Herpesvirus entry mediator (HVEM) attenuates signals mediated by the lymphotoxin β receptor (LTβR) in human cells stimulated by the shared ligand LIGHT

Signals mediated by members of the tumor necrosis factor receptor superfamily modulate a network of diverse processes including initiation of inflammatory responses and altering cell fate between pathways favoring survival and death. Although such pathways have been well-described for the TNF-α receptor, less is known about signaling induced by the TNF superfamily member LIGHT and how it is differentially altered by expression of its two receptors LTβR and HVEM in the same cell. We used cell lines with different relative expression of HVEM and LTβR to show that LIGHT-induced signals mediated by these receptors were associated with altered TRAF2 stability and RelA nuclear translocation. Production of the inflammatory chemokine CXCL10 was primarily mediated by LTβR. Higher expression of HVEM was associated with cell survival, while unopposed LTβR signaling favored pathways leading to apoptosis. Importantly, restoring HVEM expression in cells with low endogenous expression recapitulated the phenotype of cells with higher endogenous expression. Together, our data provide evidence that relative expression of HVEM and LTβR modulates canonical NF-κB and pro-apoptotic signals stimulated by LIGHT.     

Lymphotoxin and LIGHT signaling pathways and target genes

Summary: Lymphotoxins (LTα and LTβ), LIGHT [homologous to LT, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes], tumor necrosis factor (TNF), and their specific receptors LTβR, HVEM, and TNF receptor 1 (TNFR1) and TNFR2, form the immediate family of the larger TNF superfamily. These cytokines establish a critical communication system required for the development of secondary lymphoid tissues; however, knowledge of the target genes activated by these signaling pathways is limited. Target genes regulated by the LTαβ‐LTβR pathway include the tissue‐organizing chemokines, CXCL13, CCL19, and CCL21, which establish cytokine circuits that regulate LT expression on lymphocytes, leading to organized lymphoid tissue. Infectious disease models have revealed that LTαβ pathways are also important for innate and adaptive immune responses involved in host defense. Here, regulation of interferon‐β by LTβR and TNFR signaling may play a crucial role in certain viral infections. Regulation of autoimmune regulator in the thymus via LTβR implicates LT/LIGHT involvement in central tolerance. Dysregulated expression of LIGHT overrides peripheral tolerance leading to T‐cell‐driven autoimmune disease. Blockade of TNF/LT/LIGHT pathways as an intervention in controlling autoimmune diseases is attractive, but such therapy may have risks. Thus, identifying and understanding the target genes may offer an opportunity to fine‐tune inhibitory interventions.     

Molecular mechanisms of the biphasic effects of interferon-γ on osteoclastogenesis

Although interferon-γ (IFN-γ) potently inhibits osteoclastogenesis, the suppressive effect is significantly reduced when osteoclast precursors are pre-exposed to the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular mechanism underlying the biphasic effects of IFN-γ on osteoclastogenesis remains elusive. Here, we recapitulate the biphasic functions of IFN-γ in osteoclastogenesis in both tissue culture dishes and on bone slices. We further demonstrate that IFN-γ markedly suppresses the RANKL-induced expression of nuclear factor of activated T-cells c1 (NFATc1) in normal, but not RANKL-pretreated bone marrow macrophages (BMMs). Similarly, IFN-γ impairs the activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in normal, but not RANKL-pretreated, BMMs. These findings indicate that IFN-γ inhibits osteoclastogenesis partially by suppressing the expression of NFATc1 and the activation of the NF-κB and JNK pathways. Moreover, IFN-γ inhibits the RANKL-induced expression of osteoclast genes, but RANKL pretreatment reprograms osteoclast genes into a state in which they can no longer be suppressed by IFN-γ, indicating that IFN-γ inhibits osteoclastogenesis by blocking the expression of osteoclast genes. Finally, the IVVY(535-538) motif in the cytoplasmic domain of RANK is responsible for rendering BMMs refractory to the inhibitory effect of IFN-γ. Taken together, these findings provide important mechanistic insights into the biphasic effects of IFN-γ on osteoclastogenesis.     

