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Tumor necrosis factor alpha (TNF) has sleep regulatory and brain development roles. TNF promotes sleep in vivo and in vitro while TNF inhibition diminishes sleep. Transmembrane (tm) TNF and the tmTNF receptors (Rs), are cleaved by tumor necrosis factor alpha convertase to produce soluble (s) TNF and sTNFRs. Reverse signaling occurs in cells expressing tmTNF upon sTNFR binding. sTNFR administration in vivo inhibits sleep, thus we hypothesized that a wake-like state in vitro would be induced by sTNFR-tmTNF reverse signaling. Somatosensory cortical neuron/glia co-cultures derived from male and female mice lacking both TNFRs (TNFRKO), or lacking TNF (TNFKO) and wildtype (WT) mice were plated onto six-well multi-electrode arrays. Daily one-hour electrophysiological recordings were taken on culture days 4 through 14. sTNFR1 (0.0, 0.3, 3, 30, 60, and 120 ng/µL) was administered on day 14. A final one-hour recording was taken on day 15. Four measures were characterized that are also used to define sleep in vivo: action potentials (APs), burstiness index (BI), synchronization of electrical activity (SYN), and slow wave power (SWP; 0.25–3.75 Hz). Development rates of these emergent electrophysiological properties increased in cells from mice lacking TNF or both TNFRs compared to cells from WT mice. Decreased SWP, after the three lowest doses (0.3, 3 and 30 ng/µL) of the sTNFR1, indicate a wake-like state in cells from TNFRKO mice. A wake-like state was also induced after 3 ng/µl sTNFR1 treatment in cells from TNFKO mice, which express the TNFR1 ligand, lymphotoxin alpha. Cells from WT mice showed no treatment effects. Results are consistent with prior studies demonstrating involvement of TNF in brain development, TNF reverse signaling, and sleep regulation in vivo. Further, the current demonstration of sTNFR1 induction of a wake-like state in vitro is consistent with the idea that small neuronal/glial circuits manifest sleep- and wake-like states analogous to those occurring in vivo. Finally, that sTNF forward signaling enhances sleep while sTNFR1 reverse signaling enhances a wake-like state is consistent with other sTNF/tmTNF/sTNFR1 brain actions having opposing activities.

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.

The role of tumor necrosis factor (TNF) in the inflammatory response in rheumatoid arthritis (RA) is well established, whereas less is known about the role of TNF’s close homolog, lymphotoxin alpha (LTα). Increased levels of LTα are found in the serum and synovial tissue of patients with RA, and in vitro studies found that LTα-induced proliferation of RA fibroblast-like synoviocytes was at a similar level to TNF. These findings support the idea that anti-LTα treatment could be beneficial in patients with RA, but pateclizumab, an anti-LTα antibody, was not as efficacious as the anti-TNF agent adalimumab in reducing symptoms of RA in a head-to-head study, suggesting that anti-LTα therapies might not represent a valid alternative treatment option in patients with RA. However, suppression of LTα activity might be relevant in the context of RA-related comorbidities, as patients with RA have an increased risk of myocardial infarction (MI) compared with the general population, and specific polymorphisms of the LTα gene have been linked to increased MI risk. In this review, we summarize the key characteristics of LTα and the most recent findings on the role of LTα in RA.

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.

Background & Aims We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. Methods We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. Results Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc–induced tumors in mice. Conclusions MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418 ).

