thus, in our model z-VAD-fmk may lack specificity for complete suppression of caspase-1 and, through effects on caspase-8, may have promoted the survival of inflammatory cells in the lungs in SSRAAD. Our findings with z-VAD-fmk do provide further support for our proposal that excess IL-1β promotes steroid-resistant airway inflammation in SSRAAD.
To overcome these limitations, we considered using several tetra-peptide caspase inhibitors that exhibit increased target-specificity compared with tri-peptide formulations, such as z-VAD-fmk. Ac-YVAD-cho is a tetra-peptide inhibitor that potently, selectively, and specifically inhibits caspase-1 (43). Thus, we then assessed the specific role of caspase-1 in infection-induced SSRAAD using Ac-YVAD-cho treatment (43). Treatment inhibited excess IL-1β production in SSRAAD, and suppressed infection-induced, steroid-resistant neutrophilic airway inflammation (Figure 5D; see Figure E3C) and AHR (Figures 5G and 5H; see Figures E3F and E3G). These effects were similar to those with α-IL-1β suggesting that a common pathogenic caspase-1–mediated activation of IL-1β pathway exists that induces SSRAAD. Ac-YVAD-cho did not affect macrophage recruitment, nor did it completely inhibit AHR, suggesting that suppressing other factors in the pathway, such as those upstream like NLRP3, may have greater therapeutic effects.
Thus, we next assessed the contribution and potential for therapeutic targeting of excessive infection-induced, NLRP3 inflammasome–dependent, caspase-1–mediated activation of IL-1β in SSRAAD using the highly selective NLRP3 inflammasome inhibitor, MCC950. MCC950 reduces the cleavage of procaspase-1 and inhibits the release of IL-1β, but not tumor necrosis factor-α, from macrophages activated by inflammasome stimulators (37). However, it does not inhibit IL-1β release from NLRC4 or AIM2 inflammasomes activated with Salmonella or dsDNA. In the current study, treatment with MCC950 (10 mg/kg) reduced lung IL-1β levels, airway inflammation (total leukocytes and neutrophils), and AHR in SSRAAD (Figure 6; see Figure E3). Treatment had similar effects to α-IL-1β and Ac-YVAD-cho, suggesting that the key features of infection-induced SSRAAD are mediated by excessive NLRP3 inflammasome- and caspase-1–dependent activation of IL-1β in the lungs. This also suggests that treatment with α-IL-1β, Ac-YVAD-cho, and MCC950 affect a common pathogenic NLRP3/caspase-1/IL-1β pathway that promotes the key features of both Chlamydia- and Haemophilus-induced SSRAAD.
Importantly, our data demonstrate a direct contribution of an NLRP3 inflammasome/caspase-1/IL-1β axis in driving SSRAAD. Targeting infection-induced NLRP3 inflammasome activation in the lungs to inhibit the excess release of active IL-1β may have therapeutic potential in SSR asthma. This has advantages over targeting IL-1β directly, including the preservation of normal IL-1β activation and function through other inflammasomes/pathways that is required for protection against infection. Furthermore, elevated IL-1β occurs in other steroid-resistant inflammatory conditions, including chronic obstructive pulmonary disease, where it is implicated in inducing neutrophilic airway inflammation, airway remodeling, and emphysema, indicating that IL-1β-targeted therapy could also be effective in this and other inflammatory diseases (23, 46, 47). Importantly, our data demonstrate that inhibition of NLRP3 with MCC950 had more potent effects than inhibiting caspase-1 with Ac-YVAD-cho. Thus, there may be additional therapeutic benefits of inhibiting NLRP3 directly, instead of targeting inflammasome-mediated caspase-1 or IL-1β activity.
Previous studies have linked NLRP3 inflammasome–dependent, IL-1β–mediated, IL-17 responses with neutrophilic airway inflammation and AHR (48), and show that IL-17 responses play an important role in the induction of steroid-resistant disease (17). However, a recent study by Raundhal and coworkers (49) showed that type 1 (IFN-γ) rather than type 17 (IL-17) responses drive steroid-resistant AHR in a house dust mite/Th1/Th17 adjuvant-induced model of SSRAAD. Although we have previously shown that IL-17 plays an important role in Haemophilus-induced SSRAAD (35), the systemic inhibition of IL-17 (local inhibition has not been tested and may be more effective) had no effect on disease phenotype in Chlamydia-induced neutrophil-dominant AAD (28). Future detailed studies that elucidate the roles of Th1 and Th17/IL-17 in SSRAAD and asthma would be valuable.
