Lymphotoxins from distinct types of lymphoid cells differentially contribute to neuroinflammation.

Lymphotoxin α and lymphotoxin ß (LTs), TNF superfamily members, are expressed in either soluble (LTα3) or membrane-bound (LTα1ß2 or LTα2ß1) forms. In the pathological context, LT-mediated signaling is known to exacerbate autoimmunity by perpetuating inflammation and promoting the formation of tertiary lymphoid organs. Despite this understanding, the exact roles of LTα and LTß in the pathogenesis of the murine model of multiple sclerosis, and experimental autoimmune encephalomyelitis (EAE), remain controversial. Here, we employed a panel of gene-modified mice with cell-type restricted ablation of LTα (targeting both membrane-bound and soluble forms of LTs) to unravel the contributions of LTs from various lymphoid cells, namely T cells, type 3 innate lymphoid cells (ILC3) and B cells, in EAE. We found that the effects of LTα deletion were dependent on the cellular source. ILC3-derived lymphotoxins exerted a protective role in EAE by regulating the accumulation of IFN-É£- and GM-CSF-producing TH cells in the CNS. In contrast, T-cell-derived lymphotoxins promoted IL-17A- and GM-CSF-mediated TH responses in the periphery, whereas B-cell-derived lymphotoxins were pathogenic only in the autoantibody-mediated EAE model. Collectively, our findings unveil the multifaceted involvement of lymphotoxins in EAE pathogenesis and challenge the view that lymphotoxins play a solely pathogenic role in neuroinflammation.

Estrogen receptor alpha signaling in dendritic cells modulates autoimmune disease phenotype in mice.

Estrogen is a disease-modifying factor in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) via estrogen receptor alpha (ERα). However, the mechanisms by which ERα signaling contributes to changes in disease pathogenesis have not been completely elucidated. Here, we demonstrate that ERα deletion in dendritic cells (DCs) of mice induces severe neurodegeneration in the central nervous system in a mouse EAE model and resistance to interferon beta (IFNß), a first-line MS treatment. Estrogen synthesized by extragonadal sources is crucial for controlling disease phenotypes. Mechanistically, activated ERα directly interacts with TRAF3, a TLR4 downstream signaling molecule, to degrade TRAF3 via ubiquitination, resulting in reduced IRF3 nuclear translocation and transcription of membrane lymphotoxin (mLT) and IFNß components. Diminished ERα signaling in DCs generates neurotoxic effector CD4+ T cells via mLT-lymphotoxin beta receptor (LTßR) signaling. Lymphotoxin beta receptor antagonist abolished EAE disease symptoms in the DC-specific ERα-deficient mice. These findings indicate that estrogen derived from extragonadal sources, such as lymph nodes, controls TRAF3-mediated cytokine production in DCs to modulate the EAE disease phenotype.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

Interference with LTßR signaling by tick saliva facilitates transmission of Lyme disease spirochetes.

Lyme spirochetes have coevolved with ticks to optimize transmission to hosts using tick salivary molecules (TSMs) to counteract host defenses. TSMs modulate various molecular events at the tick-host interface. Lymphotoxin-beta receptor (LTßR) is a vital immune receptor and plays protective roles in host immunity against microbial infections. We found that Ltbr knockout mice were more susceptible to Lyme disease spirochetes, suggesting the involvement of LTßR signaling in tick-borne Borrelia infection. Further investigation showed that a 15-kDa TSM protein from Ixodes persulcatus (I. persulcatus salivary protein; IpSAP) functioned as an immunosuppressant to facilitate the transmission and infection of Lyme disease spirochetes. IpSAP directly interacts with LTßR to block its activation, thus inhibiting the downstream signaling and consequently suppressing immunity. IpSAP immunization provided mice with significant protection against I. persulcatus-mediated Borrelia garinii infection. Notably, the immunization showed considerable cross-protection against other Borrelia infections mediated by other ixodid ticks. One of the IpSAP homologs from other ixodid ticks showed similar effects on Lyme spirochete transmission. Together, our findings suggest that LTßR signaling plays an important role in blocking the transmission and pathogenesis of tick-borne Lyme disease spirochetes, and that IpSAP and its homologs are promising candidates for broad-spectrum vaccine development.

Electrostimulation suppresses allograft rejection via promoting lymphatic regulatory T cell migration mediated by lymphotoxin - lymphotoxin receptor ß signaling.