The signaling networks of the herpesvirus entry mediator (TNFRSF14) in immune regulation

The tumor necrosis factor (TNF) receptor superfamily member herpesvirus entry mediator (HVEM) (TNFRSF14) regulates T-cell immune responses by activating both inflammatory and inhibitory signaling pathways. HVEM acts as both a receptor for the canonical TNF-related ligands, LIGHT [lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed on T lymphocytes] and lymphotoxin-α, and as a ligand for the immunoglobulin superfamily proteins BTLA (B and T lymphocyte attenuator) and CD160, a feature distinguishing HVEM from other immune regulatory molecules. The ability of HVEM to interact with multiple ligands in distinct configurations creates a functionally diverse set of intrinsic and bidirectional signaling pathways that control both inflammatory and inhibitory responses. The HVEM system is integrated into the larger LTβR and TNFR network through extensive shared ligand and receptor usage. Experimental mouse models and human diseases indicate that dysregulation of HVEM network may contribute to autoimmune pathogenesis, making it an attractive target for drug intervention.     

Inhibition of titanium particle-induced osteoclastogenesis through inactivation of NFATc1 by VIVIT peptide

Osteoclastogenesis induced by particulate wear debris is a major pathological factor contributing to periprosthetic osteolysis. Although the nuclear factor of activated T cells c1 (NFATc1) is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In the present study, we investigated the role of NFATc1 in the regulation of osteoclast differentiation from bone marrow macrophages (BMMs) stimulated with titanium (Ti) particles. The results showed that Ti particles could stimulate BMMs to produce proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and differentiate into multinucleated osteoclasts in the presence of receptor activator of nuclear factor-κB ligand (RANKL). NFATc1 was expressed in BMMs and multinucleated cells cultured with Ti particles and RANKL. Inactivation of NFATc1 by 11R-VIVIT peptide potently impeded the Ti particle-induced osteoclastogenesis. 11R-VIVIT peptide does not have toxic effect on BMMs. Based on these data, we conclude that inactivation of NFATc1 by VIVIT peptide would provide a promising therapeutic target for the treatment of periprosthetic osteolysis.     

Pathological significance and regulatory mechanism of lymphotoxin β receptor overexpression in T cells of patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a typical autoimmune disease. Lymphotoxin β receptor (LTβR) signaling plays an important role in autoimmune inflammations. LTβR-Ig fusion protein, LTβR blocking agent, has been used to treat SLE, while its mechanism remains to be fully elucidated. In this study, to investigate the expression of LTβR in the T cells of SLE patients and its roles in the pathogenesis of SLE, we isolated the peripheral blood T cells of SLE patients and normal controls to detect expression of LTβR by flow cytometry and RNA assay. T cells were also stimulated with LIGHT, a ligand of LTβR, and then detected for their LTβR expressions and apoptosis by flow cytometry. Also, their expressions of inflammatory factors and receptors were determined by RNA assay. The results showed that LTβR positive cells were 22.75%
6.98% in CD3+ cells of SLE patients, while there were almost no LTβR positive cells in CD3+ cells of normal persons. Moreover, LTβR expression was remarkably higher in CD3, CD4 and CD8 positive T cells of active SLE patients than non/low active patients (all P < 0.05), and positively correlated with increased Ig level, decreased complement level and renal damage. Moreover, the stimulation of SLE T cells with LIGHT promoted higher expression of LTβR, IL-23R and IL-17A, and apoptosis of T cells. In conclusion, we demonstrated a high expression of LTβR in the T cells of SLE patients which may be associated with pathogenesis of SLE.     

LIGHT/TNFSF14 regulates estrogen deficiency-induced bone loss

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14^−/−) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag ^−/−Tnfsf14 ^−/−) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Lymphotoxin beta receptor-Ig fusion protein treatment blocks actively induced,but not adoptively transferred,uveitis in Lewis rats

Previous studies have shown that treatment of rodents with a lymphotoxin (LT) beta receptor-Ig fusion protein (LTbetaR-Ig), which binds to both LT and LIGHT, prevents the development of autoimmune diseases, but the mechanism involved is unclear. To explore the potential role of LT or LIGHT in the pathogenesis of autoimmune uveitis, uveitis was induced in Lewis rats either by immunization with an uveitogenic peptide, R16, derived from the interphotoreceptor retinoid-binding protein, or by adoptive transfer of R16-specific T cells. Interestingly, LTbetaR-Ig treatment completely prevented actively induced uveitis, but not the adoptively transferred disease. We also show that LTbetaR-Ig-treated R16-injected rats had a significantly decreased T cell response to R16 and that herpesvirus entry mediator (HVEM)-Ig, a fusion protein that blocks LIGHT, also inhibited disease development. Our results suggest that LT or LIGHT plays a critical role in the induction, rather than the effector, phase of the disease.     

