Cooperative interaction of interferon regulatory factor -1 and bromodomain-containing protein 4 on RNA polymerase activation for intrinsic innate immunity.

Introduction: The human orthopneumovirus, Respiratory Syncytial Virus (RSV), is the causative agent of severe lower respiratory tract infections (LRTI) and exacerbations of chronic lung diseases. In immune competent hosts, RSV productively infects highly differentiated epithelial cells, where it elicits robust anti-viral, cytokine and remodeling programs. By contrast, basal cells are relatively resistant to RSV infection, in part, because of constitutive expression of an intrinsic innate immune response (IIR) consisting of a subgroup of interferon (IFN) responsive genes. The mechanisms controlling the intrinsic IIR are not known. Methods: Here, we use human small airway epithelial cell hSAECs as a multipotent airway stem cell model to examine regulatory control of an intrinsic IIR pathway. Results: We find hSAECs express patterns of intrinsic IIRs, highly conserved with pluri- and multi-potent stem cells. We demonstrate a core intrinsic IIR network consisting of Bone Marrow Stromal Cell Antigen 2 (Bst2), Interferon Induced Transmembrane Protein 1 (IFITM1) and Toll-like receptor (TLR3) expression are directly under IRF1 control. Moreover, expression of this intrinsic core is rate-limited by ambient IRF1• phospho-Ser 2 CTD RNA Polymerase II (pSer2 Pol II) complexes binding to their proximal promoters. In response to RSV infection, the abundance of IRF1 and pSer2 Pol II binding is dramatically increased, with IRF1 complexing to the BRD4 chromatin remodeling complex (CRC). Using chromatin immunoprecipitation in IRF1 KD cells, we find that the binding of BRD4 is IRF1 independent. Using a small molecule inhibitor of the BRD4 acetyl lysine binding bromodomain (BRD4i), we further find that BRD4 bromodomain interactions are required for stable BRD4 promoter binding to the intrinsic IIR core promoters, as well as for RSV-inducible pSer2 Pol II recruitment. Surprisingly, BRD4i does not disrupt IRF1-BRD4 interactions, but disrupts both RSV-induced BRD4 and IRF1 interactions with pSer2 Pol II. Conclusions: We conclude that the IRF1 functions in two modes- in absence of infection, ambient IRF1 mediates constitutive expression of the intrinsic IIR, whereas in response to RSV infection, the BRD4 CRC independently activates pSer2 Pol II to mediates robust expression of the intrinsic IIR. These data provide insight into molecular control of anti-viral defenses of airway basal cells.

Developing a Translational Team Training Program using the Wisconsin Interventions in Team Science Framework.

The Clinical and Translational Science Awards (CTSA) Program supports a national network of medical research institutions working to improve the translational process. High-performing translational teams (TTs) are critical for advancing evidence-based approaches that improve human health. When focused on content-appropriate knowledge, skills, and attitudes, targeted training results in the substantial internalization of training content, producing new skills that can be applied to improve team outputs, outcomes, and benefits. More rigorous approaches to develop, test, and evaluate interventions are needed, and we used the Wisconsin Interventions in Team Science framework as a model to systematize our efforts. We designed, built, and tested a five-session TT Training Program for translational researchers. The 90-minute sessions were pilot-tested with 47 postdoctoral fellows and evaluated through a structured evaluation plan. Ninety-five percent of post-session survey respondents indicated that the content and skills provided would make them more effective collaborators, and one hundred percent would recommend the sessions to colleagues. Respondents' scores increased from pretest to posttest for most learning outcomes. Refinements from participant feedback are described. This work provides a foundation for the continued evolution of evidence-based training programs in the CTSA environment.

A leadership model supporting maturation of high-performance translational teams.