Conventional immunosuppressants that suppress allograft rejection cause various side effects. Although regulatory T cells (Tregs) are essential for allograft survival, the limited efficacy of Treg therapy demands improvement. Thus, it is imperative to seek new approaches to enhancing Treg suppression. Low-intensity electrostimulation (ES) has been shown to exert antiinflammatory effects without causing major adverse reactions. However, it remains unknown whether and how ES regulates alloimmunity. Here, we found that regional ES delayed murine skin allograft rejection and promoted long-term allograft survival induced by an mTOR inhibitor, rapamycin. ES also extended islet allograft survival. Mechanistically, ES enhanced the expression of lymphotoxin α (LTα) on Tregs after transplantation. Blockade of lymphotoxin ß receptor-mediated nonclassical NFκB signaling suppressed lymphatic Treg migration and largely reversed the effects of ES on allograft survival. Moreover, ES failed to extend allograft survival when recipients lacked LTα/lymph nodes or if transferred Tregs lacked LTα. Therefore, ES promoted the lymphatic migration of CD4+Foxp3+ Tregs by upregulating their surface expression of LTα. Finally, ES augmented expression of LTα on murine or human Tregs, but not conventional T cells, while promoting their calcium influx in vitro. This ES-mediated upregulation of LTα relied on calcium influx. Thus, our findings have unveiled novel mechanisms underlying ES-mediated immunoregulation.

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection.

BACKGROUND: Craniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored. METHODS: A mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity. RESULTS: TNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected. CONCLUSION: These findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection.

The transcription factor Sox4 is required for thymic tuft cell development.

Medullary thymic epithelial cells (mTECs) help shape the thymic microenvironment for T-cell development by expressing a variety of peripheral tissue-restricted antigens (TRAs). The self-tolerance of T cells is established by negative selection of autoreactive T cells that bind to TRAs. To increase the diversity of TRAs, a fraction of mTECs terminally differentiates into distinct subsets resembling atypical types of epithelial cells in specific peripheral tissues. As such, thymic tuft cells that express peripheral tuft cell genes have recently emerged. Here, we show that the transcription factor SRY-box transcription factor 4 (Sox4) is highly expressed in mTECs and is essential for the development of thymic tuft cells. Mice lacking Sox4 specifically in TECs had a significantly reduced number of thymic tuft cells with no effect on the differentiation of other mTEC subsets, including autoimmune regulator (Aire)+ and Ccl21a+ mTECs. Furthermore, Sox4 expression was diminished in mice deficient in TEC-specific lymphotoxin ß receptor (LTßR), indicating a role for the LTßR-Sox4 axis in the differentiation of thymic tuft cells. Given that Sox4 promotes differentiation of peripheral tuft cells, our findings suggest that mTECs employ the same transcriptional program as peripheral epithelial cells. This mechanism may explain how mTECs diversify peripheral antigen expression to project an immunological self within the thymic medulla.

Prognostic significance of the genetic variant of lymphotoxin alpha (p.Thr60Asn) in egyptian patients with advanced hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide in terms of mortality, and susceptibility is attributed to genetic, lifestyle, and environmental factors. Lymphotoxin alpha (LTA) has a crucial role in communicating the lymphocytes with stromal cells and provoking cytotoxic effects on the cancer cells. There are no reports on the contribution of the LTA (c.179 C>A; p.Thr60Asn; rs1041981) gene polymorphism to HCC susceptibility. The main aim of this study is to investigate the association of LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant with the HCC risk in the Egyptian population. METHODS: This case-control study included 317 participants (111 HCC patients, and 206 healthy controls). The LTA (c.179 C>A; p.Thr60Asn; rs1041981) polymorphism was assessed by tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) technique. RESULTS: The frequencies of the dominant and recessive models (CA + AA; AA) of the LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant were statistically significant among HCC patients in comparison to controls (p = 0.01; p = 0.007; respectively). The A-allele of LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant was statistically significant in HCC patients in comparison to controls (p ˂ 0.001). CONCLUSION: The LTA (c.179 C>A; p.Thr60Asn; rs1041981) polymorphism was independently associated with an increased risk for hepatocellular carcinoma in the Egyptian population.

Autoreactive B cells in SLE, villains or innocent bystanders?

