Search

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 TNFβ, 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.

Objectives: Human papillomavirus (HPV) ranks the first cause of cervical cancer. Cervical cancer has high prevalence rates in women around the world. The HPV-E7 oncoprotein is expressed in cervical cancer and is a target of developing immunotherapies against HPV-associated tumors. However, the antigenicity of this protein is low. Due to this reason, potent adjuvants are required to enhance its therapeutic efficacy. This preliminary study aims to evaluate whether lymphotoxin (LT) could act as an effective immune adjuvant for HPV infection in mice models. Material and methods: Intranasal immunization was used to explore the effect of HPV-E7 and/or LT immune response. After the third intranasal immunization, the titer for the HPV-E7 antibody was detected in serum and vaginal washing fluid. Also, we assessed the expression of chemokine ligand 13 (CXCL13) and Peripheral Node Addressin (PNAd) in the lymph nodes after intranasal immunization with immunohistochemical analysis. Results: compared to HPV-E7 immunization, intranasal immunization with HPV-E7 plus LT significantly increased HPV-E7-specific serum IgG and vaginal IgA titers. Furthermore, the combined use of HPV-E7 and LT strongly induced E7-specific CTL responses. Conclusions: LT can be effective for intranasal immunized HPV-E7 to improve E7-specific immune responses to HPV infection. It is new approach to eradicate chronic HPV infection capable of inducing an effective anti-infection method.

The members of the Tumor Necrosis Factor (TNF) superfamily, the ligand lymphotoxin α1β2 (LTα1β2) and its unique receptor lymphotoxin β receptor (LTβR), play a pivotal role in the establishment and regulation of the immune system by allowing a tight communication between lymphocytes and stromal cells. Recent advances using transgenic mice harboring a specific deletion of the Ltbr gene in distinct stromal cells have revealed important roles for LTβR signaling in the thymic function that ensures the generation of a diverse and self-tolerant T-cell repertoire. In this review, we summarize our current knowledge on this signaling axis in the thymic homing of lymphoid progenitors and peripheral antigen-presenting cells, the trafficking and egress of thymocytes, the differentiation of medullary thymic epithelial cells, and the establishment of central tolerance. We also highlight the importance of LTα1β2/LTβR axis in controlling the recovery of the thymic function after myeloablative conditioning regimen, opening novel perspectives in regenerative medicine.

Etanercept is a soluble form of the tumor necrosis factor receptor 2 (TNFR2) that inhibits pathological tumor necrosis factor (TNF) responses in rheumatoid arthritis and other inflammatory diseases. However, besides TNF, etanercept also blocks lymphotoxin- (LT), which has no clear therapeutic value and might aggravate some of the adverse effects associated with etanercept. Poxviruses encode soluble TNFR2 homologs, termed viral TNF decoy receptors (vTNFRs), that display unique specificity properties. For instance, cytokine response modifier D (CrmD) inhibits mouse and human TNF and mouse LT, but it is inactive against human LT. Here, we analyzed the molecular basis of these immunomodulatory activities in the ectromelia virus– encoded CrmD. We found that the overall molecular mechanism to bind TNF and LT from mouse and human origin is fairly conserved in CrmD and dominated by a groove under its 50s loop. However, other ligand-specific binding determinants optimize CrmD for the inhibition of mouse ligands, especially mouse TNF. Moreover, we show that the inability of CrmD to inhibit human LT is caused by a Glu-Phe-Glu motif in its 90s loop. Importantly, transfer of this motif to etanercept diminished its anti-LT activity in >60-fold while weakening its TNF-inhibitory capacity in 3-fold. This new etanercept variant could potentially be used in the clinic as a safer alternative to conventional etanercept. This work is the most detailed study of the vTNFR–ligand interactions to date and illustrates that a better knowledge of vTNFRs can provide valuable information to improve current anti-TNF therapies., Spanish Ministry of Economy and Competitiveness Grants SAF2012-38957 and SAF2015-67485-R and the European Union (European Regional Development Fund

