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.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to Pseudomonas aeruginosa infection. Recent data suggest that IL-17 contributes to pathological inflammation in the setting of abnormal mucosal immunity, and type 17 immunity-driven inflammatory responses may represent a target to block aberrant inflammation in CF. Indeed, transcriptomic analysis of the airway epithelium from CF patients undergoing clinical bronchoscopy revealed upregulation of IL-17 downstream signature genes, implicating a substantial contribution of IL-17-mediated immunity in CF lungs. Bromodomain and extraterminal domain (BET) chromatin modulators can regulate T cell responses, specifically Th17-mediated inflammation, by mechanisms that include bromodomain-dependent inhibition of acetylated histones at the IL17 locus. Here, we show that, in vitro, BET inhibition potently suppressed Th17 cell responses in explanted CF tissue and inhibited IL-17-driven chemokine production in human bronchial epithelial cells. In an acute P. aeruginosa lung infection murine model, BET inhibition decreased inflammation, without exacerbating infection, suggesting that BET inhibition may be a potential therapeutic target in patients with CF.

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.

Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of auto-immunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.

Proteins of the BET family bind to acetylated histones and promote gene expression by recruiting transcriptional activators. We developed BET inhibitors and demonstrated a strong association between biochemical inhibition of BET-chromatin binding, MYC suppression and reduced proliferation in hematopoietic cancer cells. We assessed the gene loci affected by BET inhibition by integrating ChipSeq and gene expression profiling, and identified genes that are directly regulated by BRD4 binding, most notably MYC, or genes that are targets of MYC. BET inhibition resulted in robust suppression of MYC expression in tumor xenograft models. Moreover, based on the PK/PD relationships established for these compounds we designed dosing regimens that resulted in significant anti-tumor efficacy in xenograft models of Burkitt9s lymphoma and acute leukemia. Compound doses and schedules resulting in sustained MYC suppression were most effective in inhibiting tumor growth and were well tolerated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B179.

In recent years significant progress has been made in our understanding of epigenetic control of a wide range of cellular processes. This has come about both through the concerted effort of the research community and through the development of technologies essential to the area. The importance of the epigenetic control of the immune system is becoming increasingly clear, and therefore epigenetics presents itself as an attractive, and potentially ground-breaking, entry point to tackle immune-mediated conditions. The advances in our understanding are in part due to the development of next generation sequencing technologies and chromatin immunoprecipitation. When combined, these approaches have allowed studies at the chromatin level to understand cellular responses to cell-external cues on a genome scale. This has contributed significantly to improved understanding of chromatin, its regulation through histone post-translational modification (PTM), and the enzymes and proteins involved in ‘writing’, ‘reading’ and ‘erasing’ these histone PTMs. In this review we focus specifically on the progress made in understanding the mechanisms involved in modulating histone methylation in the context of immunity and discuss the potential, and the challenges, presented by this exciting area for drug discovery.

Small molecules that perturb chromatin proteins are an emerging focus of current biomedical research. Two groups reporting in this issue have targeted bromodomain-containing BET proteins that bind acetylated lysine residues during gene activation, arriving at cell-permeable small molecule compounds with similar structures based on fused triazole-diazepine rings. James Bradner and colleagues report the development of a compound named JQ1. The BET protein BRD4, with two bromodomains, is implicated in human squamous cell carcinoma. JQ1 inhibits the growth of BRD4-dependent tumours in mouse models. Alexander Tarakhovsky and colleagues' inhibitor, I-BET, is shown to interfere with the binding of certain BET family members to acetylated histones. It inhibits activation of pro-inflammatory genes in macrophages and has immunomodulatory activity in a mouse model of inflammatory disease. Post-translationally modified histones are recognized by effector proteins which contain specific binding modules; for example, the bromodomain-containing BET proteins bind acetylated lysine residues during gene activation. Here a synthetic small molecule is described that interferes with the binding of certain BET family members to acetylated histones. The compound inhibits activation of pro-inflammatory genes in macrophages and has activity in a mouse model of inflammatory disease. Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression1,2,3,4. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression5,6,7,8,9,10. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by ‘mimicking’ acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4R alpha/IL-13R alpha 1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappa B. As a result, we hypothesized that NF-kappa B activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappa B activity. Three pharmacologic approaches were used to inhibit NF-kappa B including 1) PS1145, a small molecule inhibitor of I kappa B alpha kinase (IKK2), 2) antennapedia-linked NF-kappa B essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappa B component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappa B is an attractive target for immunotherapy of IL-13-mediated diseases.

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by ∼70% of CD4+ T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4+ T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4+ effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1 alpha/CCL3 and MIP-1 beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1 alpha/CCL3, MIP-1 beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha 1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.

Kim DC, Hsu FI, Barrett NA, et al. J Immunol. 2006;176:4440–4448 PURPOSE OF THE STUDY. The cysteinyl leukotrienes (cysLTs), derived from the 5-lipoxygenase pathway, play an important role in asthma as smooth muscle constrictors of airways and microvasculature. This study investigated a potential additional role of cysLTs in T-helper 2 (Th2) cell-dependent pulmonary inflammation using an ovalbumin-sensitization and -challenge protocol with mice that lacked LTC4 synthase (LTC4S), the terminal pathway enzyme for cysLT biosynthesis. METHODS. LTC4S-null mice and wild-type mice underwent intraperitoneal ovalbumin sensitization, followed on days 40, 43, and 46 by intranasal ovalbumin or saline challenge. Pulmonary histology was examined 48 hours after the last challenge. Total and ovalbumin-specific serum immunoglobulin (Ig) levels, cytokine mRNA expression in the lung, cytokine production by parabronchial lymph node cells after in vitro ovalbumin restimulation, delayed-type hypersensitivity, and airway hyperresponsiveness to methacholine 24 hours after the last challenge were measured also. RESULTS. In the LTC4S-null mice, antigen-induced pulmonary inflammation (eosinophil infiltration and goblet and mast cell hyperplasia) and airway hyperresponsiveness to methacholine were significantly reduced. In addition, antigen-specific serum immunoglobulin E and G1 and Th2 cell cytokine messenger RNA expression in the lung were reduced in LTC4S-null mice compared with wild-type controls. The production of Th2 cytokines by antigen-restimulated parabronchial lymph node cells from LTC4S-null mice was also significantly reduced compared with those from wild-type controls; however, there was no suppression of cutaneous delayed-type hypersensitivity in LTC4S-null mice. CONCLUSIONS. These findings support a role for cysLTs in the development and/or amplification of a pulmonary Th2 response. REVIEWER COMMENTS. Two types of leukotriene-based medications have been used to treat patients with bronchial asthma: (1) leukotriene inhibitors that block the actual synthesis of leukotrienes and (2) leukotriene antagonists that inhibit leukotriene function by blocking leukotriene-receptor sites. Previous studies suggested that the main role of cysLTs was smooth muscle constriction of airways and microvasculature. This study, as well as other growing evidence, points to a unique and broader role for cysLTs in the initiation and amplification of the Th2 response in the lung. Future studies should assess the mechanism of this role, specifically the impact of cysLTs on dendritic-cell and T-cell function. Therefore, the therapeutic benefit of leukotriene antagonists may be additionally derived from this broader physiologic role of cysLTs in pulmonary Th2 responses.