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.

Excessive CD11c+Tbet+ B cells promote aberrant TFH differentiation and affinity-based germinal center selection in lupus.

Excessive self-reactive and inadequate affinity-matured antigen-specific antibody responses have been reported to coexist in lupus, with elusive cellular and molecular mechanisms. Here, we report that the antigen-specific germinal center (GC) response-a process critical for antibody affinity maturation-is compromised in murine lupus models. Importantly, this defect can be triggered by excessive autoimmunity-relevant CD11c+Tbet+ age-associated B cells (ABCs). In B cell-intrinsic Ship-deficient (ShipΔB) lupus mice, excessive CD11c+Tbet+ ABCs induce deregulated follicular T-helper (TFH) cell differentiation through their potent antigen-presenting function and consequently compromise affinity-based GC selection. Excessive CD11c+Tbet+ ABCs and deregulated TFH cell are also present in other lupus models and patients. Further, over-activated Toll-like receptor signaling in Ship-deficient B cells is critical for CD11c+Tbet+ ABC differentiation, and blocking CD11c+Tbet+ ABC differentiation in ShipΔB mice by ablating MyD88 normalizes TFH cell differentiation and rescues antigen-specific GC responses, as well as prevents autoantibody production. Our study suggests that excessive CD11c+Tbet+ ABCs not only contribute significantly to autoantibody production but also compromise antigen-specific GC B-cell responses and antibody-affinity maturation, providing a cellular link between the coexisting autoantibodies and inadequate affinity-matured antigen-specific antibodies in lupus models and a potential target for treating lupus.

IL4 (interleukin 4) induces autophagy in B cells leading to exacerbated asthma.

Allergic asthma is a common airway inflammatory disease in which B cells play important roles through IgE production and antigen presentation. SNP (single nucleotide polymorphism) analysis showed that Atg (autophagy-related) allele mutations are involved in asthma. It has been demonstrated that macroautophagy/autophagy is essential for B cell survival, plasma cell differentiation and immunological memory maintenance. However, whether B cell autophagy participates in asthma pathogenesis remains to be investigated. In this report, we found that autophagy was enhanced in pulmonary B cells from asthma-prone mice. Autophagy deficiency in B cells led to attenuated immunopathological symptoms in asthma-prone mice. Further investigation showed that IL4 (interleukin 4), a key effector Th2 cytokine in allergic asthma, was critical for autophagy induction in B cells both in vivo and in vitro, which further sustained B cell survival and enhanced antigen presentation by B cells. Moreover, IL4-induced autophagy depended on JAK signaling via an MTOR-independent, PtdIns3K-dependent pathway. Together, our data indicate that B cell autophagy aggravates experimental asthma through multiple mechanisms.

Immune-related GTPase Irgm1 exacerbates experimental auto-immune encephalomyelitis by promoting the disruption of blood-brain barrier and blood-cerebrospinal fluid barrier.

Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), is a T cell-mediated autoimmune condition characterized by prominent inflammation in the CNS. In this model, autoreactive T cells are primed in peripheral lymph nodes and migrate into uninflamed CNS across blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB) to initiate inflammation. However, the molecular mechanism controlling T cell migration remains to be determined. In an in vivo EAE mouse model, we have shown that Irgm1 (also known as LRG-47), a member of the immunity-related GTPase family, promotes the disruption of both BCSFB and BBB, and exacerbates the phenotypes of MOG-induced EAE. During EAE, Irgm1 was up-regulated in reactive astrocytes, ependymal cells and epithelial cells of the choroids plexus, which, in turn, promotes T cell infiltration into the CNS. Electron microscopy study showed that the MOG-induced disruption of both BBB and BCSFB was protected in the Irgm1(-/-) mice. Moreover, the expression of Claudin-5 (CLN-5), a major molecular determinant of BBB, in brain microvessel endothelial cells (BMVECs) was decreased in WT EAE mice while increased in Irgm1(-/-) mice. In addition, the expression of CC-chemokine ligand 20 (CCL-20), an important chemokine mediating lymphocyte trafficking across BCSFB, in the epithelial cells of choroids plexus was significantly suppressed in naïve and EAE-induced Irgm1(-/-) mice. These data suggest that Irgm1 is an important molecular regulator for the properties of both BBB and BCSFB, and a proinflammatory factor for EAE.

IRGM1 regulates oxidized LDL uptake by macrophage via actin-dependent receptor internalization during atherosclerosis.

Macrophage derived foam cells are actively involved in the initial phase of atherosclerosis. Uptake of modified lipoprotein such as oxidized LDL (oxLDL) is a critical step for foam cell formation. CD36 is the major receptor mediating oxLDL uptake by macrophage. However, the molecular mechanism underlying CD36 mediated oxLDL uptake remains unclear. Here we reported that IRGM1 (IRGM in human), a member of immunity-related small GTPase family, is essential for the actin-dependent CD36 mediated oxLDL uptake by macrophage. IRGM/IRGM1 was highly expressed by macrophage around the atherosclerotic plaque and was up-regulated by oxLDL both in vitro and in vivo. Moreover loss of IRGM/IRMG1 significantly decreased oxLDL uptake in both mouse and human. Furthermore, the IRGM1 knock-out mice displayed impaired CD36 internalization in macrophage, which was associated with the deficiency of F-actin polymerization. These results revealed a novel function of IRGM1 in regulating oxLDL uptake by macrophage during atherosclerosis.

Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke.

Autophagy is an important cellular recycling mechanism through self-digestion in responses to cellular stress such as starvation. Studies have shown that autophagy is involved in maintaining the homeostasis of the neural system during stroke. However, molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. Previously, we and others have shown that immune-related GTPase M (IRGM; termed IRGM1 in the mouse nomenclature) can regulate the survival of immune cells through autophagy in response to infections and autoimmune conditions. Here, using a permanent middle cerebral artery occlusion (pMCAO) mouse model, we found that IRGM1 was upregulated in the ischemic side of the brain, which was accompanied by a significant autophagic response. In contrast, neuronal autophagy was almost complete lost in Irgm1 knockout (KO) mice after pMCAO induction. In addition, the infarct volume in the Irgm1-KO pMCAO mice was significantly increased as compared to wild-type mice. Histological studies suggested that, at the early stage (within 24 h) of ischemia, the IRGM1-dependent autophagic response is associated with a protection of neurons from necrosis in the ischemic core but a promotion of neuronal apoptosis in the penumbra area. These data demonstrate a novel role of IRGM1 in regulating neuronal autophagy and survival during ischemic stroke.