Crocin inhibits RANKL-induced osteoclast formation and bone resorption by suppressing NF-κB signaling pathway activation

Crocin is a dietary compound with antioxidant and anti-inflammatory properties, but its effects on bone resorption have not been well characterized. Here we address this issue by examining the direct effects of crocin on osteoclast cells in vitro. Osteoclastogenesis was induced by RANKL (receptor activator of NF-κB ligand) in mouse bone marrow-derived macrophages in the absence or presence of crocin at various concentrations. Further, the bone resorption activity of mature osteoclast treated with crocin was assessed by pit assay. Without altering cell viability, crocin was shown to inhibit the differentiation and function of osteoclast cells in a dose-dependent manner. Mechanistically, RANKL-induced NF-κB and NFATc1 activation, the critical signaling pathways for osteoclast differentiation and function, were both repressed by crocin in bone marrow-derived macrophages. Thus, crocin suppresses osteoclastogenesis through direct inhibition of intracellular molecular pathways, which may contribute to future development of anti-bone resorption treatment.     

Mineral trioxide aggregate solution inhibits osteoclast differentiation through the maintenance of osteoprotegerin expression in osteoblasts

Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity. The aim of this study was to investigate whether MTA may prevent osteoclast differentiation in vitro. MTA solution, but not other commonly used retrofilling materials, such as Dycal, Super-EBA, or intermediate restorative material (IRM) solution, dose-dependently inhibited osteoclastogenesis in cocultures of mouse bone marrow cells (BMCs) with primary osteoblast cells (POBs) induced by 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2) D(3) ]. Exogenous CaCl(2) medium supplementation did not inhibit osteoclastogenesis in cocultures. Furthermore, MTA solution did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, suggesting that POBs are targets of MTA. MTA solution suppressed the 1α,25(OH)(2) D(3) -induced reduction of osteoprotegerin (OPG) mRNA and protein production without changing RANKL expression in POBs. Consistent with this result, MTA solution did not inhibit osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Therefore, the maintenance of OPG expression in POBs appears to be critical for the inhibitory effect of MTA solution on osteoclast differentiation.     

Kaempferol Inhibits IL-1β-Stimulated,RANKL-mediated Osteoclastogenesis via Downregulation of MAPKs,c-Fos,and NFATc1

Kaempferol is one of the most common flavonoid that is present in a variety of vegetables and fruits and has effects on bone metabolism. The present study was performed to define the effects of kaempferol on interleukin (IL)-1β-stimulated receptor activator of NF-κB ligand (RANKL)-mediated osteoclast differentiation. Bone marrow cells were harvested from 6-week-old male imprinting control region mice, and the differentiation of osteoclasts from these cells was evaluated by tartrate-resistant acid phosphatase staining and resorption pit formation assay. Phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p38, phosphorylated c-Jun amino-terminal kinase, NF-κB (p65), IκBα, c-Fos, and nuclear factor of activated T cells c1 (NFATc1) expressions were examined by Western blotting and quantitative RT-PCR. Kaempferol inhibits IL-1β-stimulated, RANKL-mediated osteoclast differentiation and also inhibits IL-1β-stimulated, RANKL-mediated phosphorylation of ERK 1/2, p38 and JNK MAP kinases, and expressions of c-Fos and NFATc1. These results indicate that kaempferol has an inhibitory role in the bone loss by preventing osteoclast formation and suggest that it might be a novel therapeutic agent for the treatment of inflammatory arthritis by managing bone destruction.     

