Tissue Kallikrein-1 Suppresses Type I Interferon Responses and Reduces Depressive-Like Behavior in the MRL/lpr Lupus-Prone Mouse Model.

Excessive production and response to Type I interferons (IFNs) is a hallmark of systemic lupus erythematosus (SLE). Neuropsychiatric lupus (NPSLE) is a common manifestation of human SLE, with major depression as the most common presentation. Clinical studies have demonstrated that IFNα can cause depressive symptoms. We have shown that the kallikrein-kinin system (KKS) [comprised of kallikreins (Klks) and bradykinins] and angiotensin-converting enzyme inhibitors suppressed Type I IFN responses in dendritic cells from lupus-prone mice and human peripheral blood mononuclear cells. Tissue Klk genes are decreased in patients with lupus, and giving exogenous Klk1 ameliorated kidney pathology in mice. We retro-orbitally administered mouse klk1 gene-carrying adenovirus in the Murphy Roths Large lymphoproliferative (MRL/lpr) lupus-prone mice at early disease onset and analyzed immune responses and depressive-like behavior. Klk1 improved depressive-like behavior, suppressed interferon-responsive genes and neuroinflammation, and reduced plasma IFNα levels and proinflammatory cytokines. Klk1 also reduced IFNAR1 and JAK1 protein expression, important upstream molecules in Type I IFN signaling. Klk1 reduced bradykinin B1 receptor expression, which is known to induce proinflammatory response. Together, these findings suggest that Klk1 may be a potential therapeutic candidate to control IFNα production/responses and other inflammatory responses in SLE and NPSLE.

Evidence that inositol 1,4,5-trisphosphate 3-kinase and inositol 1,3,4,5-tetrakisphosphate are negative regulators of platelet function.

Background: Inositol 1,3,4,5-tetrakisphosphate (IP4) is formed from inositol 1,4,5-trisphosphate (IP3) by IP3 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca2+ entry, IP3 regulation, and phosphoinositide 3-kinase antagonism. Objectives: To better elucidate a function for IP4, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP4 directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to pleckstrin-homology (PH) domain-containing proteins in platelets. Methods: Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca2+ studies, or resuspended in high K+ and low Na+ buffers for permeabilization experiments. Phosphorylation of AKT-Ser473 and Rap1-GTP formation were measured by Western blotting and PIP3 binding using PIP3 beads. Results: GNF362-enhanced platelet aggregation stimulated by low concentrations of ADP, collagen, thrombin, U46619, and thrombus formation in collagen-coated capillaries. GNF362 induced a transient elevation of Ca2+ concentration, elevated basal levels of IP3, and enhanced the peak height of Ca2+ elevated by agonists. In permeabilized platelets, IP4 inhibited GTPγS induced formation of AKT-Ser473 phosphorylation and platelet aggregation. IP4 reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP3 beads. Conclusion: ITPK and IP4 are negative regulators of platelet function. IP4 regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling.

Background: Heat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. Objectives: In this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. Methods: HSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. Results: Using subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2ß1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. Conclusion: The present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

Centrally Acting Angiotensin-Converting Enzyme Inhibitor Suppresses Type I Interferon Responses and Decreases Inflammation in the Periphery and the CNS in Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE, often causing depression. Interferon-α (IFNα) is a central mediator in disease pathogenesis. Administration of IFNα to patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation, and brain functions. It is known that ACE degrades bradykinin (BK) into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells (BMDC) and human peripheral blood mononuclear cells that captopril (a centrally acting ACE inhibitor-ACEi) suppressed Type I IFN responsive gene (IRG) expression. In this report, we used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to determine the in vivo effects of captopril on Type I IFN and associated immune responses in the periphery and brain and effects on behavior. Administering captopril to MRL/lpr mice decreased expression of IRGs in brain, spleen and kidney, decreased circulating and tissue IFNα levels, decreased microglial activation (IBA-1 expression) and reduced depressive-like behavior. Serotonin levels that are decreased in depression were increased by captopril treatment. Captopril also reduced autoantibody levels in plasma and immune complex deposition in kidney and brain. Thus, ACEi's may have potential for therapeutic use for systemic and NPSLE.