The current concepts for development of autoreactive B cells in SLE (systemic lupus erythematosus) focus on extrinsic stimuli and factors that provoke B cells into tolerance loss. Traditionally, major tolerance loss pathways are thought to be regulated by factors outside the B cell including autoantigen engagement of the B-cell receptor (BCR) with simultaneous type I interferon (IFN) produced by dendritic cells, especially plasmacytoid dendritic cells (pDCs). Later, in autoreactive follicles, B-cells encounter T-follicular helper cells (Tfh) that produce interleukin (IL)-21, IL-4 and pathogenic cytokines, IL-17 and IFN gamma (IFNÉ£). This review discusses these mechanisms and also highlights recent advances pointing to the peripheral transitional B-cell stage as a major juncture where transient autocrine IFNß expression by developing B-cells imprints a heightened susceptibility to external factors favoring differentiation into autoantibody-producing plasmablasts. Recent studies highlight transitional B-cell heterogeneity as a determinant of intrinsic resistance or susceptibility to tolerance loss through the shaping of B-cell responsiveness to cytokines and other environment factors.

Ophthalmic manifestations are associated with reduced tear lymphotoxin-α levels in chronic ocular graft-versus-host disease.

PURPOSE: To determine the role tear lymphotoxin-α (LT-α) in chronic ocular graft-versus-host disease (oGVHD). METHODS: Twenty-two chronic oGVHD and 17 control tear samples were collected, and commercial test strips were used to detect LT-α concentrations. Concentration differences between patients with and without oGVHD were determined via Mann-Whitney U test. The correlation between LT-α levels and ophthalmic parameters was analyzed using Spearman's test. RESULTS: The concentration of LT-α was significantly lower in oGVHD patients than in controls. LT-α levels were significantly correlated with OSDI, NIH eye score, T-BUT, and CFS among all participants. ROC analysis revealed that the area under the curve of LT-α was 0.847, and the cutoff value for chronic oGVHD diagnosis was 0.203 ng/mL. CONCLUSION: Our study revealed the significant decrease of tear LT-α in oGVHD, and suggested LT-α as a promising marker for chronic oGVHD diagnosis.

Association of LTA and SOD Gene Polymorphisms with Cerebral White Matter Hyperintensities in Migraine Patients.

White matter hyperintensities (WMHs) in migraine could be related to inflammatory and antioxidant events. The aim of this study is to verify whether migraine patients with WMHs carry a genetic pro-inflammatory/pro-oxidative status. To test this hypothesis, we analyzed lymphotoxin alpha (LTA; rs2071590T and rs2844482G) and superoxide dismutase 1 (SOD1; rs2234694C) and 2 (SOD2; rs4880T) gene polymorphisms (SNPs) in 370 consecutive patients affected by episodic (EM; n = 251) and chronic (CM; n = 119) migraine and in unrelated healthy controls (n = 100). Brain magnetic resonance was available in 183/370 patients. The results obtained show that genotypes and allele frequencies for all tested SNPs did not differ between patients and controls. No association was found between single SNPs or haplotypes and sex, migraine type, cardiovascular risk factors or disorders. Conversely, the LTA rs2071590T (OR = 2.2) and the SOD1 rs2234694C (OR = 4.9) alleles were both associated with WMHs. A four-loci haplotype (TGCT haplotype: rs2071590T/rs2844482G/rs2234694C/rs4880T) was significantly more frequent in migraineurs with WMHs (7 of 38) compared to those without WMHs (4 of 134; OR = 8.7). We may, therefore, conclude by suggesting that that an imbalance between pro-inflammatory/pro-oxidative and antioxidant events in genetically predisposed individuals may influence the development of WMHs.

Lymphotoxin ß Receptor: a Crucial Role in Innate and Adaptive Immune Responses against Toxoplasma gondii.

The lymphotoxin ß receptor (LTßR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTßR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTßR is a key regulator required for the intricate balance of adaptive immune responses. Toxoplasma gondii-infected LTßR-deficient (LTßR-/-) mice show globally altered interferon-γ (IFN-γ) regulation, reduced IFN-γ-controlled host effector molecule expression, impaired T cell functionality, and an absent anti-parasite-specific IgG response, resulting in a severe loss of immune control of the parasites. Reconstitution of LTßR-/- mice with toxoplasma immune serum significantly prolongs survival following T. gondii infection. Notably, analysis of RNA-seq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study uncovers the decisive role of the LTßR in cytokine regulation and adaptive immune responses to control T. gondii.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTßR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-ß receptor (LTßR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTßR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTßR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTßR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and ß (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.

Lymphotoxin targeted to salivary and lacrimal glands induces tertiary lymphoid organs and cervical lymphadenopathy and reduces tear production.

