Bromodomain and Extra-Terminal Protein Inhibition Attenuates Neutrophil-dominant Allergic Airway Disease

Atopic asthma is a prevalent respiratory disease that is characterized by inflammation, mucus hypersecretion, and airway hyperresponsiveness. The complexity of this heterogeneous disorder has commanded the need to better define asthma phenotypes based on underlying molecular mechanisms of disease. Although classically viewed as a type 2-regulated disease, type 17 helper T (Th17) cells are known to be influential in asthma pathogenesis, predominantly in asthmatics with neutrophilia and severe refractory disease. Bromodomain and extra-terminal domain (BET) chromatin adaptors serve as immunomodulators by directly regulating Th17 responses and Th17-mediated pathology in murine models of autoimmunity and infection. Based on this, we hypothesized that BET proteins may also play an essential role in neutrophil-dominant allergic airway disease. Using a murine model of neutrophil-dominant allergic airway disease, we demonstrate that BET inhibition limits pulmonary inflammation and alters the Th17-related inflammatory milieu in the lungs. In addition, inhibition of BET proteins improved lung function (specifically quasi-static lung compliance and tissue elastance) and reduced mucus production in airways. Overall, these studies show that BET proteins may have a critical role in asthma pathogenesis by altering type 17 inflammation, and thus interfering with BET-dependent chromatin signaling may provide clinical benefits to patients suffering from asthma.

Based on the fundamental role of BET bromodomains in cell growth and proliferation, recent studies have focused on establishing a link between BET proteins and inflammatory responses in disease. Mele and colleagues have shown that BET proteins are critical for controlling both human and murine Th17 differentiation and activation as well as for regulating Th17-associated cytokine production (IL-17, IL-21, and GM-CSF) 16 . More importantly, their work showed that the BET family members Brd2 and Brd4 critically modulate Th17 biology by associating with the Il17 locus in a bromodomain-dependent manner, thereby directly controlling the transcription of IL-17 16 . A study by Chen et al. also showed BET inhibition repressed Th17 cell responses in explanted tissues from cystic fibrosis patients 20 . Aside from circulating immune cells, BET proteins have been shown to influence cell function and response of airway epithelial cells 20,21 , airway smooth muscle 22,23 , and lung fibroblasts 24 . Further, pharmacological inhibition of BET bromodomains was protective in murine models of autoimmunity 16 , bleomycin-induced pulmonary fibrosis 24 , LPS-induced shock 18 , and Pseudomonas aeruginosa lung infection 20 .
Although the role of epigenetic regulation in asthma pathogenesis remains unclear, evidence of altered histone acetylation, aberrant histone acetyltransferases and histone deacetylases expression, as well as abnormal expression of genes involved in pulmonary repair and inflammation has been reported in the airways of asthmatics [21][22][23][25][26][27][28][29] . Additionally, mimics of BET bromodomains have been shown to inhibit airway smooth muscle cell proliferation and cytokine release from patients with asthma 22,23 . Based on these findings and the role of BET proteins in Th17 cell function, we hypothesize that the BET inhibitor CPI-203 would limit asthma pathogenesis in a Th17-induced murine model of severe refractory asthma. Overall, this work establishes a role for BET proteins in Th17-mediated allergic airway disease and suggests that interfering with BET-dependent chromatin signaling may provide clinical benefits to patients suffering from asthma.
All mice were housed in a pathogen-free environment at the Children's Hospital of Pittsburgh of UPMC and were given food and water ad libitum. All animal experiments were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All experiments were performed in accordance with IACUC guidelines and regulations.