IL-4 is critical for differentiation of Th2 cells and antibody isotype switching, but our work demonstrated that it is produced in the peripheral LN under both Type 2, and Type 1 conditions, raising the possibility of other functions. We found that IL-4 is vital for proper positioning of hematopoietic and stromal cells in steady state, and the lack of IL-4 or IL-4Rα correlates with disarrangement of both follicular dendritic cells and CD31(+) endothelial cells. We observed a marked disorganization of B cells in these mice, suggesting that the lymphocyte-stromal cell axis is maintained by the IL-4 signaling pathway. This study showed that absence of IL-4 correlates with significant downregulation of Lymphotoxin alpha (LTα) and Lymphotoxin beta (LTβ), critical lymphokines for the development and maintenance of lymphoid organs. Moreover, immunization of IL-4 deficient mice with Type 2 antigens failed to induce lymphotoxin production, LN reorganization, or germinal center formation, while this process is IL-4 independent following Type 1 immunization. Additionally, we found that Type 1 antigen mediated LN reorganization is dependent on IFN-γ in the absence of IL-4. Our findings reveal a role of IL-4 in the maintenance of peripheral lymphoid organ microenvironments during homeostasis and antigenic challenge.

Although much is understood about the proinflammatory cytokine TNF-α, very limited data are available about TNF-β (lymphotoxin). Whether TNF-β can induce the proliferation of tumor cells, how TNF-β-induced proliferation of tumor cells is affected by natural products such as resveratrol and the role of NF-κB in this process, is not understood. In the present study, we used clonogenic and cytotoxic methods to show the effect of TNF-β on cell proliferation. We also examined the impact of resveratrol on TNF-β-promoted proliferation and on NF-κB activation in HCT116 colorectal cancer (CRC). Our findings showed that TNF-β induced the proliferation and invasion in CRC cells and this was comparable with that of TNF-α. TNF-β-stimulated proliferation of CRC cells was blocked via anti-TNF-β-receptor. We found that resveratrol reversed the TNF-β-induced proliferation and invasion of CRC cells, and this correlated with the suppression of TNF-β-stimulated NF-κB signaling. Like resveratrol, IκB-kinase (IKK) inhibitor (BMS-345541), also reversed TNF-β-stimulated proliferation, NF-κB activation and these were mediated through inhibition of IκB-kinase, phosphorylation of IκBα, suppression of phosphorylation, and nuclear translocation of the p65 subunit of NF-κB. Furthermore, resveratrol similar to BMS-345541 suppressed TNF-β-promoted NF-κB-mediated gene biomarkers linked with proliferation, apoptosis, and invasion. Overall, our findings indicate for the first time that TNF-β/TNF-β-receptor signaling is involved in proliferation of CRC cells in parallel to TNF-α, and that resveratrol down-modulates TNF-β/TNF-β-receptor-mediated inflammatory response, at least in part through down modulating NF-κB activation, thereby regulating tumor cell growth. Impact statement The mechanism by which natural products such as resveratrol suppresses TNF-β-promoted tumor cell proliferation, invasion, and colony formation is unknown. In this study, we explored for the first time the effect of resveratrol on the proinflammatory cytokine TNF-β-, compared to TNF-α-stimulated proliferative and pro-inflammatory signaling in HCT116 cells. Our findings suggest that expression of TNF-β and TNF-β-receptor, like TNF-α, can lead to activation of inflammatory transcription factor (NF-κB) and NF-κB-regulated gene biomarkers, which are involved in the promotion of cancer proliferation, invasion, metastasis, and cell survival of tumor. Resveratrol can block TNF-β/TNF-β-receptor-induced activation of NF-κB, NF-κB-modulated gene products, and inhibition of caspase-3 cleavage. These results highlight the therapeutic effect of resveratrol-mediated anti-tumor activity by multitargeting cellular signaling pathways.