Despite understanding its impact on organizational effectiveness, practical guidance on how to train translational team (TT) leaders is lacking. Previously, we developed an evolutionary learning model of TT maturation consisting of three goal-directed phases: (1). team assembly (Formation); (2). conducting research (Knowledge Generation); and (3). dissemination and implementation (Translation). At each phase, the team acquires group-level knowledge, skills, and attitudes (KSAs) that enhance its performance. Noting that the majority of team-emergent KSAs are promoted by leadership behaviors, we examine the SciTS literature to identify the relevant behaviors for each phase. We propose that effective team leadership evolves from a hierarchical, transformational model early in team Formation to a shared, functional leadership model during Translation. We synthesized an integrated model of TT leadership, mapping a generic "functional leadership" taxonomy to relevant leadership behaviors linked to TT performance, creating an evidence-informed Leadership and Skills Enhancement for Research (LASER) training program. Empirical studies indicate that leadership behaviors are stable across time; to enhance leadership skills, ongoing reflection, evaluation, and practice are needed. We provide a comprehensive multi-level evaluation framework for tracking the growth of TT leadership skills. This work provides a framework for assessing and training relevant leadership behaviors for high-performance TTs.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

Introduction: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. Methods: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. Results: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. Discussion: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

Epigenetic control of type III interferon expression by 8-oxoguanine and its reader 8-oxoguanine DNA glycosylase1.

Interferons (IFNs) are secreted cytokines with the ability to activate expression of IFN stimulated genes that increase resistance of cells to virus infections. Activated transcription factors in conjunction with chromatin remodelers induce epigenetic changes that reprogram IFN responses. Unexpectedly, 8-oxoguanine DNA glycosylase1 (Ogg1) knockout mice show enhanced stimuli-driven IFN expression that confers increased resistance to viral and bacterial infections and allergen challenges. Here, we tested the hypothesis that the DNA repair protein OGG1 recognizes 8-oxoguanine (8-oxoGua) in promoters modulating IFN expression. We found that functional inhibition, genetic ablation, and inactivation by post-translational modification of OGG1 significantly augment IFN-λ expression in epithelial cells infected by human respiratory syncytial virus (RSV). Mechanistically, OGG1 bound to 8-oxoGua in proximity to interferon response elements, which inhibits the IRF3/IRF7 and NF-κB/RelA DNA occupancy, while promoting the suppressor NF-κB1/p50-p50 homodimer binding to the IFN-λ2/3 promoter. In a mouse model of bronchiolitis induced by RSV infection, functional ablation of OGG1 by a small molecule inhibitor (TH5487) enhances IFN-λ production, decreases immunopathology, neutrophilia, and confers antiviral protection. These findings suggest that the ROS-generated epigenetic mark 8-oxoGua via its reader OGG1 serves as a homeostatic thresholding factor in IFN-λ expression. Pharmaceutical targeting of OGG1 activity may have clinical utility in modulating antiviral response.

Substrate-specific binding of 8-oxoguanine DNA glycosylase 1 (OGG1) reprograms mucosal adaptations to chronic airway injury.

Recent advances have uncovered the non-random distribution of 7, 8-dihydro-8-oxoguanine (8-oxoGua) induced by reactive oxygen species, which is believed to have epigenetic effects. Its cognate repair protein, 8-oxoguanine DNA glycosylase 1 (OGG1), reads oxidative substrates and participates in transcriptional initiation. When redox signaling is activated in small airway epithelial cells, the DNA repair function of OGG1 is repurposed to transmit acute inflammatory signals accompanied by cell state transitions and modification of the extracellular matrix. Epithelial-mesenchymal and epithelial-immune interactions act cooperatively to establish a local niche that instructs the mucosal immune landscape. If the transitional cell state governed by OGG1 remains responsive to inflammatory mediators instead of differentiation, the collateral damage provides positive feedback to inflammation, ascribing inflammatory remodeling to one of the drivers in chronic pathologies. In this review, we discuss the substrate-specific read through OGG1 has evolved in regulating the innate immune response, controlling adaptations of the airway to environmental and inflammatory injury, with a focus on the reader function of OGG1 in initiation and progression of epithelial to mesenchymal transitions in chronic pulmonary disease.

Bromodomain-containing Protein 4 regulates innate inflammation via modulation of alternative splicing.

Introduction: Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In the context of airway viral infection, BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream epithelial plasticity. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not well understood. Given BRD4's interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing. Methods: To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep and integrated coverage of the proteomic and transcriptomic landscapes of human small airway epithelial cells exposed to viral challenge and treated with BRD4i. Results: We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 (IFRD1) and X-Box Binding Protein 1 (XBP1), related to the innate immune response and the unfolded protein response (UPR). We identify requirement of BRD4 for expression of serine-arginine splicing factors, splicosome components and the Inositol-Requiring Enzyme 1 IREα affecting immediate early innate response and the UPR. Discussion: These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing via modulating splicing factor expression in virus-induced innate signaling.