To investigate the role of lymphotoxin (LT) in Sjögren's syndrome (SS) and in mucosal associated lymphoid tissue (MALT)-lymphoma, we made transgenic mice (Amy1-LTαß) that targeted LTα and LTß to the salivary and lacrimal glands. Amy1-LTαß mice developed atrophic salivary and lacrimal glands that contained tertiary lymphoid organs (TLOs) and had reduced tear production. Amy1-LTαß mice developed cervical lymphadenopathy but not MALT-lymphoma. TLO formation in the salivary and lacrimal glands of Amy1-LTαß was not sufficient to induce autoimmunity as measured by autoantibody titres.

Stromal cells and B cells orchestrate ectopic lymphoid tissue formation in nasal polyps.

BACKGROUND: Although the importance of ectopic lymphoid tissues (eLTs) in the pathophysiology of nasal polyps (NPs) is increasingly appreciated, the mechanisms underlying their formation remain unclear. OBJECTIVE: To study the role of interleukin (IL)-17A, C-X-C motif chemokine ligand 13 (CXCL13) and lymphotoxin (LT) in eLT formation in NPs. METHODS: The expression levels of CXCL13 and LT and their receptors, in addition to the phenotypes of stromal cells in NPs, were studied by flow cytometry, immunostaining, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Purified nasal stromal cells and B cells were cultured, and a murine model of nasal type 17 inflammation was established by intranasal curdlan challenge for the mechanistic study. RESULTS: The excessive CXCL13 production in NPs correlated with enhanced IL-17A expression. Stromal cells, with CD31- Pdpn+ fibroblastic reticular cell (FRC) expansion, were the major source of CXCL13 in NPs without eLTs. IL-17A induced FRC expansion and CXCL13 production in nasal stromal cells. In contrast, B cells were the main source of CXCL13 and LTα1 ß2 in NPs with eLTs. CXCL13 upregulated LTα1 ß2 expression on B cells, which in turn promoted CXCL13 production in nasal B cells and stromal cells. LTα1 ß2 induced expansion of FRCs and CD31+ Pdpn+ lymphoid endothelial cells, which were the predominant stromal cell types in NPs with eLTs. IL-17A knockout and CXCL13 and LTßR blockage diminished nasal eLT formation in the murine model. CONCLUSION: We identified an important role of IL-17A-induced stromal cell remodeling in the initiation and crosstalk between B and stromal cells via CXCL13 and LTα1 ß2 in the enlargement of eLTs in NPs.

Genetic Variation on TNF/LTA and TNFRSF1A Genes is Associated with Outcomes of Hepatitis C Virus Infection.

Hepatitis C virus (HCV) infections are a serious global-scaled public health problem. Tumor necrosis factor (TNF)/lymphotoxin alpha (LTA) has been found to play a crucial role in relation to the outcomes of HCV infection after it binds to TNF receptor superfamily member 1A (TNFRSF1A). Thus, we investigated whether or not the TNF/LTA gene cluster and TNFRSF1A gene polymorphisms were associated with the outcomes of HCV infection. 1103 control participants without HCV infection, 497 patients with spontaneous clearance of HCV infection, and 713 patients with persistent HCV infection were enrolled. Rs2229094, rs1041981, rs1799964, and rs767455 were genotyped using the ABI TaqMan allelic discrimination assay. After adjusting for age, gender, and after determining a high-risk population, we used logistic regression analyses for which results indicated that the rs767455-C allele was associated with a reduced risk of HCV infection compared to respective results for the wild-type T allele (dominant model: adjusted OR = 0.74, 95% CI = 0.60-0.92, P = .006; additive model: adjusted OR = 0.76, 95% CI = 0.62-0.91, P = .004). Results also indicated that the rs1041981-A allele was associated with a decreased risk of persistent HCV infection compared to respective results for the wild-type C allele (additive model: adjusted OR = 0.81, 95% CI = 0.68-0.96, P = .017). Genetic polymorphisms in the LTA and TNFRSF1A genes were found to have been potentially important in relation to the susceptibility and chronicity of HCV infection among Chinese Han population.

Association between tumor necrosis factor alpha and lymphotoxin alpha gene polymorphisms and migraine occurrence among Jordanians.

