Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. METHODS: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-ß)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. RESULTS: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-ß-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. CONCLUSION: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Distinct subsets of synovial fibroblasts control cartilage destruction in joint diseases.

Inflammation-induced bone destruction is the main cause of progressive joint damage in rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, depending on the tissue microenvironment stimulators, the synovium transforms into a hyperplastic invasive tissue. The synovium includes two specific subsets of fibroblasts surrounding the joints: lining and sublining synovial fibroblasts (SFs). These SFs grow and interact with immune cells invading the bone and cartilage; specifically, SFs, which are the major mesenchymal cells in the joints, develop an aggressive phenotype, thereby producing cytokines and proteases involved in arthritis pathogeneses. Transcriptomic differences in the heterogeneity of SFs reflect the joint-specific origins of the SFs interacting with immune cells. To understand the subsets of SFs that lead to joint damage in arthritis, clarifying the distinct phenotypes and properties of SFs and understanding how they influence bone cells, such as osteoclasts and chondrocytes, is crucial. This review provides an overview of the advancements in the understanding of SF subsets and features, which may aid in identifying newer therapeutic targets.

Repurposing Astragalus Polysaccharide PG2 for Inhibiting ACE2 and SARS-CoV-2 Spike Syncytial Formation and Anti-Inflammatory Effects.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to global public health. In an effort to develop novel anti-coronavirus therapeutics and achieve prophylactics, we used gene set enrichment analysis (GSEA) for drug screening and identified that Astragalus polysaccharide (PG2), a mixture of polysaccharides purified from Astragalus membranaceus, could effectively reverse COVID-19 signature genes. Further biological assays revealed that PG2 could prevent the fusion of BHK21-expressing wild-type (WT) viral spike (S) protein and Calu-3-expressing ACE2. Additionally, it specifically prevents the binding of recombinant viral S of WT, alpha, and beta strains to ACE2 receptor in our non-cell-based system. In addition, PG2 enhances let-7a, miR-146a, and miR-148b expression levels in the lung epithelial cells. These findings speculate that PG2 has the potential to reduce viral replication in lung and cytokine storm via these PG2-induced miRNAs. Furthermore, macrophage activation is one of the primary issues leading to the complicated condition of COVID-19 patients, and our results revealed that PG2 could regulate the activation of macrophages by promoting the polarization of THP-1-derived macrophages into an anti-inflammatory phenotype. In this study, PG2 stimulated M2 macrophage activation and increased the expression levels of anti-inflammatory cytokines IL-10 and IL-1RN. Additionally, PG2 was recently used to treat patients with severe COVID-19 symptoms by reducing the neutrophil-to-lymphocyte ratio (NLR). Therefore, our data suggest that PG2, a repurposed drug, possesses the potential to prevent WT SARS-CoV-2 S-mediated syncytia formation with the host cells; it also inhibits the binding of S proteins of WT, alpha, and beta strains to the recombinant ACE2 and halts severe COVID-19 development by regulating the polarization of macrophages to M2 cells.

Mesenchymal Stem Cell Secreted-Extracellular Vesicles are Involved in Chondrocyte Production and Reduce Adipogenesis during Stem Cell Differentiation.

BACKGROUND: Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity. METHODS: The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosome-specific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation. RESULTS: The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton's jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition. Moreover, EVs decreased the lipid accumulation rate during adipogenic induction. CONCLUSION: The results indicated that the production model could facilitate production of effective EVs and further demonstrated the role of MSC-derived EVs in cell differentiation. MSC-derived EVs could be successfully used in cell-free therapy to guide chondrogenic differentiation of ADSC for future clinical applications in cartilage regeneration.