Innate Immunity, Epithelial Plasticity, and Remodeling in Asthma.

Innate immune responses (IIR) of the epithelium play a critical role in the initiation and progression of asthma. The core of the IIR is an intracellular signaling pathway activated by pattern recognition receptors (PRRs) to limit the spread of infectious organisms. This chapter will focus on the epithelium as the major innate sentinel cell and its role in acute exacerbations (AEs). Although the pathways of how the IIR activates the NFκB transcription factor, triggering cytokine secretion, dendritic cell activation, and Th2 polarization are well-described, recent exciting work has developed mechanistic insights into how chronic activation of the IIR is linked to mucosal adaptive responses. These adaptations include changes in cell state, now called epithelial-mesenchymal plasticity (EMP). EMP is a coordinated, genomic response to airway injury disrupting epithelial barrier function, expanding the basal lamina, and producing airway remodeling. EMP is driven by activation of the unfolded protein response (UPR), a transcriptional response producing metabolic shunting of glucose through the hexosamine biosynthetic pathway (HBP) to protein N-glycosylation. NFκB signaling and UPR activation pathways potentiate each other in remodeling the basement membrane. Understanding of injury-repair process of epithelium provides new therapeutic targets for precision approaches to the treatment of asthma exacerbations and their sequelae.

Temporal development of high-performance translational teams.

Successful translation involves the coupled application of knowledge-generating research with product development to advance a device, drug, diagnostic, or evidence-based intervention for clinical adoption to improve human health. Critical to the success of the CTSA consortium, translation can be more effectively accomplished by training approaches that focus on improving team-emergent knowledge skills and attitudes (KSAs) linked to performance. We earlier identified 15 specific evidence-informed, team-emergent competencies that facilitate translational team (TT) performance. Here, we examine the SciTS literature describing developmental, temporal dynamics, and adaptive learning stages of interdisciplinary teams and integrate these with real-world observations on TT maturation pathways. We propose that TTs undergo ordered developmental phases, each representing a learning cycle that we call Formation, Knowledge Generation, and Translation. We identify major activities of each phase linked to development goals. Transition to subsequent phases is associated with a team learning cycle, resulting in adaptations that enabling progression towards clinical translation. We present known antecedents of stage-dependent competencies and rubrics for their assessment. Application of this model will ease assessment, facilitate goal identification and align relevant training interventions to improve performance of TTs in the CTSA context.

Competencies supporting high-performance translational teams: A review of the SciTS evidence base.

A translational team (TT) is a specific type of interdisciplinary team that seeks to improve human health. Because high-performing TTs are critical to accomplishing CTSA goals, a greater understanding of how to promote TT performance is needed. Previous work by a CTSA Workgroup formulated a taxonomy of 5 interrelated team-emergent competency "domains" for successful translation: 1). affect, 2). communication, 3). management, 4). collaborative problem-solving, and 5). leadership. These Knowledge Skills and Attitudes (KSAs) develop within teams from the team's interactions. However, understanding how practice in these domains enhance team performance was unaddressed. To fill this gap, we conducted a scoping literature review of empirical team studies from the broader Science of Team Science literature domains. We identified specific team-emergent KSAs that enhance TT performance, mapped these to the earlier "domain" taxonomy, and developed a rubric for their assessment. This work identifies important areas of intersection of practices in specific competencies across other competency domains. We find that inclusive environment, openness to transdisciplinary knowledge sharing, and situational leadership are a core triad of team-emergent competencies that reinforce each other and are highly linked to team performance. Finally, we identify strategies for enhancing these competencies. This work provides a grounded approach for training interventions in the CTSA context.

Win-win interactions: Results and implications of a user needs assessment of clinical and translational scientists.