In vitro differentiation of Th17 cells. CD4 + CD62L + naïve T cells from the spleens of DO11.10 TCR-transgenic mice were enriched using a CD4 + CD62L + T cell Isolation Kit II (Miltenyi Biotec) and were (A) BALB/c SCID mice were treated with ovalbumin (OVA) and adoptively transferred with Th17 cells to induce allergic airway disease and the therapeutic potential of CPI-203 was investigated in this model. Cellular inflammation in the airspaces was measured by (B) total cells in the BAL fluid and (C) BAL fluid cell differential counts. Mice in control and CPI-203 groups received all OVA challenges, but phosphate buffered saline retro-orbitally at time of cell transfer. Graphs show data for control (n = 5), CPI-203 (n = 6), Th17 (n = 8), Th17+ CPI-203 (n = 8) combined from three independent experiments. *p < 0.05 when compared to all other groups, †p < 0.05 when compared to the CPI-203 group. cultured for 6 days with antiCD3/antiCD28 mouse Dynabeads (Invitrogen) under Th17 cell polarizing conditions (10 ng/mL IL-23, 1 ng/mL TGFβ , 2 ng/mL IL-6, 10 μ g/mL anti-IL-4, and 10 μ g/mL anti-IFNγ purchased from R&D Systems) as previously described 3 .
Th17-induced, neutrophil-dominant allergic airway disease model. BALB/c SCID mice were challenged with 50 μ g of ovalbumin (Sigma-Aldrich) via oropharyngeal aspiration (OA) on Day 0. On Day 1, 1 × 10 6 Th17 cells were adoptively transferred by retro-orbital injection. Mice were subsequently challenged with 50 μ g ovalbumin OA daily for three consecutive days after cell transfer (Days 2-4). Twenty-four hours after the last challenge, mice were euthanized (Day 5) and allergic airway disease was assessed as detailed below. Control mice received all ovalbumin challenges, but received phosphate buffered saline retro-orbitally at time of cell transfer. To inhibit BET bromodomains, mice were treated with 2.5 mg/kg BET inhibitor CPI-203 (Constellation Pharmaceuticals) via intraperitoneal injection twice daily on Days 1-4.
Gene expression analysis. RNA was isolated from lung tissue using an Absolutely RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA). One μ g of RNA was then converted to cDNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Gene expression analysis was performed using Taqman Fast Mastermix and Assay On Demand TaqMan primer and probe sets for the following murine genes of interest: Muc5ac, Muc5b, Clca3, Spp1 (Life Technologies, Grand Island, NY). in the lung, standard H&E-stained lung sections were scored by observers (JFA and MLM), who were blinded to the sample group identity. The entire lung section was observed with a light microscope (× 40 magnification) and peribronchial, perivascular, and parenchymal inflammation were scored as previously described 7,31 . Briefly, cellular inflammation present in each area was scored according to the following scale: 0 = no inflammation, 1 = up to 25%, 2 = 25-50%, 3 = 50-75%, and 4 = 75-100%. The inflammation score was reported as the mean of the scores for each sample group. Mucus production was quantified through the observation of Periodic Acid-Schiff (PAS) stained lung sections by observers (JFA and MLM) blinded to the identity of the sample groups. The entire lung section was observed with a light microscope (100x magnification) and the amount of PAS staining in the entire lung section was scored using the following scale: 0 = no staining present, 1 = light staining in large airway only, 2 = staining present in all large airways and some small bronchioles, 3 = staining present in large and small airways, and 4= dense staining in all airways. The PAS score was reported as the mean of the scores for each sample group.
Experiments involving 2 variables were analyzed by two-way analysis of variance with a Bonferonni post-hoc test. Data with one variable were analyzed using one-way analysis of variance with Tukey's post-hoc test. Data comparing two groups were analyzed using an unpaired t-test. Data was log transformed for statistical analyses where indicated. Data shown are mean ± SEM. A value of p < 0.05 was considered to be statistically significant.

