A novel highly selective allosteric inhibitor of tyrosine kinase 2 (TYK2) can block inflammation- and autoimmune-related pathways.

BACKGROUND: As a member of the Janus kinase (JAK) family, which includes JAK1, JAK2 and JAK3, tyrosine kinase 2 (TYK2) plays an important role in signal transduction and immune system regulation. Moreover, it is also involved in the development of many types of inflammatory and autoimmune diseases, such as psoriasis and systemic lupus erythematosus (SLE). TYK2 is an attractive therapeutic target, and selective inhibition of TYK2 over other JAK family members is critical for the development of TYK2 small molecule inhibitors. However, targeting the catalytic region of the TYK2 ATP-binding site is a major challenge due to the high structural homology between the catalytic regions of the JAK family proteins. RESULTS: In this study, we developed a novel small molecule inhibitor (QL-1200186) by targeting the pseudokinase regulatory domain (Janus homology 2, JH2) of the TYK2 protein. The binding sites of QL-1200186 were predicted and screened by molecular docking. The inhibitory effects on IFNα, IL-12 and IL-23 signaling were tested in cell lines, human peripheral blood cells and human whole blood. The pharmacokinetic (PK) and pharmacodynamic properties of QL-1200186 were verified in mice. QL-1200186 showed high affinity for TYK2 JH2 and had no apparent selectivity for the TYK2 and JAK homologous kinase domains; these effects were demonstrated using biochemical binding, signaling pathway transduction (JAK1/2/3) and off-target effect assays. More importantly, we revealed that QL-1200186 was functionally comparable and selectivity superior to two clinical-stage TYK2 inhibitors (BMS-986165 and NDI-034858) in vitro. In the PK studies, QL-1200186 exhibited excellent exposure, high bioavailability and low clearance rates in mice. Oral administration of QL-1200186 dose-dependently inhibited interferon-γ (IFNγ) production after interleukin-12 (IL-12) challenge and significantly ameliorated skin lesions in psoriatic mice. CONCLUSION: These findings suggest that QL-1200186 is a highly selective and potent inhibitor of TYK2. QL-1200186 could be an appealing clinical drug candidate for the treatment of psoriasis and other autoimmune diseases. Video Abstract.

CD47 deficiency ameliorates autoimmune nephritis in Fas(lpr) mice by suppressing IgG autoantibody production.

CD47, a self-recognition marker, plays an important role in both innate and adaptive immune responses. To explore the potential role of CD47 in activation of autoreactive T and B cells and the production of autoantibodies in autoimmune disease, especially systemic lupus erythematosus (SLE), we have generated CD47 knockout Fas(lpr) (CD47(-/-) -Fas(lpr) ) mice and examined histopathological changes in the kidneys, cumulative survival rates, proteinuria, extent of splenomegaly and autoantibodies, serum chemistry and immunological parameters. In comparison with Fas(lpr) mice, CD47(-/-) -Fas(lpr) mice exhibit a prolonged lifespan and delayed autoimmune nephritis, including glomerular cell proliferation, basement membrane thickening, acute tubular atrophy and vacuolization. CD47(-/-) -Fas(lpr) mice have lower levels of proteinuria, associated with reduced deposition of complement C3 and C1q, and IgG but not IgM in the glomeruli, compared to age-matched Fas(lpr) mice. Serum levels of antinuclear antibodies and anti-double-stranded DNA antibodies are significantly lower in CD47(-/-) -Fas(lpr) than in Fas(lpr) mice. CD47(-/-) -Fas(lpr) mice also display less pronounced splenomegaly than Fas(lpr) mice. The mechanistic studies further suggest that CD47 deficiency impairs the antigenic challenge-induced production of IgG but not IgM, and that this effect is associated with reduction of T follicular cells and impairment of germinal centre development in lymphoid tissues. In conclusion, our results demonstrate that CD47 deficiency ameliorates lupus nephritis in Fas(lpr) mice via suppression of IgG autoantibody production.

Control of secondary granule release in neutrophils by Ral GTPase.