Medullary thymic epithelial cells (mTEC) purge the T cell repertoire of autoreactive thymocytes. Although dendritic cells (DC) reinforce this process by transporting innocuous peripheral self-antigens, the mechanisms that control their thymic entry remain unclear. Here we show that mTEC-CD4+ thymocyte crosstalk regulates the thymus homing of SHPS-1+ conventional DCs (cDC), plasmacytoid DCs (pDC) and macrophages. This homing process is controlled by lymphotoxin α (LTα), which negatively regulates CCL2, CCL8 and CCL12 chemokines in mTECs. Consequently, Ltα-deficient mice have increased expression of these chemokines that correlates with augmented classical NF-κB subunits and increased thymic recruitment of cDCs, pDCs and macrophages. This enhanced migration depends mainly on the chemokine receptor CCR2, and increases thymic clonal deletion. Altogether, this study identifies a fine-tuning mechanism of T cell repertoire selection and paves the way for therapeutic interventions to treat autoimmune disorders., Autoreactive T cells are removed during their development in the thymus through the functions of medullary thymic epithelial cells (mTEC) and dendritic cells (DC), a process termed negative selection. Here the authors show that mTEC-T cell crosstalk and lymphotoxin α signalling are essential for the proper recruitment of DCs into the thymus.

Background Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and results En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/− mice, ApoE−/− mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15 weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/−mice. Anti-LTα preventive treatment was initiated in ApoE−/− mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/− mice exhibited atherosclerotic lesions at any developmental stage. Significance ApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

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.

Lymphotoxin expressed by RORγt+ innate lymphoid cells is critical for natural killer cell development., Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1β2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORγt+ innate lymphoid cells (ILCs), and not CD3−NK1.1+ cells, express LT to drive NK development. Similar to LT−/− or RORγt−/− mice, the mice conditionally lacking LTα1β2 on RORγt+ ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1+ ILCs from BM or purified RORγt+ ILCs from lamina propria lymphocytes into LT-deficient RORγt+ BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORγt+ ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development.

CD169-positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169-positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115+ F4/80(lo) CD11c(lo) macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll-like receptor signalling in mucosal homeostasis, the presence of CD169+ macrophages was not affected in mice that were deficient in MyD88-mediated Toll-like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169+ macrophages was dependent on lymphotoxin α, colonic CD169+ macrophages were present in normal numbers in lymphotoxin α-deficient mice. In contrast, reduced numbers of CD169+ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169+ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169+ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.

The journey from the discoveries of lymphotoxin (LT) and tumor necrosis factor (TNF) to the present day age of cytokine inhibitors as therapeutics has been an exciting one with many participants and highs and lows; the saga is compared to that in "The Wizard of Oz". This communication summarizes the contributions of key players in the discovery of the cytokines and their receptors, the changes in nomenclature, and the discovery of the LT family's crucial role in secondary and tertiary lymphoid organs. The remarkable advances in therapeutics are detailed as are remaining problems. Finally, special tribute is paid to two pioneers in the field who have recently passed away: Byron H. Waksman and Lloyd Old.

The tumor necrosis factor superfamily (TNFSF) consists of more than 20 members that can modulate cellular and immunological functions, including cell survival and the stimulation of an inflammatory response. Many TNF superfamily members display potent anticancer activity when used as recombinant proteins in vitro and in vivo. While TNF, TRAIL and FasL have already been used as payloads in antibody-based pharmacodelivery strategies, most TNF superfamily members have not yet been investigated as antibody payloads. Here, we report the cloning, production and characterization of eight novel antibody fusion proteins based on CD40L, FasL, TRAIL, LiGHT, VEGI, lymphotoxin alpha, lymphotoxin beta and lymphotoxin alpha1/beta2. The monoclonal antibody F8 was chosen as fusion partner of proven tumor targeting performance, which recognizes the alternatively-spliced EDA domain of fibronectin, a marker of angiogenesis. A quantitative biodistribution analysis performed with radioiodinated protein preparations in tumor-bearing mice revealed that TRAIL and lymphotoxin alpha1/beta2 were able to selectively accumulate at the tumor site, while all other members of the TNF superfamily abrogated the selective tumor targeting performance of the parental antibody or accumulated also in healthy tissues. The study indicates that even cytokines, which are closely related in terms of structure and function, may have a substantially different impact on the biodistribution and functional properties of the corresponding fusions with disease-homing antibodies.