Though lymphotoxin (LT) is highly expressed by type I helper T (Th1) cells, its contribution to CD4+ T cell differentiation during infections and diseases remains a mystery. In HSV-1 infection, we observed that LTβR signaling is required to limit the Th1 response. Using bone marrow chimeric mice, mixed-T-cell chimeric mice, and LTβR in vivo blockades, we unexpectedly observed that LT, especially T cell-derived LT, played an indispensable role in limiting the Th1 response. The LTβR-Ig blockade promoted the Th1 response by increasing infiltration of monocytes and monocyte-derived DCs and up-regulating IL-12 secretion in the lymphoid environment. Our findings identified a novel role for T cell-derived LT in manipulating Th1 differentiation.

Conventional dendritic cells (cDCs) bridge antigen detection to the induction of adaptive immunity, playing crucial roles in host defense. Vanderkerken, Baptista, et al. show that LTα1β2-expressing ILC3s, together with B cells, control the size of the splenic cDC compartment and cDC2 differentiation., The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1β2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTβR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1β2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis.

We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

The mouse lymph node (LN) can provide a niche to grow metanephric kidney to maturity. Here, we show that signaling through the lymphotoxin-β receptor (LTβR) is critical for kidney organogenesis both in the LN and the omentum. By transplanting kidney rudiments either in the LNs of mice undergoing LTβR antagonist treatment or in the omenta of Ltbr knockout (Ltbr(−/−)) mice, the host LTβR signals were found to be crucial for obtaining a well-vascularized kidney graft. Indeed, defective LTβR signaling correlated with decreased expression of endothelial and angiogenic markers in kidney grafts as well as structural alterations. Because the number of glomerular endothelial cells expressing the LTβR target nuclear factor κB–inducing kinase (NIK) decreased in the absence of a functional LTβR, it was speculated that an LTβR/NIK axis mediated the angiogenetic signals required for successful ectopic kidney organogenesis, given the established role of NIK in neovascularization. However, the transplantation of kidney rudiments in omenta of Nik(−/−) mice revealed that NIK is dispensable for ectopic kidney vascular integration and maturation. Finally, defective LTβR signaling impaired compensatory glomerular adaptation to renal mass reduction, indicating that kidney regeneration approaches, besides whole kidney reconstruction, might benefit from the presence of LTβR signals.

Lymphotoxin-a (LTA) may be associated with the pathogenesis of inflammatory diseases. To assess the association of the LTA rs909253 A/G polymorphism with plasma level and risk of ankylosing spondylitis (AS) in a Chinese Han population. Genotyping and LTA plasma were tested by mass spectroscopy and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that the average plasma level of LTA in AS was significantly lower than in the controls (P = 0.000). Our results also indicated that LTA rs909253 A/G was associated with a decreased risk of AS (G vs. A: P = 0.014). Significant differences were also found between the rs909253 A/G genotype and down-regulated plasma level in AS patients, compared with controls. After stratification analysis, a decreased risk of AS was associated with the LTA rs909253 G allele (G vs. A) among female patients, younger patients (Yr. LTA rs909253 A/G genotype has a significant relationship with decreased susceptibility to AS.

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood‐borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1(BKO)) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B‐cell loss, we have analysed the spleen MZ architecture of TSC1(BKO) mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTβ) and lymphotoxin receptor (LTβR) expression indicated that LTβR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTβR is sensitive to proteolysis, we analysed cathepsin activity in TSC1(BKO). A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan‐cathepsin inhibitor restored LTβR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with unclear etiology. Several loci associated with genetic susceptibility for lupus have been described. However, it lacks reports on cytokine gene-gene interactions among SLE patients from Asian population. Epistasis interaction among single nucleotide polymorphisms (SNPs) of cytokine genes in Indian SLE patients was tested using multifactor-dimensionality reduction (MDR) analysis. A total of fourteen SNPs lacking linkage disequilibrium among different cytokines genes were genotyped in a cohort of 200 SLE patients and 201 healthy individuals as controls of Indian origin. A high degree of synergism among Lymphotoxin-α (LT-α), Interleukin-1β (IL-1β) and Interleukin-10 (IL-10) gene polymorphisms was detected in our SLE patients. Furthermore, by virtue of biological inter-relations among different cytokines, a high strength of interactions was observed among pro-inflammatory (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokine gene SNPs. Also, among studied pro-inflammatory cytokines and chemokines, a strong synergistic effect among Tumor Necrosis Factor-α (TNF-α), LT-α and Monocyte Chemo-attractant Protein-1 (MCP-1) SNPs was occurred. A nature of strong interaction among the candidate cytokine genes may speculate a proactive role in causing genetic susceptibility to the disease in SLE patients with Indian origin.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-β receptor (LTβR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTβR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTβR signaling affects mucosal immune responses during adulthood.