Adipose-Derived Stem Cell Exosomes as a Novel Anti-Inflammatory Agent and the Current Therapeutic Targets for Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA), a chronic inflammatory joint disorder, may not respond adequately to current RA treatments. Mesenchymal stem cells (MSCs) elicit several immunomodulatory and anti-inflammatory effects and, thus, have therapeutic potential. Specifically, adipose-derived stem cell (ADSC)-based RA therapy may have considerable potency in modulating the immune response, and human adipose tissue is abundant and easy to obtain. Paracrine factors, such as exosomes (Exos), contribute to ADSCs' immunomodulatory function. ADSC-Exo-based treatment can reproduce ADSCs' immunomodulatory function and overcome the limitations of traditional cell therapy. ADSC-Exos combined with current drug therapies may provide improved therapeutic effects. Using ADSC-Exos, instead of ADSCs, to treat RA may be a promising cell-free treatment strategy. This review summarizes the current knowledge of medical therapies, ADSC-based therapy, and ADSC-Exos for RA and discusses the anti-inflammatory properties of ADSCs and ADSC-Exos. Finally, this review highlights the expanding role and potential immunomodulatory activity of ADSC-Exos in patients with RA.

Radiographic outcomes of the treatment of complex femoral shaft fractures (AO/OTA 32-C) with intramedullary nailing: a retrospective analysis of different techniques.

OBJECTIVES: To assess the results of open versus closed reduction in intramedullary nailing (IMN) for complex femoral fractures (Arbeitsgemeinschaft für Osteosynthesefragen Foundation/Orthopaedic Trauma Association [AO/OTA]: 32-C) and to determine the factors involved in bone healing. METHODS: This retrospective study involved 47 consecutive patients with complex femoral diaphyseal fractures who underwent reduction and fixation. RESULTS: All open-reduction and 12 closed-reduction patients (52.17%) had an anatomical-to-small gap. The closed-small group had the highest bone union rate (100%), followed by the open-reduction (79.17%) and closed-large groups (72.73%); intergroup differences were significant. The closed-small group had the shortest mean union time (7.31 months), followed by the open-reduction group (7.58 months). The closed-large group had a significantly longer union time (9.75 months) than those in the closed-small and open-reduction groups. Femoral radiographic union scores in the closed-small and open-reduction groups were similar at three timepoints; scores were higher than those in the closed-large group, with a significant difference 6 and 9 months post-operatively. CONCLUSION: IMN with closed reduction for complex femoral shaft fractures had better outcomes and fewer complications versus open reduction. For unsatisfactory closed reduction outcomes (i.e., residual gap >10 mm), minimally invasive techniques or open reduction with minimal stripping should be considered.

Increased HIF-1α expression in T cells and associated with enhanced Th17 pathway in systemic lupus erythematosus.

BACKGROUND/PURPOSE: Recent emerging evidence indicates that dysfunction of metabolic remodeling underlies aberrant T cell immune responses in systemic lupus erythematosus (SLE). This study was undertaken to investigate the expression of HIF-1α, a regulator of metabolic reprogramming, in T cells from SLE. METHODS: HIF-1α expression in T lymphocytes from SLE patients was examined by quantitative polymerase chain reaction (PCR) and the protein expression was analyzed with intracellular staining in flow cytometry. HIF-1α was overexpressed in murine CD4 T cells via transducing T cells with HIF-1α containing lentivirus. The expression of HIF-1α, metabolic- and Th17-associated genes in T cells from SLE patients and its association with clinical manifestation was analyzed. RESULTS: HIF-1α expression is increased in CD4 T cells from SLE patients both in intracellular staining and quantitative PCR analysis. In addition, there is enhanced HIF-1α expression in Th17-skewing murine T cells, and lentivirus-mediated HIF-1α overexpression promotes Th17 differentiation. Moreover, HIF-1α gene expression is positively correlated with the expression of glycolysis- and IL-17-associated genes in SLE patients. CONCLUSION: HIF-1α expression is increased in T cells from SLE patients, and is positively correlated with glycolysis- and Th17- associated pathway, implicating HIF-1α contributes to the activation of Th17 cells in SLE, and represents a potential novel therapeutic target.

Delivery of Mesenchymal Stem Cell in Dialdehyde Methylcellulose-Succinyl-Chitosan Hydrogel Promotes Chondrogenesis in a Porcine Model.