In the current study, we show that IL-1β responses correlate with increased neutrophilic inflammation and disease severity in patients with asthma (Figure 2), and are increased in two models of infection-induced SSRAAD (Figures 3–6; see Figure E2). Also, therapeutic targeting of NLRP3 and IL-1β responses effectively inhibits infection-induced SSRAAD (Figures 3–6; see Figures E2 and E3), and increasing IL-1β responses in naive and allergic mice without infection induces steroid-resistant neutrophilic inflammation and AHR (Figure 7; see Figure E7). Furthermore, depletion of neutrophils in IL-1β–induced SSRAAD suppresses disease (Figure 7). Collectively this provides strong evidence for mechanistic roles for NLRP3-dependent, IL-1β–mediated, neutrophil responses in the pathogenesis of SSR asthma (Figure 8). Previous studies have also highlighted the role of macrophages in driving steroid-resistant AHR in AAD (30). Our current findings demonstrate for the first time, that IL-1β responses drive steroid-resistant AHR through the induction of neutrophilic inflammatory responses, although macrophages are likely still involved. The precise mechanisms by which IL-1β responses drive neutrophilic inflammation and whether neutrophils directly induce AHR or do this by affecting macrophage responses are yet to be determined in future studies.
Recent evidence suggests that fungi may also play a role in the pathogenesis of SSR asthma (50). Experimentally the administration of LPS and fungal β-glucan during house dust mite–induced AAD induced neutrophilic inflammation, involving Dectin-1 and TLR4, which was steroid resistant (50). It would be interesting to study the role of NLRP3-dependent, IL-1β responses in that context.
We also show that the inhibition of IL-1β results in the suppression of airway inflammation and AHR in steroid-sensitive AAD (see Figure E7). This demonstrates the potential importance of IL-1β–mediated responses in not only the pathogenesis of neutrophil-dominant forms of SSR asthma but also in eosinophilic steroid-sensitive disease. These findings suggest that targeting NLRP3-mediated and/or IL-1β responses in the lung may have widespread efficacy for the treatment of asthma, regardless of severity of disease, steroid responsiveness, or inflammatory phenotype. Together, our findings highlight the complexity of the roles of NLRP3 and IL-1β in the pathogenesis of asthma and provide the impetus for further examination of the role of NLRP3-dependent, IL-1β responses in asthma pathogenesis in future studies.
Although the current study has important ramifications for the role and potential therapeutic targeting of NLRP3 responses in infection-associated SSR asthma, future studies are required to identify and assess the roles of other upstream drivers of NLRP3 inflammasome assembly and activation in the context of SSR asthma. Such studies need to consider that NLRP3 inflammasome-activating stimuli can be derived from many environmental sources and pathophysiologic conditions other than infections, including pollution (51, 52), and obesity (48).
Our study demonstrates for the first time that the NLRP3 inflammasome is important in promoting steroid-resistant airway inflammation and AHR in respiratory infection–induced SSRAAD. We have identified functional roles for an infection-induced NLRP3 inflammasome/caspase-1/IL-1β signaling axis in promoting these disease features. Our data show that therapeutic inhibition of the NLRP3 inflammasome and this signaling axis with MCC950 may be effective in SSR asthma in humans (Figure 8), and is likely to be more beneficial than inhibition of caspase-1 or global targeting of IL-1β. Targeting the NLRP3 inflammasome/caspase-1/IL-1β signaling axis with compounds, such as MCC950, may also potentially be applicable to the treatment of other steroid-resistant inflammatory conditions.