Progranulin (proepithelin, granulin precursor) has been recently suggested to exhibit anti-inflammatory properties by directly binding to tumour necrosis factor (TNF) receptors and thereby inhibiting TNF signalling by Tang et al. This finding was challenged by Chen et al. and no interaction between progranulin and TNF receptor (TNFR) 1 or 2 was observed. We tested the ability of recombinant progranulin from different commercial sources to inhibit TNF- or lymphotoxin-α-induced signalling through TNFR1. We observed that progranulin does not affect signalling and cell death induction downstream of TNF or lymphotoxin-α. Our results suggest that the anti-inflammatory role of progranulin is not mediated through direct inhibition of TNFR1.

Recent clinical observations demonstrate adiponectin (APN), an adipocytokine with potent cardioprotective actions, is significantly reduced following myocardial ischemia/reperfusion (MI/R). However, mechanisms responsible for MI/R-induced hypoadiponectinemia remain incompletely understood. Adult male mice were subjected to 30-min MI followed by varying reperfusion periods. Adipocyte APN mRNA and protein expression and plasma APN and TNFα concentrations were determined. APN expression/production began to decline 3 h after reperfusion (reaching nadir 12 h after reperfusion), returning to control levels 7 days after reperfusion. Plasma TNFα levels began to increase 1 h after reperfusion, peaking at 3 h and returning to control levels 24 h after reperfusion. TNFα knockout significantly increased plasma APN levels 12 h after reperfusion but failed to improve APN expression/production 72 h after reperfusion. In contrast, TNF receptor-1 (TNFR1) knockout significantly restored APN expression 12 and 72 h after reperfusion, suggesting that other TNFR1 binding cytokines contribute to MI/R-induced APN suppression. Among many cytokines increased after MI/R, lymphotoxin-α (LTα) was the only cytokine remaining elevated 24–72 h after reperfusion. LTα knockout did not augment APN levels 12 h post-reperfusion, but did so by 72 h. Finally, in vitro treatment of adipocytes with TNFα and LTα at concentrations seen in MI/R plasma additively inhibited APN expression/production in TNFR1-dependent fashion. Our study demonstrates for the first time that LTα is a novel suppressor of APN expression and contributes to the sustained hypoadiponectinemia following MI/R. Combining anti-TNFα with anti-LTα strategies may achieve the best effects restoring APN in MI/R patients.

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTα3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTα have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTα3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTα is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTα- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta−/− donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTα were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTα3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

B cell responses are required for resistance to Toxoplasma gondii ; however, the events that lead to production of class-switched antibodies during T. gondii infection have not been defined. Indeed, mice challenged with the parasite exhibited an expansion of T follicular helper cells and germinal center B cells in the spleen. Unexpectedly, this was not associated with germinal center formation and was instead accompanied by profound changes in splenic organization. This phenomenon was transient and was correlated with a decrease in expression of effector proteins that contribute to splenic organization, including lymphotoxins α and β. The importance of lymphotoxin was confirmed, as the use of a lymphotoxin β receptor agonist results in partial restoration of splenic structure. Splenectomized mice were used to test the splenic contribution to the antibody response during T. gondii infection. Analysis of splenectomized mice revealed delayed kinetics in the production of parasite-specific antibody, but the mice did eventually develop normal levels of parasite-specific antibody. Together, these studies provide a better understanding of how infection with T. gondii impacts the customized structures required for the optimal humoral responses to the parasite and the role of lymphotoxin in these events.

This review focuses on the biological functions and signalling pathways activated by Lymphotoxin α (LTα)/Lymphotoxin β (LTβ) and their receptor LTβR. Genetic mouse models shed light on crucial roles for LT/LTβR to build and to maintain the architecture of lymphoid organs and to ensure an adapted immune response against invading pathogens. However, chronic inflammation, autoimmunity, cell death or cancer development are disorders that occur when the LT/LTβR system is twisted. Biological inhibitors, such as antagonist antibodies or decoy receptors, have been developed and used in clinical trials for diseases associated to the LT/LTβR system. Recent progress in the understanding of cellular trafficking and NF-κB signalling pathways downstream of LTα/LTβ may bring new opportunities to develop therapeutics that target the pathological functions of these cytokines.