Due to the limitation in the current treatment modalities, such as secondary surgery in ACI and fibrocartilage formation in microfracture surgery, various scaffolds or hydrogels have been developed for cartilage regeneration. In the present study, we used sodium periodate to oxidize methylcellulose and formed dialdehyde methylcellulose (DAC) after dialysis and freeze-drying process, DAC was further mixed with succinyl-chitosan (SUC) to form an DAC-SUC in situ forming hydrogel. The hydrogel is a stiffness, elastic-like and porous hydrogel according to the observation of SEM and rheological analysis. DAC-SUC13 hydrogel possess well cell-compatibility as well as biodegradability. Most bone marrow mesenchymal stem cells (BM-pMSCs) were alive in the hydrogel and possess chondrogenesis potential. According to the results of animal study, we found DAC-SUC13 hydrogel can function as a stem cell carrier to promote glycosaminoglycans and type II collagen synthesis in the osteochondral defects of porcine knee. These findings suggested that DAC-SUC13 hydrogel combined with stem cell is a potential treatment for cartilage defects repair in the future.

Potential of Using Infrapatellar-Fat-Pad-Derived Mesenchymal Stem Cells for Therapy in Degenerative Arthritis: Chondrogenesis, Exosomes, and Transcription Regulation.

Infrapatellar fat pad-derived mesenchymal stem cells (IPFP-MSCs) are a type of adipose-derived stem cell (ADSC). They potentially contribute to cartilage regeneration and modulation of the immune microenvironment in patients with osteoarthritis (OA). The ability of IPFP-MSCs to increase chondrogenic capacity has been reported to be greater, less age dependent, and less affected by inflammatory changes than that of other MSCs. Transcription-regulatory factors strictly regulate the cartilage differentiation of MSCs. However, few studies have explored the effect of transcriptional factors on IPFP-MSC-based neocartilage formation, cartilage engineering, and tissue functionality during and after chondrogenesis. Instead of intact MSCs, MSC-derived extracellular vesicles could be used for the treatment of OA. Furthermore, exosomes are increasingly being considered the principal therapeutic agent in MSC secretions that is responsible for the regenerative and immunomodulatory functions of MSCs in cartilage repair. The present study provides an overview of advancements in enhancement strategies for IPFP-MSC chondrogenic differentiation, including the effects of transcriptional factors, the modulation of released exosomes, delivery mechanisms for MSCs, and ethical and regulatory points concerning the development of MSC products. This review will contribute to the understanding of the IPFP-MSC chondrogenic differentiation process and enable the improvement of IPFP-MSC-based cartilage tissue engineering.

T Cell-Specific Deletion of TRAIL Receptor Reveals Its Critical Role for Regulating Pathologic T Cell Activation and Disease Induction in Experimental Autoimmune Encephalomyelitis.

Recent evidence from several autoimmune animal models has demonstrated that TRAIL suppresses the activation of T cells and inhibits autoimmune inflammation via an apoptosis-independent pathway. However, it remains unclear whether the immunosuppressive effects of TRAIL are dependent on its direct effects on T cells or on other immune cells to regulate T cells for the induction of disease. Therefore, we generated mice with T cell-specific TRAIL receptor (TRAIL-R) conditional knockout to investigate the impact of TRAIL on autoimmune inflammation and disease induction in experimental autoimmune encephalomyelitis (EAE). T cell-specific TRAIL-R knockout mice were found to completely reverse the TRAIL-mediated suppression of inflammation and disease induction, indicating that TRAIL-R on T cells is essential for TRAIL-mediated suppression of inflammation and disease induction in EAE. Moreover, the immune suppression effects were not due to the induction of cell apoptosis, but to the direct inhibition of T cell activation. In addition, RNA sequencing and transcriptome analysis revealed that TRAIL-R signaling significantly downregulated the genes involved in TCR signaling pathways, T cell differentiation, and proinflammatory cytokines. These results indicate that TRAIL-R on T cells is critical for pathologic T cell activation and induction of inflammation in EAE, suggesting that TRAIL-R serves as a novel immune checkpoint receptor in T cell-mediated autoimmune diseases.

Tissue microenvironment dictates inflammation and disease activity in rheumatoid arthritis.