Results
Inhibition of BET proteins decreases pulmonary inflammation and alters cytokine and chemokine production in the lungs during Th17-induced allergic airway disease. Previous work has shown that adoptive transfer of OVA-specific Th17 cells into OVA-treated BALB/c SCID mice is sufficient to promote neutrophilic inflammation, AHR, and mucus metaplasia, which are not affected by glucocorticoid treatment 3 . Utilizing this model, we investigated the therapeutic potential of CPI-203, a highly selective and potent inhibitor of BET proteins (Fig. 1A). Similar to previous work 3,7 , adoptive transfer of OVA-specific Th17 cells into OVA-treated BALB/c SCID mice increased cellular inflammation in the airspaces, resulting in higher levels of macrophages, neutrophils, and lymphocytes (Fig. 1B,C). CPI-203 treatment during Th17-driven allergic airway disease attenuated inflammation in the airspaces of the lungs and resulted in significantly lower levels of macrophages, lymphocytes, and neutrophils (Fig. 1B,C). Overall, these results suggest that the steroid-insensitive neutrophilia in the airspaces that was induced by Th17-mediated allergic airway disease, was reduced by CPI-203 treatment.  In addition to reducing cellular inflammation present in the airspaces, inhibition of BET proteins during Th17-induced allergic airway disease also decreased inflammation present in the lung tissue. Histological assessment of hematoxylin and eosin-stained lung sections showed that perivascular, peribronchial, and parenchymal inflammation were all lessened by CPI-203 treatment in this murine model of Th17-induced allergic airway disease (Fig. 2). Furthermore, treatment with CPI-203 altered cytokine and chemokine levels in the lungs of Th17 adoptive transfer, OVA challenged mice. Specifically, IL-1α , IL-1β , IL-2, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, and eotaxin increased in response to CPI-203 treatment, while G-CSF, CXCL1, MIP-1β , and CCL5 decreased (Table 1). Several cytokines and chemokines were also unaffected by CPI-203 treatment, which included IL-3, IL-4, IL-5, IL-9, GM-CSF, IFNγ , MCP-1, MIP-1α and TNFα . These data demonstrate that inhibition of BET proteins alters Th17 cell-driven inflammation by modulating downstream pro-inflammatory mediators, which promote neutrophil recruitment into the lungs.

BET bromodomain inhibition with CPI-203 alters respiratory mechanics in mice with
Th17-induced allergic airway disease. To further characterize the role of BET proteins in Th17-dominant allergic airway disease, lung function was assessed by measuring Newtonian resistance (Rn), tissue damping (G), and tissue elastance (H), in response to increasing doses of methacholine as well as by measuring quasi-static lung compliance. Inhibition of BET proteins with CPI-203 treatment did not affect Rn, a parameter that is representative of central or conducting airway resistance, and G, a parameter that relates to tissue or parenchymal resistance, in this model of Th17-driven allergic airway disease (Fig. 3A,B). However, blocking BET bromodomains significantly lowered tissue elastance (H), a parameter related to parenchymal recoil, in Th17 cell adoptive transfer, OVA challenge mice (Fig. 3C). CPI-203 treatment also trended to improve quasi-static lung compliance in mice with Th17-induced allergic airway disease (Fig. 3D).
Inhibition of BET bromodomains limits Th17-mediated mucus production in the lungs of mice during allergic airway disease. As previously reported 3 , adoptive transfer of Th17 cells into OVA-treated BALB/c SCID mice increased pulmonary mRNA expression of mucus-associated genes, Muc5ac and Clca3, and increased mucus production as evident by PAS staining present in the airways (Fig. 4). In this study, inhibition of BET bromodomains during Th17-induced allergic airway disease significantly reduced PAS-positive airways, while not significantly altering Muc5ac and Clca3 mRNA expression (Fig. 4). These results suggest that BET proteins are important drivers of mucus production in the airways during allergic airway disease, independent of promoting mucin gene expression.

Discussion
In this study, we show that BET inhibition limits neutrophilia and Th17-driven cytokine and chemokine release in the lungs in a murine model of severe, steroid-insensitive asthma. Inhibition of BET proteins also improved lung function, specifically quasi-static lung compliance and tissue-related AHR, and reduced mucus production in the airways. To our knowledge, these results are novel and demonstrate that BET proteins are important regulators of asthma pathogenesis, and more significantly can affect severe, steroid-insensitive disease. Therefore, our work herein demonstrates that BET inhibitors may provide therapeutic benefit to patients with asthma, especially those with neutrophilia and/or severe refractory disease.
