Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling.

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.

Modeling and measuring extracellular matrix alterations in fibrosis: challenges and perspectives for antifibrotic drug discovery.

An imbalance of extracellular matrix (ECM) deposition and turnover is a hallmark of fibrotic pathologies as opposed to normal repair response to injury across several organs. Antifibrotic approaches to date have targeted multiple mechanisms and pathways involved in inflammation, angiogenesis, injury, wound repair, ECM biosynthesis, assembly, crosslinking and degradation. Many of these approaches have been unsuccessful which may in part be due to suboptimal models and the lack of validated functional ECM end points relevant to fibrosis. In addition, drug discovery and development for fibrotic diseases has been challenging due to the lack of translatability from in vivo models to the clinic. Targeting growth factor signaling pathways such as transforming growth factor beta (TGFß), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) are possible in simple recombinant cell models and the approval of the tyrosine kinase inhibitor, nintedanib (Ofev) is testament to the approach. However, drug targets directly impacting ECM synthesis, assembly or degradation have proven clinically intractable to date. The reasons for a lack of progress are many and include; non-traditional drug targets, lack of suitable high throughput screening assays and translational models, incomplete understanding of the role of the target. Here, we review the role of ECM in fibrosis, the challenges of ECM-targeted antifibrotic approaches, progress in the development of functional and biomarker-related ECM assays and where new translational models of fibrotic ECM remodeling could support drug discovery for fibrotic diseases.

Assessment of Brd4 inhibition in idiopathic pulmonary fibrosis lung fibroblasts and in vivo models of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of high unmet medical need. Although bromodomain (Brd) and extra terminal domain isoforms have recently been implicated in mediating inflammatory and oncologic indications, their roles in lung fibrosis have not been comprehensively assessed. We investigated the role of Brd on the profibrotic responses of lung fibroblasts (LFs) in patients with rapidly progressing IPF and a mouse bleomycin model of lung fibrosis. The enhanced migration, proliferation, and IL-6 release observed in LFs from patients with rapidly progressing IPF are attenuated by pharmacologic inhibition of Brd4. These changes are accompanied by enhanced histone H4 lysine5 acetylation and association of Brd4 with genes involved in the profibrotic responses in IPF LFs as demonstrated using chromatin immunoprecipitation and quantitative PCR. Oral administration of 200 mg/kg per day Brd4 inhibitor JQ1 in a therapeutic dosing regimen substantially attenuated lung fibrosis induced by bleomycin in C57BL/6 mice. In conclusion, this study shows that the Brd4 inhibitor JQ1, administered in a therapeutic dosage, is capable of inhibiting the profibrotic effects of IPF LFs and attenuates bleomycin-induced lung fibrosis in mice. These results suggest that Brd4 inhibitors may represent a novel therapy for the treatment of rapidly progressing IPF.

Natural regulatory T cells are resistant to calcium release-activated calcium (CRAC/ORAI) channel inhibition.

Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.

BET bromodomain proteins mediate downstream signaling events following growth factor stimulation in human lung fibroblasts and are involved in bleomycin-induced pulmonary fibrosis.

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Comparing the differential effects of LPA on the barrier function of human pulmonary endothelial cells.

Lysophosphatidic acid (LPA) is a class of bioactive lyso-phospholipid that mediates most of its biological effects through a family of G protein-coupled receptors of which six have been identified. The role of the LPA pathway in driving chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) has gained considerable academic and industry attention. Modulation of the pulmonary artery endothelial barrier function by the LPA1 receptor has been shown to drive pulmonary fibrosis in murine models of disease. The purpose of this study was (i) to assess the effect of LPA on the barrier function of human pulmonary arterial (HPAEC) and microvascular (HMVEC) endothelial cells and (ii) to identify the LPA receptor subtype(s) responsible for changes in human pulmonary endothelial cell permeability using LPA receptor antagonists and siRNA technology. Analysis of the LPA receptor subtype expression demonstrated predominant expression of LPA2 and LPA6 receptor subtypes in both HPAECs and HMVECs. HPAECs also exhibit low expression of LPA1, LPA3, and LPA4 receptor subtypes. Treatment of cells with increasing concentrations of LPA caused loss of barrier function in HPAECs but not HMVECs, despite both cell types exhibiting very similar LPA receptor expression profiles. The LPA-mediated loss of barrier function in HPAECs appears to be independent of the LPA1 receptor and likely to be mediated via the LPA6 receptor although we cannot exclude an additional role for the LPA2 and LPA4 receptors in mediating these effects. These results suggest cell-specific mechanisms exist in human pulmonary endothelial cells to permit regulation of barrier function downstream of LPA receptors. More importantly, our data indicate that selective LPA1 receptor antagonism may be insufficient for therapeutic use in pulmonary diseases where impaired endothelial barrier function is related to disease initiation and progression.

Combined inhibition of tumor necrosis factor α and interleukin-17 as a therapeutic opportunity in rheumatoid arthritis: development and characterization of a novel bispecific antibody.

OBJECTIVE: Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. METHODS: Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. RESULTS: TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. CONCLUSION: Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.