The recent advance in treatments for rheumatoid arthritis (RA) has significantly improved the prognosis of RA patients. However, these novel therapies do not work well for all RA patients. The unmet need suggests that the current understanding about how inflammatory response arises and progresses in RA is limited. Recent accumulating evidence reveals an important role for the tissue microenvironment in the pathogenesis of RA. The synovium, the main tissue where the RA activity occurs, is composed by a unique extracellular matrix (ECM) and residing cells. The ECM molecules provide environmental signals that determine programmed site-specific cell behavior. Improved understanding of the tissue microenvironment, especially how the synovial architecture, ECM molecules, and site-specific cell behavior promote chronic inflammation and tissue destruction, will enhance deciphering the pathogenesis of RA. Moreover, in-depth analysis of tissue microenvironment will allow us to identify potential therapeutic targets. Research is now undertaken to explore potential candidates, both cellular and ECM molecules, to develop novel therapies. This article reviews recent advances in knowledge about how changes in cellular and ECM factors within the tissue microenvironment result in propagation of chronic inflammation in RA.

TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment.

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between members of the tumor necrosis factor (TNF) ligand superfamily and their receptors. Recent evidence indicated that TNF-α-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation via a TRAF6-dependent signaling pathway; but paradoxically, it inhibits RANK ligand (RANKL)-induced osteoclast differentiation. Although a number of signaling pathways were linked to the RANK and osteoclastogenesis, it is not known how TRAIL regulates RANK signaling. In this study, we demonstrate that TRAIL regulates RANK-induced osteoclastogenesis in terms of the assembly of lipid raft-associated signaling complexes. RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of lipid raft-associated signaling complexes and TRAF6 recruitment was abolished in the presence of TRAIL. TRAIL-induced dissociation of RANKL-induced lipid raft signaling complexes was reversed by treatment with TRAIL receptor (TRAIL-R) siRNA or an anti-TRAIL-R blocking antibody, indicating that TRAIL mediates suppression of RANKL-induced lipid raft signaling via interactions with TRAIL-R. Finally, we demonstrated that TRAIL suppressed inflammation-induced bone resorption and osteoclastogenesis in a collagen-induced arthritis (CIA) rat animal model. Our results provide a novel apoptosis-independent role of TRAIL in regulating RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment.

An apoptosis-independent role of TRAIL in suppressing joint inflammation and inhibiting T-cell activation in inflammatory arthritis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been implicated in the regulation of inflammation in rheumatoid arthritis (RA), primarily due to its ability to promote apoptosis in synoviocytes and infiltrating lymphocytes. The aim of this study was to investigate the immunomodulatory mechanism and role of TRAIL in inflammatory arthritis. We created an animal model of inflammatory arthritis and demonstrated that TRAIL significantly inhibited joint inflammation and reduced the severity of arthritis. The suppression of joint inflammation was not due to the TRAIL-mediated induction of apoptosis in T cells, macrophages or synovial fibroblasts. In contrast, TRAIL directly inhibited T-cell proliferation and suppressed the production of cytokines, which indicated that TRAIL exerted its anti-inflammatory effects by direct inhibition of T-cell activation. Moreover, TRAIL receptor (TRAIL-R)-knockout mice developed more severe disease, and the protective effects of TRAIL were abolished in the experimental arthritis model in TRAIL-R knockout mice. From these results, we conclude that TRAIL suppresses joint inflammation via an apoptosis-independent pathway and directly inhibits T-cell activation. Our results provide a novel apoptosis-independent, immune regulatory role for TRAIL in suppressing inflammatory arthritis and shed light on the development of effective new therapies for autoimmune inflammatory diseases.

Galectin-3 suppresses mucosal inflammation and reduces disease severity in experimental colitis.