Neutrophil (polymorphonuclear leukocyte; PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocytosis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, we found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules but not primary or tertiary granules and caused dysregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, whereas chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Overexpression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, we found that Ral, which was associated with the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral translocation intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattractant stimulation.

Role of junctional adhesion molecule-like protein in mediating monocyte transendothelial migration.

OBJECTIVE: Monocyte migration across the vascular endothelium of blood vessels is a key early event in atherosclerosis. The mechanisms underlying monocyte transendothelial migration (TEM), however, are still not completely understood. Here we studied the role of junctional adhesion molecule-like protein (JAML) in regulating monocyte TEM. METHODS AND RESULTS: Firstly, by Western blot and flow cytometry, we showed that JAML was strongly expressed in monocytes and monocyte surface expression of JAML was upregulated by monocyte chemotaxis protein-1 stimulation. Both monocyte adhesion to and migration across tumor necrosis factor-alpha (TNFalpha) preactivated human microvascular endothelial cell (HMEC-1) monolayers were dose-dependently reduced by anti-JAML antiserum or soluble extracellular JAML recombinant. Secondly, short-term exposure of human monocytes and THP-1 cells to advanced glycation end products increased cell surface JAML expression, which was correlated with enhanced cell adhesion and TEM. In contrast, knockdown of JAML in THP-1 monocytes decreased both adhesion and transmigration of THP-1 monocytes. Finally, direct binding assay of the soluble JAML to HMEC-1 monolayers suggested that endothelial coxsackie and adenovirus receptor (CAR) may serve as one of the ligands for JAML. CONCLUSIONS: Monocytic JAML plays a critical role in regulating monocyte TEM probably via binding to the endothelial CAR and other tight junction-associated adhesive molecules.

The heparan sulfate proteoglycan form of epithelial CD44v3 serves as a CD11b/CD18 counter-receptor during polymorphonuclear leukocyte transepithelial migration.

Leukocyte beta2-integrin CD11b/CD18 mediates the firm adhesion and subsequent transepithelial migration of polymorphonuclear leukocytes, but the identity of its counter-receptor(s) on epithelia remains elusive. Here we identified a monoclonal antibody, clone C3H7, which strongly bound to the basolateral membranes of epithelial cells and inhibited both the adhesion of epithelial cells to immobilized CD11b/CD8 and the transepithelial migration of PMNs in a physiologically relevant basolateral-to-apical direction. C3H7 antigen expression in epithelial monolayers was significantly increased by treatment with proinflammatory cytokine interferon-gamma or a combination of interferon-gamma and tumor necrosis factor-alpha. Up-regulation of C3H7 antigen was also observed in the epithelium of inflamed human colon tissues. Microsequencing and Western blotting of the purified antigen showed it to be CD44 variant 3 (CD44v3), a approximately 160-kDa membrane glycoprotein. Further studies demonstrated that this epithelial CD44v3 specifically binds to CD11b/CD18 through its heparan sulfate moieties. In summary, our study demonstrates for the first time that the heparan sulfate proteoglycan form of epithelial CD44v3 plays a critical role in facilitating PMN recruitment during inflammatory episodes via directly binding to CD11b/CD18.

Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium.

Receptor for advanced glycation endproducts (RAGE) is an Ig superfamily cell surface receptor that interacts with a diverse array of ligands associated with inflammatory responses. In this study, we provide evidence demonstrating that RAGE is involved in inflammatory responses in the intestines. We showed that RAGE is expressed in intestinal epithelial cells, primarily concentrated at the lateral membranes close to the apical cell junction complexes. Although RAGE expression was low in epithelium under normal conditions, this protein was up-regulated after treatment with the inflammatory cytokines IFN-gamma and/or TNF-alpha. RAGE expression was also elevated in colon tissue samples from patients with inflammatory bowel diseases. Using in vitro transmigration assays, we found that RAGE mediates neutrophil (polymorphonuclear leukocytes (PMN)) adhesion to, and subsequent migration across, intestinal epithelial monolayers. This activity appears to be mediated by the binding of RAGE to the PMN-specific beta(2) integrin CD11b/CD18. Thus, these results provide a novel mechanism for the regulation of PMN transepithelial migration and may suggest a new therapeutic target for intestinal inflammation.

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.