IL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton’s tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

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Mast cells are important effector cells in asthma and atopic disease. Upon IgE receptor cross‐linking, they release potent inflammatory mediators, cytokines, and chemokines. Mast cells can also be activated by IgE‐independent signals, including the anaphylatoxin C5a, adenosine, and other agents. The human mast cell line, HMC‐1, expresses the Gαi‐coupled C5a receptor, along with Gαi‐coupled adenosine receptors A1R (low levels) and A3R, the Gαs coupled A2aR, and the Gαs/Gαq coupled A2bR. We found that HMC‐1 cells exhibited a potent synergistic induction of chemokine release, coupled to NFAT activation, when exposed to C5a in combination with the adenosine analog, NECA. To elucidate the receptor(s) mediating this response, two strategies were employed. First, a panel of specific adenosine receptor agonists was tested in combination with C5a for synergy of chemokine release. Agents selective for A1R or A2aR did not stimulate chemokine production, while an A3R selective agonist induced low levels of chemokine production only in combination with C5a. To investigate the role of A2bR, for which no selective agonist was available, an siRNA approach was employed. Knockdown of A2bR, but not A2aR, significantly reduced NECA‐induced second messenger accumulation (cAMP and IP3) and chemokine production, and inhibited the synergy with C5a. To identify downstream signaling molecules important for this response, a broad‐based phospho‐protein screen was performed, which revealed signaling to SRC phosphorylation is enhanced upon C5a and NECA costimulation. These data suggest a cooperative signaling node in adenosine and C5a costimulation of mast cells resulting from the activation of their respective GPCRs coupled to Gαq and Gαi.

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