| 1. | Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 2006;355:2226–2235. |
| 2. | Hansbro PM, Scott GV, Essilfie AT, Kim RY, Starkey MR, Nguyen DH, Allen PD, Kaiko GE, Yang M, Horvat JC, et al. Th2 cytokine antagonists: potential treatments for severe asthma. Expert Opin Investig Drugs 2013;22:49–69. |
| 3. | Kim HY, DeKruyff RH, Umetsu DT. The many paths to asthma: phenotype shaped by innate and adaptive immunity. Nat Immunol 2010;11:577–584. |
| 4. | Thorburn AN, Hansbro PM. Harnessing regulatory T cells to suppress asthma: from potential to therapy. Am J Respir Cell Mol Biol 2010;43:511–519. |
| 5. | Fajt ML, Wenzel SE. Asthma phenotypes and the use of biologic medications in asthma and allergic disease: the next steps toward personalized care. J Allergy Clin Immunol 2015;135:299–310, quiz 311. |
| 6. | Simpson JL, Scott R, Boyle MJ, Gibson PG. Inflammatory subtypes in asthma: assessment and identification using induced sputum. Respirology 2006;11:54–61. |
| 7. | Wang W, Li JJ, Foster PS, Hansbro PM, Yang M. Potential therapeutic targets for steroid-resistant asthma. Curr Drug Targets 2010;11:957–970. |
| 8. | Yang M, Kumar RK, Hansbro PM, Foster PS. Emerging roles of pulmonary macrophages in driving the development of severe asthma. J Leukoc Biol 2012;91:557–569. |
| 9. | Barnes PJ, Adcock IM. Glucocorticoid resistance in inflammatory diseases. Lancet 2009;373:1905–1917. |
| 10. | Chung KF, Godard P, Adelroth E, Ayres J, Barnes N, Barnes P, Bel E, Burney P, Chanez P, Connett G, et al.; European Respiratory Society. Difficult/therapy-resistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. ERS Task Force on Difficult/Therapy-Resistant Asthma. Eur Respir J 1999;13:1198–1208. |
| 11. | Kim RY, Pinkerton JW, Gibson PG, Cooper MA, Horvat JC, Hansbro PM. Inflammasomes in COPD and neutrophilic asthma. Thorax 2015;70:1199–1201. |
| 12. | Schroder K, Tschopp J. The inflammasomes. Cell 2010;140:821–832. |
| 13. | Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Transcriptional phenotypes of asthma defined by gene expression profiling of induced sputum samples. J Allergy Clin Immunol 2011;127:153–160. |
| 14. | Hastie AT, Moore WC, Meyers DA, Vestal PL, Li H, Peters SP, Bleecker ER; National Heart Lung, Blood Institute Severe Asthma Research P. Analyses of asthma severity phenotypes and inflammatory proteins in subjects stratified by sputum granulocytes. J Allergy Clin Immunol 2010;125:1028–1036. |
| 15. | Simpson JL, Phipps S, Baines KJ, Oreo KM, Gunawardhana L, Gibson PG. Elevated expression of the NLRP3 inflammasome in neutrophilic asthma. Eur Respir J 2014;43:1067–1076. |
| 16. | Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, Ma L, Watowich SS, Jetten AM, Tian Q, et al. Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 2009;30:576–587. |
| 17. | McKinley L, Alcorn JF, Peterson A, Dupont RB, Kapadia S, Logar A, Henry A, Irvin CG, Piganelli JD, Ray A, et al. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol 2008;181:4089–4097. |
| 18. | Cho YS, Kim TB, Lee TH, Moon KA, Lee J, Kim YK, Lee KY, Moon HB. Chlamydia pneumoniae infection enhances cellular proliferation and reduces steroid responsiveness of human peripheral blood mononuclear cells via a tumor necrosis factor-alpha-dependent pathway. Clin Exp Allergy 2005;35:1625–1631. |
| 19. | Patel KK, Vicencio AG, Du Z, Tsirilakis K, Salva PS, Webley WC. Infectious Chlamydia pneumoniae is associated with elevated interleukin-8 and airway neutrophilia in children with refractory asthma. Pediatr Infect Dis J 2010;29:1093–1098. |
| 20. | Wark PA, Johnston SL, Simpson JL, Hensley MJ, Gibson PG. Chlamydia pneumoniae immunoglobulin A reactivation and airway inflammation in acute asthma. Eur Respir J 2002;20:834–840. |
| 21. | Wood LG, Simpson JL, Hansbro PM, Gibson PG. Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia. Free Radic Res 2010;44:146–154. |
