Fibronectin-LILRB4/gp49B interaction negatively regulates osteoclastogenesis through inhibition of RANKL-induced TRAF6/TAK1/NF-kB/MAPK signaling.

Dysregulation of osteoclasts, the multinucleated cells responsible for bone resorption, contributes to several degenerative bone disorders. Previously, we showed that blocking the leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4), a kind of inhibitory receptor that plays an important role in immune regulation, promotes osteoclast differentiation in vitro. Here, we explored whether gp49B, the murine ortholog of LILRB4, regulates osteoclastogenesis in vivo, and whether fibronectin (FN), a ligand of LILRB4/gp49B, certainly contributes to LILRB4/gp49B-mediated osteoclastogenesis. In comparison with wild-type mice, gp49B deficiency mice exhibited a loss of trabecular bone number and an increase in osteoclast formation. Gp49B knockout improved the bone resorptive capacity of osteoclasts derived from murine Raw264.7 cells by increasing osteoclast formation. We further revealed that gp49B deficiency increased the receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced signaling transduction by increasing the phosphorylation of transforming growth factor (TGF)-activated kinase 1 (TAK1), NF-κB and mitogen-activated protein kinases (MAPKs). Furthermore, the N-terminal 30 kDa proteolytic fragments of FN promoted gp49B-mediated inhibition of osteoclastogenesis by increasing Src homology-2-containing tyrosine phosphatase 1 (SHP-1) phosphorylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-SHP-1 association. In summary, the FN-LILRB4/gp49B interaction negatively regulates RANKL-induced TRAF6/TAK1/NF-κB/MAPK signaling in osteoclastogenesis.

Myeloid immune checkpoint ILT3/LILRB4/gp49B can co-tether fibronectin with integrin on macrophages.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin ß 1 in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49-integrin ß 1 signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin ß 1 become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B-FN-integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice.

The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4-FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4-FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

Gp49B is a pathogenic marker for auto-antibody-producing plasma cells in lupus-prone BXSB/Yaa mice.

AbstractImmune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the 'paradoxical' up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.

Deletion of N-myc downstream-regulated gene 2 attenuates reactive astrogliosis and inflammatory response in a mouse model of cortical stab injury.

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the CNS. In this study, we examined the expression and the role of Ndrg2 after cortical stab injury. We observed that Ndrg2 expression was elevated in astrocytes surrounding the wounded area as early as day 1 after injury in wild-type mice. Deletion of Ndrg2 resulted in lower induction of reactive astroglial and microglial markers in the injured cortex. Histological analysis showed reduced levels of hypertrophic changes in astrocytes, accumulation of microglia, and neuronal death in Ndrg2(-/-) mice after injury. Furthermore, activation of the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway, including the expression of IL-6 family cytokines and phosphorylation of STAT3, was markedly reduced in Ndrg2(-/-) mice after injury. In a culture system, both of Il6 and Gfap were up-regulated in wild-type astrocytes treated with forskolin. Deletion of Ndrg2 attenuated induction of these genes, but did not alter proliferation or migration of astrocytes. Adenovirus-mediated reexpression of Ndrg2 rescued the reduction of IL-6 expression after forskolin stimulation. These findings suggest that Ndrg2 plays a key role in reactive astrogliosis after cortical stab injury through a mechanism involving the positive regulation of IL-6/STAT3 signaling.

A case of reinitiation of modified electroconvulsive therapy 2 weeks after modified electroconvulsive therapy-induced Takotsubo cardiomyopathy in a male patient with major depressive disorder.