OBJECTIVE: Interleukin-14α-transgenic (IL-14αTG) mice develop an autoimmune exocrinopathy with characteristics similar to Sjögren’s syndrome, including sialadenitis and hyposalivation. The P2Y(2) receptor (P2Y(2)R) for extracellular ATP and UTP is up-regulated during salivary gland inflammation (i.e., sialadenitis) where it regulates numerous inflammatory responses. This study investigated the role of P2Y(2)Rs in autoimmune sialadenitis in the IL-14αTG mouse model of Sjögren’s syndrome. MATERIALS AND METHODS: IL-14αTG mice were bred with P2Y(2)R(−/−) mice to generate IL-14αTG × P2Y(2)R(−/−) mice. P2Y(2)R expression, lymphocytic focus scores, B- and T-cell accumulation, and lymphotoxin-α expression were evaluated in the submandibular glands (SMG) along with carbachol-stimulated saliva secretion in IL-14αTG, IL-14αTG × P2Y(2)R(−/−), and C57BL/6 control mice at 9 and 12 months of age. RESULTS: Genetic ablation of P2Y(2)Rs in IL-14αTG mice significantly reduced B and T lymphocyte infiltration of SMGs. However, reduced sialadenitis did not restore saliva secretion in IL-14αTG × P2Y(2)R(−/−) mice. Decreased sialadenitis in IL-14αTG × P2Y(2)R(−/−) mice correlated with decreased lymphotoxin-α levels, a critical proinflammatory cytokine associated with autoimmune pathology in IL-14αTG mice. CONCLUSIONS: The results of this study suggest that P2Y(2)Rs contribute to the development of salivary gland inflammation in IL-14αTG mice and may also contribute to autoimmune sialadenitis in humans.

Lymphotoxin (LT) is a member of the tumor necrosis factor (TNF) superfamily of cytokines which serves multiple functions, including the control of lymphoid organ development and maintenance, as well as regulation of inflammation and autoimmunity. Although the role of LT in organogenesis and maintenance of lymphoid organs is well established, the contribution of LT pathway to homeostasis of lymphoid organs during the immune response to pathogens is less understood. In this review, we highlight recent advances on the role of LT pathway in antiviral immune responses. We discuss the role of LT signaling in lymphoid organ integrity, type I IFN production and regulation of protection and immunopathology during viral infections. We further discuss the potential of therapeutic targeting LT pathway for controlling immunopathology and antiviral protection.

This case–control study was aimed to assess the effect of genetic variants of tumor necrosis factor (TNF) α−308 and lymphotoxin (LT) α+252 on development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Their gene–gene interaction was also investigated. We enrolled 200 pairs of age- and sex-matched patients with cirrhotic HBV-HCC and unrelated patients with HBV-cirrhosis alone. Polymorphisms of TNFα −308 and LTα +252 were genotyped. Synergy index was used to calculate interaction between the variant genotypes. The results indicated that the frequency distribution of the variant genotypes ( TNFα −308 G/A and LTα +252 G/G) in patients with HCC were significantly higher than those in patients with cirrhosis alone. Multivariate analysis indicated that TNFα −308 G/A (odds ratio [OR], 2.34) and LTα +252 G/G (OR, 2.04) were independent risk factors for HCC. By the clinical characteristics of study population, multivariate analysis demonstrated that independent factors associated with harboring the variant genotypes included cirrhosis with Child-Pugh C (OR = 6.47 in cases and OR = 11.56 in controls) and thrombocytopenia (OR = 8.86 in cases and OR = 7.74 in controls). Calculation of synergy index (SI) indicated that there are additive interaction between TNFα −308 G/A and LTα +252 G/G on risk of HCC (SI = 1.29). In conclusion: There are independent and additive interactions between TNFα −308 G/A and LTα +252 G/G on risk for HBV-HCC. They correlated with advanced hepatic fibrosis and severe liver damage, which might contribute to a higher risk for HCC.