| 22. | He X, Mekasha S, Mavrogiorgos N, Fitzgerald KA, Lien E, Ingalls RR. Inflammation and fibrosis during Chlamydia pneumoniae infection is regulated by IL-1 and the NLRP3/ASC inflammasome. J Immunol 2010;184:5743–5754. |
| 23. | Rotta Detto Loria J, Rohmann K, Droemann D, Kujath P, Rupp J, Goldmann T, Dalhoff K. Nontypeable Haemophilus influenzae infection upregulates the NLRP3 inflammasome and leads to caspase-1-dependent secretion of interleukin-1β: a possible pathway of exacerbations in COPD. PLoS One 2013;8:e66818. |
| 24. | Essilfie AT, Horvat JC, Kim RY, Mayall JR, Pinkerton JW, Beckett EL, Starkey MR, Simpson JL, Foster PS, Gibson PG, et al. Macrolide therapy suppresses key features of experimental steroid-sensitive and steroid-insensitive asthma. Thorax 2015;70:458–467. |
| 25. | Kim RY, Horvat JC, Pinkerton JW, Starkey MR, Essilfie AT, Mayall JR, Nair PM, Hansbro NG, Jones B, Haw TJ, et al. MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying phosphoinositide 3-kinase-mediated suppression of histone deacetylase 2. J Allergy Clin Immunol 2017;139:519–532. |
| 26. | Beckett EL, Stevens RL, Jarnicki AG, Kim RY, Hanish I, Hansbro NG, Deane A, Keely S, Horvat JC, Yang M, et al. A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis. J Allergy Clin Immunol 2013;131:752–762. |
| 27. | Horvat JC, Beagley KW, Wade MA, Preston JA, Hansbro NG, Hickey DK, Kaiko GE, Gibson PG, Foster PS, Hansbro PM. Neonatal chlamydial infection induces mixed T-cell responses that drive allergic airway disease. Am J Respir Crit Care Med 2007;176:556–564. |
| 28. | Horvat JC, Starkey MR, Kim RY, Beagley KW, Preston JA, Gibson PG, Foster PS, Hansbro PM. Chlamydial respiratory infection during allergen sensitization drives neutrophilic allergic airways disease. J Immunol 2010;184:4159–4169. |
| 29. | Horvat JC, Starkey MR, Kim RY, Phipps S, Gibson PG, Beagley KW, Foster PS, Hansbro PM. Early-life chlamydial lung infection enhances allergic airways disease through age-dependent differences in immunopathology. J Allergy Clin Immunol 2010;125:617–625. |
| 30. | Li JJ, Wang W, Baines KJ, Bowden NA, Hansbro PM, Gibson PG, Kumar RK, Foster PS, Yang M. IL-27/IFN-γ induce MyD88-dependent steroid-resistant airway hyperresponsiveness by inhibiting glucocorticoid signaling in macrophages. J Immunol 2010;185:4401–4409. |
| 31. | Starkey MR, Essilfie AT, Horvat JC, Kim RY, Nguyen DH, Beagley KW, Mattes J, Foster PS, Hansbro PM. Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease. Mucosal Immunol 2013;6:569–579. |
| 32. | Starkey MR, Kim RY, Beckett EL, Schilter HC, Shim D, Essilfie AT, Nguyen DH, Beagley KW, Mattes J, Mackay CR, et al. Chlamydia muridarum lung infection in infants alters hematopoietic cells to promote allergic airway disease in mice. PLoS One 2012;7:e42588. |
| 33. | Starkey MR, Nguyen DH, Essilfie AT, Kim RY, Hatchwell LM, Collison AM, Yagita H, Foster PS, Horvat JC, Mattes J, et al. Tumor necrosis factor-related apoptosis-inducing ligand translates neonatal respiratory infection into chronic lung disease. Mucosal Immunol 2014;7:478–488. |
| 34. | Essilfie AT, Simpson JL, Dunkley ML, Morgan LC, Oliver BG, Gibson PG, Foster PS, Hansbro PM. Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma. Thorax 2012;67:588–599. |
| 35. | Essilfie AT, Simpson JL, Horvat JC, Preston JA, Dunkley ML, Foster PS, Gibson PG, Hansbro PM. Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease. PLoS Pathog 2011;7:e1002244. |
| 36. | Thorburn AN, Foster PS, Gibson PG, Hansbro PM. Components of Streptococcus pneumoniae suppress allergic airways disease and NKT cells by inducing regulatory T cells. J Immunol 2012;188:4611–4620. |
| 37. | Coll RC, Robertson AA, Chae JJ, Higgins SC, Muñoz-Planillo R, Inserra MC, Vetter I, Dungan LS, Monks BG, Stutz A, et al. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med 2015;21:248–255. |