Background: Takotsubo cardiomyopathy (TCM) is a left ventricular dysfunction resembling acute coronary syndrome. Its prognosis is generally favorable; however, a subset of patients may present with severe complications. TCM is a rare side-effect of modified electroconvulsive therapy (ECT); it has been reported in 22 female and two male patients. Eight cases of ECT reinitiation after TCM have been reported (all females), with the shortest duration being 3 weeks. Case Presentation: We report the case of a 61-year-old man with a history of major depressive disorder and no history of heart disease or previous ECT treatment. Antidepressants had been ineffective, and ECT was indicated. After the third ECT session, the patient complained of chest pain and shortness of breath. Electrocardiography revealed ST elevation, and catheter angiography was used to diagnose TCM. The patient had mild heart failure but remained stable. Recognizing that ECT was effective, the patient asked for it to be reinitiated. We confirmed that the cardiac function had been normalized, applied a bisoprolol fumarate patch as a preventive measure, and reinitiated ECT 14 days after the onset of TCM. ECT was performed five times, with no recurrence of TCM and a marked improvement in depression. Conclusion: We describe a male patient with major depressive disorder who underwent reinitiation of ECT 2 weeks after ECT-induced TCM. Therefore, TCM should be recognized as a side-effect of ECT, even in men. Moreover, depending on whether the patient's condition is stable, ECT can be successfully performed in patients with TCM.

Behavioral stress and antidepressant treatments altered hippocampal expression of Nogo signal-related proteins in rats.

Some immune molecules including neurite outgrowth inhibitor (Nogo) ligands and their receptor（Nogo receptor-1: NgR1）are expressed at the neuronal synaptic sites. Paired immunoglobulin-like receptor B (PirB) is another Nogo receptor that also binds to major histocompatibility complex I and ß-amyloid and suppresses dendritic immune cell functions and neuronal plasticity in the central nervous system. Augmenting structural and functional neural plasticity by manipulating the Nogo signaling pathway is a novel promising strategy for treating brain ischemia and degenerative processes such as Alzheimer's disease. In recent decades psychiatric research using experimental animals has focused on the attenuation of neural plasticity by stress loadings and on the enhanced resilience by psychopharmacological treatments. In the present study, we examined possible expressional alterations in Nogo signal-related proteins in the rat hippocampus after behavioral stress loadings and antidepressant treatments. To validate the effectiveness of the procedures, previously reported increase in brain-derived neurotrophic factor (BDNF) by ECS or ketamine administration and decrease of BDNF by stress loadings are also shown in the present study. Significant increases in hippocampal NgR1 and PirB expression were observed following chronic variable stress, and a significant increase in NgR1 expression was observed under a single prolonged stress paradigm. These results indicate a possible contribution of enhanced Nogo signaling to the attenuation of neural plasticity in response to stressful experiences. Additionally, the suppression of hippocampal NgR1 expression using electroconvulsive seizure treatment and administration of subanesthetic dose of ketamine supported the increased neural plasticity induced by the antidepressant treatments.

Deletion of Atf6α enhances kainate-induced neuronal death in mice.

Excessive amount of L-glutamate in the brain causes neuronal damage in various pathological conditions including epilepsy and stroke. We previously reported that the 150-kDa oxygen-regulated protein (ORP150), a molecular chaperone in the endoplasmic reticulum (ER), inhibited the L-glutamate-induced neuronal death, at least partly, by improving Ca(2+) homeostasis in the ER. In the present study, we analyzed the role of activating transcription factor 6α (ATF6α), an upstream transcriptional factor critical for the operation of the ER, using mouse intrahippocampal kainate (KA) injection model. Expression of Hspa5, which encodes the molecular chaperone 78 kDa glucose-regulated protein (GRP78), increased after KA injection in the wild type (WT) mice. Comparative analysis using WT and Atf6α(-/-) mice revealed that KA induced pronounced neuronal death in the CA3 region of Atf6α(-/-) mice. The enhanced neuronal death in Atf6α(-/-) mice was associated with reduced expression of molecular chaperones in the ER and significant induction of c-fos in the hippocampal neurons. Furthermore, an injection of dantrolene, an inhibitor of ryanodine receptor, partially rescued these effects in Atf6α(-/-) mice after KA injection. Our results suggest that ATF6α plays an important role in neuronal survival after KA-induced excitotoxicity through the regulation of Ca(2+) response and neuronal activity.