Introduction: This study describes a needs assessment of clinical and translational research (CTR) scientists at a large, distributed, School of Medicine within a public university and affiliated clinics. Method: We performed an Exploratory Conversion Mixed-Methods analysis using a quantitative survey and qualitative interviews with CTR scientists across the training continuum, from early-career scholars, mid-career mentors, and senior administrators at the University of Wisconsin and Marshfield Clinics. Qualitative findings were confirmed using epistemic network analysis (ENA). A survey was distributed to CTR scientists in training. Results: Analyses supported that early-career and senior-career scientists have unique needs. Scientists who identified as non-White or female reported needs that differed from White male scientists. Scientists expressed the needs for educational training in CTR, for institutional support of career development, and trainings for building stronger relationships with community stakeholders. The tension between meeting tenure clocks and building deep community connections was particularly meaningful for scholars who identified as under-represented, including based on race, gender, and discipline. Conclusions: This study yielded clear differences in support needs between scientists based upon their years in research and diversity of identities. The validation of qualitative findings, through quantification with ENA, enables robust identification of unique needs of CTR investigators. It is critically important to the future of CTR that scientists are provided with supports throughout the career. Delivery of that support in efficient and timely ways improves scientific outcomes. Advocacy at the level of the institution for under-represented scientists is of utmost importance.

Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters.

8-Oxoguanine targeted by 8-oxoguanine DNA glycosylase 1 (OGG1) is central to fibrogenic gene activation upon lung injury.

Reactive oxygen species (ROS) are implicated in epithelial cell-state transition and deposition of extracellular matrix upon airway injury. Of the many cellular targets of ROS, oxidative DNA modification is a major driving signal. However, the role of oxidative DNA damage in modulation profibrotic processes has not been fully delineated. Herein, we report that oxidative DNA base lesions, 8-oxoG, complexed with 8-oxoguanine DNA glycosylase 1 (OGG1) functions as a pioneer factor, contributing to transcriptional reprogramming within airway epithelial cells. We show that TGFß1-induced ROS increased 8-oxoG levels in open chromatin, dynamically reconfigure the chromatin state. OGG1 complexed with 8-oxoG recruits transcription factors, including phosphorylated SMAD3, to pro-fibrotic gene promoters thereby facilitating gene activation. Moreover, 8-oxoG levels are elevated in lungs of mice subjected to TGFß1-induced injury. Pharmacologic targeting of OGG1 with the selective small molecule inhibitor of 8-oxoG binding, TH5487, abrogates fibrotic gene expression and remodeling in this model. Collectively, our study implicates that 8-oxoG substrate-specific binding by OGG1 is a central modulator of transcriptional regulation in response to tissue repair.

Bromodomain-containing Protein 4 Regulates Innate Inflammation in Airway Epithelial Cells via Modulation of Alternative Splicing.

Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In airway viral infection, non-toxic BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream remodeling. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not as well understood. Based on its interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing. To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep and integrated coverage of the proteomic and transcriptomic landscapes of human small airway epithelial cells exposed to viral challenge and treated with BRD4i. The transcript-level data was further interrogated for alternative splicing analysis, and the resulting data sets were correlated to identify pathways subject to post-transcriptional regulation. We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 ( IFRD1 ) and X-Box Binding Protein 1 ( XBP1 ), related to the innate immune response and the unfolded protein response, respectively. These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing in innate signaling.

The IRE1α-XBP1s Arm of the Unfolded Protein Response Activates N-Glycosylation to Remodel the Subepithelial Basement Membrane in Paramyxovirus Infection.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections (LRTI) associated with decreased pulmonary function, asthma, and allergy. Recently, we demonstrated that RSV induces the hexosamine biosynthetic pathway via the unfolded protein response (UPR), which is a pathway controlling protein glycosylation and secretion of the extracellular matrix (ECM). Because the presence of matrix metalloproteinases and matricellular growth factors (TGF) is associated with severe LRTI, we studied the effect of RSV on ECM remodeling and found that RSV enhances the deposition of fibronectin-rich ECM by small airway epithelial cells in a manner highly dependent on the inositol requiring kinase (IRE1α)-XBP1 arm of the UPR. To understand this effect comprehensively, we applied pharmacoproteomics to understand the effect of the UPR on N-glycosylation and ECM secretion in RSV infection. We observe that RSV induces N-glycosylation and the secretion of proteins related to ECM organization, secretion, or proteins integral to plasma membranes, such as integrins, laminins, collagens, and ECM-modifying enzymes, in an IRE1α-XBP1 dependent manner. Using a murine paramyxovirus model that activates the UPR in vivo, we validate the IRE1α-XBP1-dependent secretion of ECM to alveolar space. This study extends understanding of the IRE1α-XBP1 pathway in regulating N-glycosylation coupled to structural remodeling of the epithelial basement membrane in RSV infection.