| 38. | Wang CC, Fu CL, Yang YH, Lo YC, Wang LC, Chuang YH, Chang DM, Chiang BL. Adenovirus expressing interleukin-1 receptor antagonist alleviates allergic airway inflammation in a murine model of asthma. Gene Ther 2006;13:1414–1421. |
| 39. | Park JW, Taube C, Swasey C, Kodama T, Joetham A, Balhorn A, Takeda K, Miyahara N, Allen CB, Dakhama A, et al. Interleukin-1 receptor antagonist attenuates airway hyperresponsiveness following exposure to ozone. Am J Respir Cell Mol Biol 2004;30:830–836. |
| 40. | Salvana EM, Salata RA. Infectious complications associated with monoclonal antibodies and related small molecules. Clin Microbiol Rev 2009;22:274–290. |
| 41. | Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J, et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992;356:768–774. |
| 42. | Lipinska K, Malone KE, Moerland M, Kluft C. Applying caspase-1 inhibitors for inflammasome assays in human whole blood. J Immunol Methods 2014;411:66–69. |
| 43. | Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, Thornberry NA. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 1998;273:32608–32613. |
| 44. | Vats V, Agrawal T, Salhan S, Mittal A. Characterization of apoptotic activities during chlamydia trachomatis infection in primary cervical epithelial cells. Immunol Invest 2010;39:674–687. |
| 45. | Wang Z, Watt W, Brooks NA, Harris MS, Urban J, Boatman D, McMillan M, Kahn M, Heinrikson RL, Finzel BC, et al. Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors. Biochim Biophys Acta 2010;1804:1817–1831. |
| 46. | Churg A, Zhou S, Wang X, Wang R, Wright JL. The role of interleukin-1beta in murine cigarette smoke-induced emphysema and small airway remodeling. Am J Respir Cell Mol Biol 2009;40:482–490. |
| 47. | Gibson PG, Simpson JL. The overlap syndrome of asthma and COPD: what are its features and how important is it? Thorax 2009;64:728–735. |
| 48. | Kim HY, Lee HJ, Chang YJ, Pichavant M, Shore SA, Fitzgerald KA, Iwakura Y, Israel E, Bolger K, Faul J, et al. Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity. Nat Med 2014;20:54–61. |
| 49. | Raundhal M, Morse C, Khare A, Oriss TB, Milosevic J, Trudeau J, Huff R, Pilewski J, Holguin F, Kolls J, et al. High IFN-γ and low SLPI mark severe asthma in mice and humans. J Clin Invest 2015;125:3037–3050. |
| 50. | Hadebe S, Kirstein F, Fierens K, Chen K, Drummond RA, Vautier S, Sajaniemi S, Murray G, Williams DL, Redelinghuys P, et al. Microbial ligand costimulation drives neutrophilic steroid-refractory asthma. PLoS One 2015;10:e0134219. |
| 51. | Gold MJ, Hiebert PR, Park HY, Stefanowicz D, Le A, Starkey MR, Deane A, Brown AC, Liu G, Horvat JC, et al. Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization. Mucosal Immunol 2016;9:809–820. |
| 52. | Hirota JA, Gold MJ, Hiebert PR, Parkinson LG, Wee T, Smith D, Hansbro PM, Carlsten C, VanEeden S, Sin DD, et al. The nucleotide-binding domain, leucine-rich repeat protein 3 inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under 10 μm. Am J Respir Cell Mol Biol 2015;52:96–105. |
*These authors contributed equally to this manuscript.
Supported by grants and fellowships from the National Health and Medical Research Council of Australia, the University of Newcastle, and the Hunter Medical Research Institute.
Author Contributions: R.Y.K., J.W.P., J.C.H., and P.M.H. designed and performed experiments and analyzed the data. R.Y.K., L.A.J.O’N., M.A.C., J.C.H., and P.M.H. conceptualized studies and wrote the manuscript. K.J.B., L.G.W., J.L.S., and P.G.G. analyzed clinical data. A.T.E., A.C.B., M.K.A., and J.R.M. assisted in performing experiments. A.A.B.R. synthesized MCC950. M.R.S. and N.G.H. helped analyze data and edited the manuscript. J.A.H., D.A.K., P.A.W., and L.A.J.O’N. helped interpret data, and edited and revised the manuscript. J.C.H. and P.M.H. supervised studies and edited and revised the manuscript.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201609-1830OC on March 2, 2017
Author disclosures are available with the text of this article at www.atsjournals.org.