BACKGROUND Recombinant human lymphotoxin-α derivative (rhLTα-Da) is a lymphotoxin-α derivative that is missing 27 N-terminal amino acid residues. Previous studies indicated a benefit from the addition of rhLTα-Da to cisplatin-based treatment in patients with metastatic esophageal squamous cell carcinoma. The current study was conducted to evaluate the efficacy and safety of rhLTα-Da plus cisplatin and fluorouracil (PF) in patients with mESCC. METHODS Patients from 15 centers in China were randomly assigned (1:1:1) to 3 arms (arm A, PF plus 10 μg/m2 daily rhLTα-Da; arm B, PF plus 20 μg/m2 daily rhLTα-Da; arm C, PF alone). The primary endpoints included progression-free survival (PFS) and the confirmed overall response rate (ORR). An exploratory analysis was performed to evaluate the role of serum tumor necrosis factor receptor II (TNFR II) in predicting the efficacy of rhLTα-Da. RESULTS Between September 2010 and May 2013, 150 patients were enrolled. No significant differences in either PFS or ORR were observed between the 3 arms. However, in a small subset of patients who had low serum TNFR II levels, the median PFS was significantly longer for those in arm B than for these in other 2 arms (7.2 months [95% confidence interval, 5.1-8.6 months] for arm B vs 3.5 months [95% confidence interval, 1.7-5.5 months] for arm A [P = .022] and 4.0 months [95% confidence interval, 3.2-6.3 months] for arm C [P = .027]). The addition of rhLTα-Da significantly increased the incidence of chills (P < .001). CONCLUSIONS rhLTα-Da combined with the PF regimen failed to improve PFS and ORR in patients with mESCC, except in a small subset that had low serum TNFR II concentrations. Cancer 2017. © 2017 American Cancer Society.

Acute or chronic damage to the liver may occur through alcohol, drugs, viruses, genetic disorders, and toxicity. In this study, we planned to investigate the protective and therapeutic effects of melatonin (Mel) by causing damage to the liver with thioacetamide (TAA). Thirty-five rats were used. Group I: control group (seven pieces), group II: Mel group (seven pieces) the single dose on the first day of the experiment was 10 mg/kg, group III: TAA (seven pieces) 300 mg/kg with 24-hour intervals, two doses, group IV: Mel + TAA group (seven pieces) 10 mg/kg single dose Mel was applied 24 hours before TAA application, group V: TAA + Mel group (seven pieces) single dose (24th hour) of 10 mg/kg Mel was administered after TAA (300 mg/kg) two doses. The liver histology was evaluated. Apoptosis, autophagy, and necrosis markers in tissue were determined by immunohistochemistry. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels in blood serum samples and transforming growth factor-ß (TGF-ß) and tumor necrosis factor-? (TNF-?) levels were determined in liver tissue. TAA affected histologically the classical lobule structure both in cell cords and sinusoids. Caspase-3, RIP3, and LC3 levels were increased in group III compared with the control group. TAA did not cause a statistically significant change in TNF-? level but decreased the TGF-ß level significantly. AST and ALT levels were statistically significant in group II and V compared with group I, the ALP level was significant in group IV compared with group II. The results of this study showed that TAA caused significant damage to tissues and increased cell death, Mel was found to have more therapeutic than the protective effect on tissues. © 2020 Wiley Periodicals, Inc.