RELA∙8-Oxoguanine DNA Glycosylase1 Is an Epigenetic Regulatory Complex Coordinating the Hexosamine Biosynthetic Pathway in RSV Infection.

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.

Selective Inhibition of Bromodomain-Containing Protein 4 Reduces Myofibroblast Transdifferentiation and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a lethal disease driven by myofibroblast expansion. Currently no therapies exist that target the epigenetic mechanisms controlling myofibroblast transdifferentiation, which is responsible for unregulated extracellular matrix (ECM) production. We have recently shown that bromodomain-containing protein 4 (BRD4), an epigenetic regulator that forms a scaffold for nuclear activators and transcription factors, is essential for TGFß-induced myofibroblast transdifferentiation. However, its role in the development and progression of pulmonary fibrosis in vivo has not been established. Here, we evaluate the hypothesis that BRD4 bromodomain interactions mediate myofibroblast expansion and fibrosing disease in vivo. C57BL/6J mice challenged with intratracheal bleomycin were systemically treated with a selective allosteric inhibitor of the BRD4 bromodomain 1 (BD1), ZL0591 (10 mg/kg), during the established fibrotic phase (14 days post-bleomycin) in a rigorous therapeutic paradigm. Eleven days after initiation of ZL0591 treatment (25 days post-bleomycin), we detected a significant improvement in blood O2 saturation compared to bleomycin/vehicle control. Twenty-eight days post-bleomycin, we observed a reduction in the volumetric Hounsfield Unit (HU) density by micro computed tomography (µCT) in the ZL0591-treated group, as well as a reduction in collagen deposition (hydroxyproline content) and severity of injury (Ashcroft scoring). Myofibroblast transdifferentiation was measured by smooth muscle α-actin (αSMA) staining, indicating a loss of this cell population in the ZL0591-treated group, and corresponded to reduced transcript levels of myofibroblast-associated extracellular matrix genes, tenascin-C and collagen 1α1. We conclude that BRD4 BD1 interactions are critical for myofibroblast transdifferentiation and fibrotic progression in a mouse model of pulmonary fibrosis.

Innate Immune Responses to RSV Infection Facilitated by OGG1, an Enzyme Repairing Oxidatively Modified DNA Base Lesions.

The primary cause of morbidity and mortality from infection with respiratory syncytial virus (RSV) is the excessive innate immune response(s) (IIR) in which reactive oxygen species (ROS) play key role(s). However, the mechanisms for these processes are not fully understood. We hypothesized that expressions of IIR genes are controlled by the ROS-generated epigenetic-like mark 7,8-dihydro-8-oxo(d)guanine (8-oxo(d)Gua) and 8-oxoguanine DNA glycosylase1 (OGG1). Here, we report that ROS not only generates intrahelical 8-oxo(d)Gua, but also enzymatically disables OGG1 in RSV-infected human airway epithelial cells and mouse lungs. OGG1 bound to 8-oxo(d)Gua in gene regulatory sequences promotes expression of IIR genes, and consequently exacerbates lung inflammation, histological changes, and body weight loss of experimental animals. Pharmacological inhibition of OGG1 substrate binding decreased expression of RSV-induced chemokine and cytokines and significantly lessened clinical symptoms. Results of mechanistic studies show that OGG1 binding at 8-oxo(d)Gua promoter regions modulated loading of transcription factors via transient cooperative interactions in RSV-infected lungs and airway epithelial cells. Other base specific DNA repair proteins had no effects. Collectively, this study identifies unprecedented roles of ROS-generated DNA base lesion(s) and cognate repair protein as a determinant of RSV-induced exuberant inflammation. Pharmaceutical inhibition of OGG1 interaction with its DNA substrate may represent a novel strategy in prevention/intervention of respiratory viral infections.