Mitogen- and stress-activated protein kinase 1 activates osteoclastogenesis in vitro and affects bone destruction in vivo

Mitogen- and stress-activated protein kinase (MSK) 1 is an important regulator of immune response and mitogenic signaling. In this study, we report for the first time that MSK1 was activated by the osteoclast differentiation factor receptor activator of nuclear factor kappa B ligand (RANKL) in osteoclast precursor cells. Inhibition of upstream kinases ERK1/2 and p38, but not JNK, suppressed MSK activation upon RANKL stimulation. An MSK1 inhibitor efficiently prevented the induction of c-Fos and NFATc1 and CREB phosphorylation by RANKL. Inhibition of MSK1 also successfully blocked RANKL-induced osteoclastogenesis. MSK knockdown with small interfering RNA significantly inhibited osteoclast differentiation and bone resorption. MSK1 did not affect osteoclast survival. The induction of c-Fos and NFATc1 and the phosphorylation of CREB and ATF2 were also inhibited by MSK1 knockdown. Moreover, knockdown of MSK1 significantly blocked recruitment of c-Fos to the NFATc1 promoter upon RANKL stimulation. Therefore, NFATc1-inducible osteoclast-specific genes were downregulated by MSK1 blockade. NFATc1 retrovirus transduction almost completely rescued the differentiation defect of MSK1-silenced cells. In vivo knockdown of MSK1 reduced RANKL-induced bone resorption as well as osteoclast formation. Thus, our results suggested that MSK1 is an important novel molecule involved in RANKL signaling and osteoclast differentiation.     

Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways

Summary: Cytokines mediate key communication pathways essential for regulation of immune responses. Full activation of antigen-responding lymphocytes requires cooperating signals from the tumor necrosis factor (TNF)-related cytokines and their specific receptors. LIGHT, a lymphotoxin-β (LTβ)-related TNF family member, modulates T-cell activation through two receptors, the herpesvirus entry mediator (HVEM) and indirectly through the LT-β receptor. An unexpected finding revealed a non-canonical binding site on HVEM for the immunoglobulin superfamily member, B and T lymphocyte attenuator (BTLA), and an inhibitory signaling protein suppressing T-cell activation. Thus, HVEM can act as a molecular switch between proinflammatory and inhibitory signaling. The non-canonical HVEM–BTLA pathway also acts to counter LTβR signaling that promotes the proliferation of antigen-presenting dendritic cells (DCs) within lymphoid tissue microenvironments. These results indicate LTβ receptor and HVEM–BTLA pathways form an integrated signaling circuit. Targeting these cytokine pathways with specific antagonists (antibody or decoy receptor) can alter lymphocyte differentiation and activation. Alternately, agonists directed at their cell surface receptors can restore homeostasis and potentially reset immune and inflammatory processes, which may be useful in treating autoimmune and infectious diseases and cancer.     

Metabolic activation and colitis pathogenesis is prevented by lymphotoxin β receptor expression in neutrophils

Inflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTβR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTβR and the mechanism critical for exacerbation of colitis. Specific deletion of LTβR in neutrophils (LTβR^ΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTβR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTβR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis.     

破骨细胞分化成熟调节的分子机理研究进展

破骨细胞 (ｏｓｔｅｏｃｌａｓｔ ,ＯＣ)来源于骨髓中的粒 -巨噬细胞克隆形成单位 (ｇｒａｎｕｌｏｃｙｔｅａｎｄｍａｃｒｏｐｈａｇｅｃｏｌｏｎｙｆｏｒｍｉｎｇｃｅｌｌｓ ,ＣＦＵ -ＧＭ)。ＯＣ的分化和 /或功能变化所导致的骨再建失衡是多种代谢性骨病 ,如骨质疏松症、骨质硬化症和畸形性骨炎 (Ｐａｇｅｔ骨病 )等形成的病理基础。目前已经证明多种激素和局部细胞因子如活性维生素Ｄ3 (1,2 5 (ＯＨ) 2 Ｄ3 )、糖皮质激素、甲状旁腺素 (ＰＴＨ)、前列腺素 (ＰＧＥ2 )、ＩＬ - 4、ＩＬ -6、ＩＬ - 11、ＩＬ - 12等不仅能调节破骨细胞生成…