Background & Aims Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. Methods We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. Results A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. Conclusions We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

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

Summary: Adaptive type 2 immune responses against the intestinal helminth Heligmosomoides polygyrus (Hp) require the interaction of follicle-associated CXCR5+ dendritic cells with naive T cells in the draining mesenteric lymph nodes (mLNs). However, the source of CXCL13 responsible for attracting CXCR5+ dendritic cells has remained unclear. Using multiplex imaging combined with deep tissue analysis, we observed new CXCL13+ fibroblastic reticular cells surrounding paracortical and cortical B cell follicles in the mLNs of infected mice. CXCL13+ fibroblasts expressed markers of marginal reticular cells (MRCs), and their expansion required lymphotoxin (LT)-dependent interactions between IL-4Rα-expressing B cells and CCL19+ fibroblasts. Infection-induced follicles did not necessarily contain follicular dendritic cells (FDCs), indicating that CXCL13+ fibroblasts may instead drive their formation. These data reveal a role for lymphotoxin signaling to CCL19+ fibroblasts in the development of CXCL13+ MRC-like cells and adaptive type 2 immunity in response to helminth infection. : MRCs are a specialized, CXCL13-producing, stromal population located beneath the subcapsular sinus of lymph nodes. Dubey et al. demonstrate that intestinal helminth infection drives the remodeling and development of MRCs toward the paracortical region within the draining mLNs and requires the expression of type 2 cytokines and intimate B cell-stromal cell interactions. Keywords: marginal reticular cells, fibroblastic reticular cells, IL-4Rα, helminth infection, Heligmosomoides polygyrus, interfollicular region, CXCL13, mesenteric lymph node, MAdCAM-1, intestinal infection

Ulcerative colitis and Crohn's disease are the major forms of inflammatory bowel disease. Cytokines of the tumor necrosis factor (TNF) family play an important role in the regulation of intestinal inflammation. In this review, we discuss the function of key cytokines of this family - TNF and lymphotoxin (LT) - in mucosal healing, IgA production, and in control of innate lymphoid cells (ILCs), novel regulators of mucosal homeostasis in the gut. TNF plays a central role in the pathogenesis of inflammatory bowel diseases (IBD). LT regulates group 3 of ILCs and IL-22 production and protects the epithelium against damage by chemicals and mucosal bacterial pathogens. In addition, we discuss major mouse models employed to study the mechanism of intestinal inflammation, their advantages and limitations, as well as application of TNF blockers in the therapy for IBD.

Cerebral malaria (CM) has a high mortality rate and incidence of neurological sequelae in survivors. Hypoxia and cytokine expression in the brain are two mechanisms thought to contribute to the pathogenesis of CM. The cytokines interferon (IFN)-γ and lymphotoxin (LT)-α and the chemokine CXCL10 are essential for the development of CM in a mouse model. Furthermore, serum IFN-γ protein levels are higher in human CM than in controls, and CXCL10 is elevated in both serum and cerebrospinal fluid in Ghanaian paediatric CM cases. Astrocytes actively participate in CNS pathologies, becoming activated in response to various stimuli including cytokines. Astrocyte activation also occurs in murine and human CM. We here determined the responsiveness of mouse and human astrocytes to IFN-γ and LT-α, with the aim of further elucidating the role of astrocytes in CM pathogenesis. Initially we confirmed that Ifn-γ and Cxcl10 are expressed in the brain in murine CM, and that the increased Cxcl10 expression is IFN-γ-dependant. IFN-γ induced CXCL10 production in human and murine astrocytes in vitro. The degree of induction was increased synergistically in the presence of LT-α. IFN-γ induced the expression of receptors for LT-α, while LT-α increased the expression of the receptor for IFN-γ, in the astrocytes. This cross-induction may explain the synergistic effect of the two cytokines on CXCL10 production. Expression of these receptors also was upregulated in the brain in murine CM. The results suggest that astrocytes contribute to CM pathogenesis by producing CXCL10 in response to IFN-γ and LT-α.

SummaryTertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αβ (LTαβ) on Th17 cells and LTβR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTβR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.