Type I interferons (IFN-αβ) are pleiotropic cytokines critical for antiviral host defense, and the timing and magnitude of their production involve a complex interplay between host and pathogen factors. Mouse cytomegalovirus (a β-herpesvirus) is a persistent virus that induces a biphasic IFN-αβ response during the first days of infection. The cell types and molecular mechanisms governing these 2 phases are unique, with splenic stromal cells being a major source of initial IFN-αβ, requiring communication with B cells expressing lymphotoxin, a tumor necrosis factor family cytokine. Here we review the factors that regulate this lymphotoxin-IFN-αβ "axis" during cytomegalovirus infection, highlight how stroma-derived IFN-αβ contributes in other models, and discuss how deregulation of this axis can lead to pathology in some settings.

Substance P (SP) elicits numerous potent neuroimmunomodulatory effects, increasing the release of tumor necrosis factor alpha (TNF-α). The study aimed to investigate immunoneural communication in experimentally-induced gastritis in rats. SP-containing nerve fibers and lymphocytes and mast cells were counted in the mucosa of the stomachs of rats using double immunohistochemical and confocal laser microscopic methods, proving colocalization of SP and TNF-α in the lymphocytes and mast cells. In controls, only the nerve fibers showed SP immunoreactivity (IR). However, in gastritis the number of SP-IR fibers and TNF-α IR lymphocytes and mast cells increased significantly (P

Lymphotoxin-α (LTα), lymphotoxin-β (LTβ), and tumor necrosis factor-α (TNFα) are inflammatory mediators that play crucial roles in lymphoid organ development. We demonstrate here that LTα also contributes to the function of lymphatic vessels and to lymphangiogenesis during inflammation. LTα−/− mice exhibited reduced lymph flow velocities and increased interstitial fluid pressure. Airways of LTβ−/− mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα−/− mice, as did the skin draining immunization sites of LTβ−/− mice. Macrophages, B cells, and T cells, known sources of LT and TNFα, were apparent in the skin surrounding the immunization sites as were LTα, LTβ, and TNFα mRNAs. Ectopic expression of LTα led to the development of LYVE-1 and Prox1-positive lymphatic vessels within tertiary lymphoid organs (TLOs). Quantification of pancreatic lymphatic vessel density in RIPLTαLTβ−/− and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTβ was not required for this process. Kidneys of inducible LTα transgenic mice developed lymphatic vessels before the appearance of obvious TLOs. These data indicate that LTα plays a significant role in lymphatic vessel function and in inflammation-associated lymphangiogenesis.

To study gene expression within the mammary glands of dairy goats with mastitis, mRNA was collected from milk somatic cells (MSCs) of left udder halves challenged with Staphylococcus aureus and right udder halves infused with PBS, as control, at different time points (0, 12, 24 and 48 h post-infection). Transcriptional profiles were investigated using bovine cDNA microarrays; of the total 288 differentially expressed genes identified with ANOVA analysis (False Discovery Rate = 0.05, 1.5-fold change), 26, 36 and 16 genes were down-regulated at 12, 24 and 48 h post-infection, respectively, while 60, 141 and 9 genes were up-regulated at the same corresponding time points. The expression profiles clearly changed at 24 h post-infection with 177 genes significantly altered, corresponding to a 10-fold increase of S. aureus bacterial count in milk from infected udders. Differential expression of selected genes (CD2BP2, BCAP31, MHCII, FOSL2, MAPK13, ILT5 and JUNB) was also confirmed by real-time PCR at the different time points considered, showing high correlation with the microarray measurements and high reliability of the microarray analyses. The most readily inducible classes of genes in caprine MSCs infected with S. aureus were pro-inflammatory cytokines, chemokines and their receptors; IL-1α, lymphotoxin alpha, granulocyte chemotactic protein (CXCL6), and IL-2 receptor gamma were all up-regulated in infected udders versus healthy controls. This study identified a number of differentially expressed genes induced by S. aureus intramammary infection and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.

