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Background and Purpose: Most inhibitors of histone deacetylases (HDACs) are not selective and are cytotoxic. Some have anti‐inflammatory activity in disease models, but cytotoxicity prevents long‐term uses in non‐fatal diseases. Inhibitors selective for class IIa HDACs are much less cytotoxic and may have applications in management of chronic inflammatory diseases. Experimental Approach: LL87 is a novel HDAC inhibitor examined here for HDAC enzyme selectivity. It was also investigated in macrophages for cytotoxicity and for inhibition of lipopolysaccharide (LPS)‐stimulated cytokine secretion. In a rat model of collagen‐induced arthritis, LL87 was investigated for effects on joint inflammation in Dark Agouti rats. Histological, immunohistochemical, micro‐computed tomography and molecular analyses characterise developing arthritis and anti‐inflammatory efficacy. Key Results: LL87 was significantly more inhibitory against class IIa than class I or IIb HDAC enzymes. In macrophages, LL87 was not cytotoxic and reduced both LPS‐induced secretion of pro‐inflammatory cytokines, and IL6‐induced class IIa HDAC activity. In rats, LL87 attenuated paw swelling and clinical signs of arthritis, reducing collagen loss and histological damage in ankle joints. LL87 decreased immune cell infiltration, especially pro‐inflammatory macrophages and osteoclasts, into synovial joints and significantly reduced expression of pro‐inflammatory cytokines and tissue‐degrading proteases. Conclusion and Implications: A novel inhibitor of class IIa HDACs has been shown to have an anti‐inflammatory and anti‐arthritic profile distinct from current therapies. It is efficacious in reducing macrophage infiltration and joint inflammation in a chronic model of rat arthritis. [ABSTRACT FROM AUTHOR]

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR‐inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR‐inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7‐deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild‐type or an enzyme‐dead mutant of Hdac7 enhanced LPS‐inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il‐1β and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR‐inducible production of specific inflammatory mediators, whereas it acts in an enzyme‐independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses. [ABSTRACT FROM AUTHOR]

Background and Purpose: Specific cellular functions mediated by GPCRs are often associated with signalling through a particular G protein or β‐arrestin. Here, we examine signalling through a GPCR, protease‐activated receptor 2 (PAR2), in a high‐grade serous ovarian cancer cell line (OV90). Experimental Approach Human ovarian cancer tissues (n = 1,200) and nine human ovarian cancer cell lines were assessed for PAR2 expression. PAR2 signalling mechanisms leading to cell migration and invasion were dissected using cellular assays, western blots, CRISPR‐Cas9 gene knockouts, pharmacological inhibitors of PAR2 and downstream signalling proteins in OV90 cancer cells. Key Results: PAR2 was significantly overexpressed in clinical ovarian cancer tissues and in OV90 ovarian cancer cells. PAR2 agonists, an endogenous protease (trypsin) and a synthetic peptide (2f‐LIGRL‐NH2), induced migration and invasion of OV90 ovarian cancer cells through activating a combination of Gαq/11, Gα12/13 and β‐arrestin1/2, but not Gαs or Gαi. This novel cooperative rather than parallel signalling resulted in downstream serial activation of Src kinases, then transactivation of epidermal growth factor receptor (EGFR), followed by downstream MEK–ERK1/2–FOS/MYC/STAT3–COX2 signalling. Either a PAR2 antagonist (I‐191), CRISPR‐Cas9 gene knockouts (PAR2 or Gα proteins or β‐arrestin1/2), or inhibitors of each downstream protein attenuated human ovarian cancer cell motility. Conclusion and Implications: This study highlights a novel shared signalling cascade, requiring each of Gαq/11, Gα12/13 and β‐arrestin1/2 for PAR2‐induced ovarian cancer cell migration and invasion. This mechanism controlling a cellular function is unusual in not being linked to a specific individual G protein or β‐arrestin‐mediated signalling pathway. [ABSTRACT FROM AUTHOR]

Mammalian survival depends on metabolizing nutrients, storing energy, and combating infection. Complement activation in blood triggers energy-depleting immune responses to fight infections. Here we identify surprising energy-conserving roles for complement proteins C5a and C3a and their receptors, C5aR and C3aR, roles that are contraindicated in complement biology. Rats fed a high-carbohydrate high-fat diet developed obesity, visceral adiposity, adipose inflammation, glucose/insulin intolerance, and cardiovascular dysfunction that correlated with increased plasma C3a, adipose C5aR, and C3aR. These in vivo changes were dramatically attenuated by receptor-selective antagonists of either C5aR (5 mg/kg/d p.o.) or C3aR (30 mg/kg/d p.o.), which both reduced proinflammatory adipokines and altered expression of inflammatory genes in adipose tissue. In vitro C5a and C3a (100 nM) exhibited novel insulin-like effects on 3T3-L1 adipocytes, promoting energy conservation by increasing glucose and fatty acid uptake while inhibiting cAMP signaling and lipolysis, and induced PGE(2) release from macrophages, effects all blocked by each respective antagonist (10 μM). These studies reveal important new links between complement signaling and metabolism, highlight new complement functions on adipocytes and in adipose tissue, demonstrate how aberrant immune responses may exacerbate obesity and metabolic dysfunction, and show that targeting C3aR or C5aR with antagonists is a new strategy for treating metabolic dysfunction.

Human complement is a cascading network of plasma proteins important in immune defense, cooperatively effecting recognition, opsonization, destruction, and removal of pathogens and infected/damaged cells. Overstimulated or unregulated complement activation can result in immunoinflammatory diseases. Key serine proteases in this cascade are difficult to study due to their multiprotein composition, short lifetimes, formation on membranes, or serum circulation as inactive zymogens. Factor B is inactive at pH 7, but a catalytically active serine protease under alkaline conditions, enabling structure-activity relationship studies for 63 substrate-based peptide inhibitors with 4-7 residues and a C-terminal aldehyde. A potent factor B inhibitor was hexpeptide Ac-RLTbaLAR-H (IC(50) 250 nM, pH 9.5), which at pH 7 also blocked formation of membrane attack complex via the "alternative pathway" of complement activation and inhibited human complement mediated lysis of rabbit erythrocytes. Inhibitors of factor B may be valuable probes and drug leads for complement mediated immunity and disease.