UNLABELLED: Galectin-3, a member of the ß-galactoside-binding lectin family, expresses in many different immune cells and modulates broad biological functions including cell adhesion, cell activation, cell growth, apoptosis, and inflammation. However, the role of galectin-3 in mucosal immunity or inflammatory bowel diseases is still not clear. We demonstrate here that galectin-3 knockout mice have more severe disease activity in the dextran sulfate sodium (DSS)-induced colitis model, indicating that galectin-3 may protect from inflammation in DSS-induced colitis. Furthermore, treating with galectin-3 reduced body weight loss, shortened colonic length, and ameliorated mucosal inflammation in mice having DSS-induced colitis. However, the protective effects of galectin-3 were eliminated by the administration of anti-CD25 mAb. In addition, primary T cells treated with galectin-3 ex vivo induced the expression of FOXP3, ICOS, and PD-1 with a Treg cell phenotype having a suppression function. Moreover, adoptive transfer of galectin-3-treated T cells reduced bowel inflammation and colitis in the T cell transfer colitis model. In conclusion, our results indicate that galectin-3 inhibited colonic mucosa inflammation and reduced disease severity by inducing regulatory T cells, suggesting that it is a potential therapeutic approach in inflammatory bowel disease. KEY MESSAGES: Galectin-3 offers protection from inflammation in experimental colitis. Galectin-3 knockout mice have more severe disease activity in DSS-induced colitis. Adoptive transfer of galectin-3-treated T cells reduced bowel inflammation. Galectin-3 inhibited colonic mucosa inflammation by inducing regulatory T cells. Galectin-3 is a potential therapeutic approach in inflammatory bowel disease.

Increased neutrophil infiltration, IL-1 production and a SAPHO syndrome-like phenotype in PSTPIP2-deficient mice.

OBJECTIVE: Proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) is involved in macrophage activation, neutrophil motility and osteoclast differentiation. However, the role of PSTPIP2 in inflammation and autoinflammatory diseases is still not clear. In this study, we generated PSTPIP2 knockout (Pstpip2(-/-)) mice to investigate its phenotype and role in autoinflammatory diseases. METHODS: We constructed a Pstpip2-targeting vector and generated Pstpip2(-/-) mice. The phenotype and immunopathology of Pstpip2(-/-) mice were analysed. RESULTS: All Pstpip2(-/-) mice developed paw swelling, synovitis, hyperostosis and osteitis, resembling SAPHO syndrome, an inflammatory disorder of the bone, skin and joints. Multifocal osteomyelitis was found in inflamed paws, with increased macrophage and marked neutrophil infiltrations in the bone, joint and skin. Profound osteolytic lesions with markedly decreased bone volume density developed in paws and limbs. Neutrophil-attracting chemokines and IL-1ß were markedly elevated in inflamed tissues. CONCLUSION: Our study suggests that PSTPIP2 could play a role in innate immunity and development of autoinflammatory bone disorders, and may be associated with the pathogenesis of human SAPHO syndrome.

Tumor necrosis factor-alpha induced by hepatitis B virus core mediating the immune response for hepatitis B viral clearance in mice model.

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-α/ß receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.

Simvastatin enhances human osteoblast proliferation involved in mitochondrial energy generation.

Simvastatin has been shown to stimulate osteogenic cell differentiation. Our previous study showed osteoblasts on trabecular surface are increased by simvastatin treatment in animal study. However, whether simvastatin stimulates osteoblast proliferation and by what molecular mechanism have not been adequately investigated. Because the mitochondrial function is crucial for cell survival and proliferation, we hypothesize that simvastatin may promote human osteoblast (hOBs) proliferation and it may be related to mitochondrial function. Our results showed that simvastatin significantly enhanced proliferation and increased both mRNA and protein levels of cyclin D2, Bcl-2 and the ratio of Bcl-2 to Bax (Bcl-2/Bax). Furthermore, simvastatin increased mitochondrial activity and ATP content of hOBs. Most importantly, treatment with ATP synthase blocker, oligomycin, significantly decreased both simvastatin-stimulated ATP content and cell proliferation, and completely reversed the simvastatin-induced up-regulation of cyclin D2 and Bcl-2 expression in hOBs. On the other hand, rotenone, the complex I blocker, also partially blocked simvastatin-stimulated ATP content and cell proliferation, but the blocker did not suppress the effect of simvastatin on cyclin D2 and Bcl-2 expression. These results indicate that the up-regulation of cyclin D2 and Bcl-2/Bax by simvastatin depends on the intact function of ATP synthase in the mitochondria of hOBs. It suggests that simvastatin may promote hOB proliferation, at least partly, via up-regulating mitochondrial function and subsequently cyclin D2 and Bcl-2/Bax expression. The findings provide new information for the basic medical science in bone physiology and for new therapy strategy of simvastatin on bone formation in future.

PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model.

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.

TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation.

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.