CD8 memory T cells can play a critical role in protection against repeated exposure to infectious agents such as viruses, yet can also contribute to the immunopathology associated with these pathogens. Understanding the mechanisms that control effective memory responses has important ramifications for vaccine design and in the management of adverse immune reactions. Recent studies have implicated several members of the tumor necrosis factor receptor (TNFR) family as key stimulatory and inhibitory molecules involved in the regulation of CD8 T cells. In this review, we discuss their control of the generation, persistence, and reactivation of CD8 T cells during virus infection.

B and T lymphocyte associated (BTLA) is an Ig domain superfamily protein with cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Its ligand, herpesvirus entry mediator (HVEM), is a tumor necrosis factor receptor superfamily member. The unique interaction between BTLA and HVEM allows for a system of bidirectional signaling that must be appropriately regulated to balance the outcome of the immune response. HVEM engagement of BTLA produces inhibitory signals through SH2 domain–containing protein tyrosine phosphatase 1 (Shp-1) and Shp-2 association, whereas BTLA engagement of HVEM produces proinflammatory signals via activation of NF-κB. The BTLA-HVEM interaction is intriguing and quite complex given that HVEM has four other ligands that also influence immune responses, the conventional TNF ligand LIGHT and lymphotoxin α, as well as herpes simplex virus glycoprotein D and the glycosylphosphatidylinositol-linked Ig domain protein CD160. BTLA-HVEM interactions have been shown to regulate responses in several pathogen and autoimmune settings, but our understanding of this complex system of interactions is certainly incomplete. Recent findings of spontaneous inflammation in BTLA-deficient mice may provide an important clue.

The identification of individuals genetically susceptible to cancer calls for preventive measures to minimize the cancer risk in these high-risk populations. Immune prevention is made necessary by the anticipated health threat, but lack of enough high-affinity T cells against tumor-associated antigens and the unpredictability of tumor antigens make antigen-based immune prevention untenable for cancer. To address this issue, we explored a non-antigen-based cancer immune prevention strategy using the transgenic adenocarcinoma of mouse prostate model that spontaneously develops prostate cancer with 100% penetrance. We show that targeted mutation of the lymphotoxin α ( LT α) gene efficiently rescued tumor-reactive T cells, drastically reduced cancer incidence, and almost completely ablated metastasis. Remarkably, short-term treatments with the fusion protein consisting of constant region of IgG and extracellular domain of lymphotoxin β receptor (LTβRIg) interrupted clonal deletion, reduced the size of the primary cancer, and completely prevented metastasis later in life. Our data demonstrated the value of non-antigen-based immune prevention for those with a genetic predisposition to cancer.

Herpes virus entry mediator (HVEM) plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha and also serves as one of the entry receptors of herpes simplex virus (HSV). The purpose of this study was to better understand the expression of HVEM in the cornea and trigeminal ganglia (TG), which are important targets of HSV infection.Immunohistochemistry was used to define HVEM expression in the cornea and TG of normal and HSV-1 infected mice euthanized 2 to 5 days or 7 months following corneal inoculation of virus.We found that HVEM is widely expressed in the normal corneal epithelium and endothelium, is weakly and focally expressed in the corneal stroma, and is expressed in a portion of neurons and non-neuronal cells in the TG. Acute HSV-1 keratitis and ganglionitis were associated with increased HVEM expression in the corneal epithelium and stroma and in neurons and non-neuronal cells of TG, and many inflammatory cells in these tissues also expressed HVEM. TG derived from mice 7 months after virus inoculation demonstrated latent HSV-1 infection that was associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues. Latent TG also contained focal infiltrates of mononuclear inflammatory cells, many of which expressed HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis, with no evidence of virus replication or increased HVEM expression in the corneal epithelium, and inflammatory cells present showed only weak HVEM expression.HVEM is expressed in the cornea and TG and therefore may serve as an HSV entry receptor in these tissues. Furthermore, these findings raise the possibility that changes in HVEM expression following ocular HSV-1 infection can modulate HSV spread and infection-induced inflammation in the cornea and TG.