Background & Aims Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice. Methods We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf–/–, Lta–/–, and Ltb–/– mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times. Results Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp–/– mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte–derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC. Conclusions Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Persistent NF-κB activation is a hallmark of the malignant Hodgkin/Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). Genomic lesions, Epstein-Barr virus infection, soluble factors, and tumor–microenvironment interactions contribute to this activation. Here, in an unbiased approach to identify the cHL cell-secreted key factors for NF-κB activation, we have dissected the secretome of cultured cHL cells by chromatography and subsequent mass spectrometry. We identified lymphotoxin-α (LTA) as the causative factor for autocrine and paracrine activation of canonical and noncanonical NF-κB in cHL cell lines. In addition to inducing NF-κB, LTA promotes JAK2/STAT6 signaling. LTA and its receptor TNFRSF14 are transcriptionally activated by noncanonical NF-κB, creating a continuous feedback loop. Furthermore, LTA shapes the expression of cytokines, receptors, immune checkpoint ligands and adhesion molecules, including CSF2, CD40, PD-L1/PD-L2, and VCAM1. Comparison with single-cell gene-activity profiles of human hematopoietic cells showed that LTA induces genes restricted to the lymphoid lineage, as well as those largely restricted to the myeloid lineage. Thus, LTA sustains autocrine NF-κB activation, impacts activation of several signaling pathways, and drives expression of genes essential for microenvironmental interactions and lineage ambiguity. These data provide a robust rationale for targeting LTA as a treatment strategy for cHL patients.

B cell-derived lymphotoxin (LT) is required for the development of follicular dendritic cell clusters for the formation of primary and secondary lymphoid follicles, but the role of T cell-derived LT in antibody response has not been well demonstrated. We observed that lymphotoxin β-receptor (LTβR) signaling is essential for optimal humoral immune response and protection against an acute herpes simplex virus 1 (HSV-1) infection. Blocking the LTβR pathway caused poor maintenance of germinal center B (GC-B) cells and follicular helper T (Tfh) cells. Using bone marrow chimeric mice and adoptive transplantation, we determined that T cell-derived LT played an indispensable role in the humoral immune response to HSV-1. Upregulation of gamma interferon by the LTβR-Ig blockade impairs the sustainability of Tfh-like cells, leading to an impaired humoral immune response. Our findings have identified a novel role of T cell-derived LT in the humoral immune response against HSV-1 infection. IMPORTANCE Immunocompromised people are susceptible to HSV-1 infection and lethal recurrence, which could be inhibited by anti-HSV-1 humoral immune response in the host. This study sought to explore the role of T cell-derived LT in the anti-HSV-1 humoral immune response using LT-LTβR signaling-deficient mice and the LTβR-Ig blockade. The data indicate that the T cell-derived LT may play an essential role in sustaining Tfh-like cells and ensure Tfh-like cells' migration into primary or secondary follicles for further maturation. This study provides insights for vaccine development against infectious diseases.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1β (IL-1β), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-β, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-β (transforming growth factor β). GI eosinophils expressed a relatively high level of IL-1β, and IL-1β-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1β in the small intestine.

T and B cell compartmentalization is a hallmark of secondary lymphoid organs and is maintained by chemokine-expressing stromal cells. How this stromal cell network initially develops and differentiates into two distinct subsets is poorly known, especially for the splenic white pulp (WP). Here, we show that perivascular fibroblast precursors are triggered by LTα1β2 signals to expand, express CCL19/21, and then differentiate into two functionally distinct fibroblast subsets responsible for B and T cell clustering and WP compartmentalization. Failure to express or sense CCL19 leads to impaired T zone development, while lack of B cells or LTα1β2 leads to an earlier and stronger impairment in WP development. We therefore propose that WP development proceeds in multiple steps, with LTα1β2 + B cells acting as major inducer cells driving the expansion and gradual differentiation of perivascular fibroblasts into T and B zone organizer cells.

Tertiary lymphoid structures (TLS) often develop at sites of persistent inflammation, including cancers and autoimmune diseases. In most cases, the presence of TLS correlates with active immune responses. Because of their proximity to pathological loci, TLS are an intriguing target for the manipulation of immune responses. For several years, it has become clear that lymphotoxin (LT) signalling plays critical roles in lymphoid tissue organogenesis and maintenance. In the current review, we will discuss the role of LT signalling in the development of TLS. With a focus on cancers and autoimmune diseases, we will highlight the correlations between TLS and disease progression. We will also discuss the current efforts and potential directions for manipulating TLS for immunotherapies.