Inflammatory reactions in the body have been shown to contribute to migraine development. Therefore, genes involved in the inflammatory pathways might play a role in the susceptibility and development of migraine. In this study, polymorphisms in tumor necrosis factor alpha (TNFα) and lymphotoxin alpha (LTA) genes were tested for association with migraine. A total of 398 participants (198 migraine patients and 200 controls) were recruited in the study. Serum TNF level was measured using a sandwich ELISA kit. Lymphocytes' and monocytes' counts were obtained from a differential complete blood count profile. Participants' DNA was extracted and genotyped for rs1800629 and rs1799724 in TNFα, and rs909253 in LTA. Controls had a significantly higher mean lymphocyte count (P = 0.018), while the mean monocyte count and serum TNFα levels did not differ between the two groups (P > 0.05). With respect to gene polymorphisms, the rs1800629 and rs1799724 variants showed significant association with migraine in all subjects, and in males and females when analyzed separately (P < 0.001). The rs909253 did not show any statistical difference in frequencies among the two groups (P > 0.05). Having the A allele in rs1800629 was associated with a higher risk of migraine in both male (OR, 95%; CI, G/A = 3.79 [1.87-7.69]; A/A = 14.22 [1.67-121.14]; P < 0.01) and female (OR, 95%; G/A = 2.54 [1.47-4.38]; A/A = 2.52 [1.12-5.69]; P < 0.001) subjects. In conclusion, rs1800629 and rs1799724 in TNFα showed significant association with migraine among the Jordanian population.

Elevated lymphotoxin-α (TNFß) is associated with intervertebral disc degeneration.

BACKGROUND: Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFß, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB. RESULTS: The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 µg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels. CONCLUSIONS: This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

Association of lymphotoxin-alpha gene polymorphisms (rs909253, rs1800683 and rs2229094) and risk of large-artery atherosclerosis stroke in Iranian population.

BACKGROUND: Lymphotoxin-alpha (LTA), a proinflammatory cytokine, is significantly associated with the progression of atherosclerosis as an independent hazard factor for stroke. According to new genetic studies, polymorphisms in the LTA gene that influence its expression or biological function may play a role in the progress of stroke; thus, the present case-control study investigated LTA gene polymorphisms (rs909253, rs1800683 and rs2229094) and the risk of large artery atherosclerosis stroke (LAA) in an Iranian population. METHODS: For 211 large artery atherosclerosis patients and 186 ischemic stroke-free controls, genotypes were determined using the tetra-primer amplification-refractory mutation system polymerase chain reaction method. Linkage disequilibrium and estimated haplotypes were analyzed using SNP Analyzer 2 software. The strength of the link between LTA gene polymorphisms (rs1800683, rs909253, and rs2229094) and the risk of stroke was determined using conditional logistic regression. RESULTS: Analysis revealed that the patterns of the rs1800683, rs909253 and rs2229094 genotypes showed no significant difference between the LAA and control group, although the distribution of the GAT (rs1800683G, rs909253A and rs2229094T) haplotype was significantly higher in the control group (odds ratio = 0.707, 95% confidence interval = 0.53-0.942, p = 0.0355). CONCLUSIONS: Our results indicate that the GAT haplotype in LTA gene is associated with a decreased risk of LAA incidence in a northeastern Iranian population.

Evidence that TNF-ß suppresses osteoblast differentiation of mesenchymal stem cells and resveratrol reverses it through modulation of NF-κB, Sirt1 and Runx2.

It has been established that inflammation plays an important role in bone formation and bone loss. Although a lot is known about the role of TNF-α in bone health, very little is understood about TNF-ß, also called lymphotoxin. In this report, we examine the effect of TNF-ß on osteogenic differentiation of mesenchymal stem cells (MSCs) and its modulation by resveratrol. Monolayer and high-density cultures of MSCs were treated with osteogenic induction medium with/without TNF-ß, Sirt1 inhibitor nicotinamide (NAM), antisense oligonucleotides against Sirt1 (ASO) and/or Sirt1 stimulator resveratrol. We found that TNF-ß inhibits, in a similar way to NAM or Sirt1-ASO, the early stage of osteogenic differentiation of MSCs and this was accompanied with downregulation of bone-specific matrix, ß1-integrin, Runx2 and with upregulation of NF-κB phosphorylation and NF-κB-regulated gene products involved in the inflammatory, degradative processes and apoptosis. However, resveratrol reversed TNF-ß- and NAM-suppressed MSCs osteogenesis by activation of Sirt1 and Runx2 that led to osteoblast differentiation. Furthermore, downregulation of Sirt1 by mRNA inhibited the effect of resveratrol, highlighting the important impact of this enzyme in the TNF-ß signaling pathway. Finally, resveratrol was able to manifest its effect both by suppression of TNF-ß-induced NF-κB and through direct activation of the Sirt1 and Runx2 pathway. Thus, through these studies, we present a mechanism by which a T cell-derived cytokine, TNF-ß can affect bone formation through modulation of MSCs differentiation that involves NF-κB, Sirt1, Runx2 and resveratrol reversed TNF-ß-promoted impairments in MSCs osteogenesis.