To study the specific role of transmembrane tumor necrosis factor (tmTNF) in protective and pathological responses against the gastrointestinal helminth Trichinella spiralis , we compared the immune responses of TNF-α/lymphotoxin alpha (LTα) −/− mice expressing noncleavable transgenic tmTNF to those of TNF-α/LTα −/− and wild-type mice. The susceptibility of TNF-α/LTα −/− mice to T. spiralis infection was associated with impaired induction of a protective Th2 response and the lack of mucosal mastocytosis. Although tmTNF-expressing transgenic (tmTNF-tg) mice also had a reduced Th2 response, the mast cell response was greater than that observed in TNF-α/LTα −/− mice and was sufficient to induce the expulsion of the parasite. T. spiralis infection of tmTNF-tg mice resulted in significant intestinal pathology characterized by villus atrophy and crypt hyperplasia comparable to that induced following the infection of wild-type mice, while pathology in TNF-α/LTα −/− mice was significantly reduced. Our data thus indicate a role for tmTNF in host defense against gastrointestinal helminths and in the accompanying enteropathy. Furthermore, they also demonstrate that TNF-α is required for the induction of Th2 immune responses related to infection with gastrointestinal helminth parasites.

Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.

Lymphoid tissue inducer cells express a diverse array of tumor necrosis family ligands, including those that bind CD30 and the lymphotoxin β receptor. Both of these signaling pathways have been linked with B/T segregation in the spleen. In this study, we have dissected a lymphotoxin-independent CD30-dependent signal for the induction of expression of the T zone chemokine, CCL21. Reduced expression of CCL21 due to CD30 deficiency was functionally significant: mice deficient in both lymphotoxin and CD30 (dKO) signals had significantly smaller accumulations of lymphocytes in their splenic white pulp areas, with no evidence of focal aggregation of T cells. Furthermore, recruitment of wild-type CD4 T cells was poor in dKO mice compared with both wild-type or lymphotoxin-deficient mice. Phylogeny suggests that CD30 signals predated those through the lymphotoxin β receptor. We suggest that CD30 signals from lymphoid tissue inducer cells were a primitive mechanism to recruit and prime CD4 T cells. This would have been a stepping stone in the evolution of the highly organized lymphotoxin dependent B and T white pulp areas within which CD4-dependent memory Ab responses now develop.

Development of NKT cells was shown to depend on lymphotoxin (LT) and IL-15 signaling pathways as well as on cytokine receptor common gamma chain. After positive selection, NKT-cell precursors transit through progressive maturation stages including proliferative expansion within the NK1.1(-) window. The transcription factors that integrate different signaling pathways into different stages of NKT-cell development are not well characterized. Here, we show that the Rel/NF-kappaB family member RelA regulates the NK1.1(-) to NK1.1(+) transition during NKT-cell development. RelA is also required for both IL-15- and IL-7-induced proliferation of CD44(hi)NK1.1(-) NKT-cell precursors. Activation of the invariant NKT-cell receptor induces both IL-15 receptor alpha and gamma chains' expression in an NF-kappaB-dependent manner, suggesting a molecular mechanism by which NF-kappaB regulates NKT-cell development. NF-kappaB also regulates TCR-induced expression of LT-alpha and LT-beta within NKT cells. In contrast to previous reports, however, we show that LT signaling is dispensable for thymic NKT-cell development but is essential for their colonization of peripheral organs such as liver.

