BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection

Abstract

The airway mucosa expresses protective interferon (IFN) and inflammatory cytokines in response to respiratory syncytial virus (RSV) infection. In this study, we examine the role of bromodomain containing 4 (BRD4) in mediating this innate immune response in human small airway epithelial cells. We observe that RSV induces BRD4 to complex with NF-B/RelA. BRD4 is functionally required for expression of the NF-Bdependent inflammatory gene regulatory network (GRN), including the IFN response factor 1 (IRF1) and IRF7, which mediate a cross talk pathway for RIG-I upregulation. Mechanistically, BRD4 is required for cyclin-dependent kinase 9 (CDK9) recruitment and phospho-Ser 2 carboxy-terminal domain (CTD) RNA polymerase (Pol) II formation on the promoters of IRF1, IRF7, and RIG-I, producing their enhanced expression by transcriptional elongation. We also find that BRD4 independently regulates CDK9/ phospho-Ser 2 CTD RNA Pol II recruitment to the IRF3-dependent IFN-stimulated genes (ISGs). In vivo, poly(I·C)-induced neutrophilia and mucosal chemokine production are blocked by a small-molecule BRD4 bromodomain inhibitor. Similarly, BRD4 inhibition reduces RSV-induced neutrophilia, mucosal CXC chemokine expression, activation of the IRF7-RIG-I autoamplification loop, mucosal IFN expression, and airway obstruction. RSV infection activates BRD4 acetyltransferase activity on histone H3 Lys (K) 122, demonstrating that RSV infection activates BRD4 in vivo. These data validate BRD4 as a major effector of RSV-induced inflammation and disease. BRD4 is required for coupling NF-B to expression of inflammatory genes and the IRF-RIG-I autoamplification pathway and independently facilitates antiviral ISG expression. BRD4 inhibition may be a strategy to reduce exuberant virus-induced mucosal airway inflammation. IMPORTANCEIn the United States, 2.1 million children annually require medical attention for RSV infections. A first line of defense is the expression of the innate gene network by infected epithelial cells. Expression of the innate response requires the recruitment of transcriptional elongation factors to rapidly induce innate response genes through an unknown mechanism. We discovered that RSV infection induces a complex of bromodomain containing 4 (BRD4) with NF-B and cyclindependent kinase 9 (CDK9). BRD4 is required for stable CDK9 binding, phosphoSer 2 RNA Pol II formation, and histone acetyltransferase activity. Inhibition of BRD4 blocks Toll-like receptor 3 (TLR3)-dependent neutrophilia and RSV-induced inflammation, demonstrating its importance in the mucosal innate response in vivo. Our study shows that BRD4 plays a central role in inflammation and activation of the IRF7-RIG-I amplification loop vital for mucosal interferon expression. BRD4 inhibition may be a strategy for modulating exuberant mucosal airway inflammation.

Pollen exposure weakens innate defense against respiratory viruses

“…The immune response to RSV infection differs between mouse strains . Within C57BL/6 mice, there is a clear role for Type I IFNs as well as IFNAR in RSV infection . We also chose the C57BL/6 background to exclude any pollen effect mediated by allergic sensitization.…”

RSV Reprograms the CDK9•BRD4 Chromatin Remodeling Complex to Couple Innate Inflammation to Airway Remodeling

“…Although IRF3 activation is largely NFκB-independent, sustained IRF action on IFN and ISG expression requires ongoing NFκB signaling in epithelial cells. Some key elements of NFκB-IRF pathway cross-talk are that: (1) the virus infection-activated IFNβ enhanceosome is activated by NFκB (note that IFNβ is a major type I IFN, responsible for the majority of mucosal anti-viral effect); (2) the induction of inducible IRF-1 and -7 subunits controlling type I IFN expression are dependent on direct NFκB transactivation [33]; and, (3) NFκB is required for the IRF-IFN-RIG-I amplification loop, described above [34]. Consequently, sustained epithelial IFN response is substantially blunted in the absence of NFκB [33,34].…”

“…The essential functional role of CDK9•BRD4 in RSV-induced IIR has been demonstrated by disruption of P-TEFb recruitment by RelA Ser 276 site mutation [52], siRNA-mediated silencing of CDK9 and BRD4 [24,52,53,61], and small-molecule inhibition of CKD9 and BRD4 [34,48,53]. Consequently, CDK9•BRD4 are molecules receiving significant attention as a target for pharmacological manipulation of viral and allergen-inducible mucosal inflammation [48,[62][63][64][65][66].…”

“…We recently found that the RSV induced RelA•BRD4 association also induced the atypical histone acetyl transferase (HAT) activity of BRD4 and acetylating histone H3 on Lys (K) 122, a modification that destabilizes nucleosomes, enhancing transcription through GC-rich gene bodies [34,74]. To illustrate the inducible formation of RelA and BRD4 complexes in the airway mucosa in vivo, we conducted proximity ligation assays (PLAs).…”

Transcription Elongation Factor P-TEFb Is Involved in IL-17F Signaling in Airway Smooth Muscle Cells

“…Recent reports demonstrated that Brd4 plays a major role in the pathogenesis of airway inflammation. Indeed, Brd4 activation in bronchial epithelial cells was induced by several inflammatory stimuli, such as RS virus, and diesel exhaust particles that are able to exacerbate airway inflammation [28, 29]. In addition, Brd4 regulates IL-1β-induced IL-8 expression in airway epithelial cells [30].…”