Previous work has suggested that cellular infiltration into the airspaces during Th17-induced allergic airway disease correlated with quasi-static lung compliance, whereas AHR was associated with tissue-based inflammation 7 . The findings of this current study support these results, which demonstrate that when Th17-induced cellular inflammation is limited via BET protein inhibition, quasi-static lung compliance is also improved. Similarly, we also observe a marked reduction in Th17-induced tissue inflammation following BET protein inhibition and lower tissue-related AHR parameters. Although quasi-static lung compliance and tissue-related AHR may be associated with pulmonary inflammation, our results imply that central airway resistance is likely an inflammation-independent process. Alterations in airway resistance have been attributed to direct effects on airway remodeling and contractility. Specifically, IL-17 is known to induce airway smooth muscle cell contraction, thereby altering airway hyperresponsiveness [34][35][36] . Overall, these results highlight the complexity of the underlying biology that contributes to asthma pathologies. Further work is needed to establish how histone modifications affect tissue responses in asthma. IL-17 is a cytokine mainly produced by Th17 cells and is thought to regulate neutrophilic inflammation and steroid resistance in severe asthmatics. BET chromatin modulators directly regulate Th17 responses and critically mediate Th17 biology via bromodomain-dependent association with acetylated histones at the IL17 locus in Th17 cells 16 . Further, BET inhibition was shown to limit Th17-mediated autoimmunity and pulmonary infection 16,20 . Thus, we hypothesized that BET inhibition would alter Th17 cell-driven allergic airway disease. In this study, we did not observe a direct change in IL-17 protein following CPI-203 treatment, but did find lower levels of IL-17-driven cytokines and chemokines (G-CSF, CXCL1), neutrophils in the airspaces, and tissue inflammation in the lungs. The murine model of asthma utilized for this work is a neutrophil-dominant allergic airway disease model induced by antigen-specific Th17 cells, which presumably produce IL-17 as early as the first antigen challenge (Day 2, Fig. 1A). The lung tissue is collected for analyses several days after this initial challenge and thus may account for the unchanged pulmonary levels of IL-17 observed. The reduction of pulmonary neutrophilia and IL-17-driven cytokines implies however that the inhibition of BET proteins reduced the activity of IL-17 in the lungs in this murine model of severe, steroid-insensitive asthma.
Our data provides evidence that the inhibition of BET proteins lessens neutrophilic inflammation likely by altering downstream pro-inflammatory mediators, which promote neutrophil recruitment into the lungs. Other studies using BET mimics have also established that BET proteins can modulate neutrophilia by regulating IL-6 and CXCL8 release from human bronchial epithelial cells and airway smooth muscle cells from healthy controls and asthmatics 21,22 . BET protein function was also shown to be required for murine macrophage inflammatory processes 18 , which may influence their phenotype and role in asthma pathogenesis. Analyses of the cytokine and chemokine milieu following BET inhibition in mice with Th17 cell-driven allergic airway disease revealed a complex inflammatory environment even when cellular inflammation in the airspace and interstitium was significantly reduced. Overall, BET proteins clearly function as modulators of the inflammatory responses in this murine model of severe, steroid-insensitive asthma.
JQ1 and CPI-203, an analog of JQ1, are well-characterized BET bromodomain inhibitors that are extremely selective, with no off-target effects at biologically relevant or suprapharmalogical concentrations [37][38][39] . Clinical trials using BET bromodomain inhibitors for treatment of leukemias, lymphomas, myelomas, and other cancers are ongoing 40 . Recent studies have suggested the utility of BET mimics in Th17 cell-driven autoimmunity, pulmonary infection, and idiopathic pulmonary fibrosis 16,20,24 . Our original work herein demonstrates a pivotal role of BET proteins in asthma and more importantly, provides evidence that BET inhibition may be beneficial in a steroid-insensitive disease setting. As some asthmatics with severe glucocorticoid resistance show abnormal histone acetylation patterns 26 , pharmacological intervention targeting BET adaptor proteins may be efficacious to lessen disease burden for patients with refractory disease.