Lymph nodes (LNs) are important sentinel organs where antigen-presenting cells interact with T cells to induce adaptive immune responses. In cutaneous infection of mice withLeishmania major, resistance depends on the induction of a T-helper-cell-1 (Th1)-mediated cellular immune response in draining, peripheral LNs. We investigated whether draining, peripheral LNs are absolutely required for resistance againstL. majorinfection. We investigated the course of experimental leishmaniasis in wild-type (wt) mice lacking peripheral LNs (pLNs), which we generated by in utero blockade of membrane-bound lymphotoxin, and in mice lacking pLNs or all LNs due to genetic deletion of lymphotoxin ligands or receptors. wt mice of the resistant C57BL/6 strain without local skin-draining LNs were still able to generate specific T-cell responses, but this yielded Th2 cells. This switch to a Th2 response resulted in severe systemic infection. We also confirmed these results with mice lacking pLNs due to genetic depletion of lymphotoxin-β. The complete absence of LNs due to a genetic depletion of the lymphotoxin-β receptor also resulted in a marked deterioration of disease and a Th2 response. Thus, in the absence of pLNs, anL. major-specific Th2 response is induced in the remaining secondary lymphoid organs, such as the spleen and non-skin-draining LNs. This indicates a critical requirement for pLNs to induce protective Th1 immunity and suggests that whether Th1 or Th2 priming to the same antigen occurs depends on the site of the primary antigen recognition.

The TNF family is critical for development of lymphoid organs and plays significant roles in regulation of innate and adoptive immune responses. Here, we describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutant strain, HLB382, with a point mutation in the Lta gene, which encodes lymphotoxin (LT) α, a member of the TNF family. Mutant mice had no lymph nodes and no Peyer’s patches. Microscopically, the spleens had disordered follicles and no germinal centers or discernible marginal zones (MZ). While the development of T cells and follicular B cells was normal, the numbers of NK and MZ B cells were significantly reduced. Interestingly, the numbers of peritoneal B1b cells were significantly increased. Genomic DNA sequences revealed a single base pair insertion in the coding region of Lta resulting in a frame shift and a premature stop codon. This new strain provides opportunities for understanding the full range of Lta gene function on a pure C57BL/6 background.

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Tertiary lymphoid tissues are lymph node-like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin-alpha(RIP-LT alpha), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP-LT alpha allografts that harbor tertiary lymphoid tissues are rejected, while wild-type allografts that lack tertiary lymphoid tissues are accepted. Wild-type allografts transplanted at the same time as RIP-LT alpha skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP-LT alpha skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T-cell activation.

Infiltration of lymphocytes into the thyroid gland and formation of lymph node-like structures is a hallmark of Hashimoto's thyroiditis. Here we demonstrate that lymphatic vessels are present within these infiltrates. Mice overexpressing the chemokine CCL21 in the thyroid (TGCCL21 mice) developed similar lymphoid infiltrates and lymphatic vessels. TGCCL21 mice lacking mature T and B cells (RAGTGCCL21 mice) did not have cellular infiltrates or increased number of lymphatic vessels compared with controls. Transfer of CD3+CD4+T cells into RAGTGCCL21 mice promoted the development of LYVE-1+podoplanin+Prox-1+vessels in the thyroid. Genetic deletion of lymphotoxin β receptor or lymphotoxin α abrogated development of lymphatic vessels in the inflamed areas in the thyroid but did not affect development of neighboring lymphatics. These results define a model for the study of inflammatory lymphangiogenesis in the thyroid and implicate lymphotoxin β receptor signaling in this process.

Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (FDCs) in the GALT. Studies in mice have shown that this accumulation is obligatory for the efficient delivery of the TSE agent to the brain. However, which GALTs are crucial for disease pathogenesis is uncertain. Mice deficient in specific GALT components were used here to determine their separate involvement in scrapie agent neuroinvasion from the intestine. In the combined absence of the GALTs and FDCs (lymphotoxin (LT)α−/− mice and LTβ−/− mice), scrapie agent transmission was blocked. When FDC maturation was induced in remaining lymphoid tissues, mice that lacked both Peyer’s patches (PPs) and mesenteric lymph nodes (wild-type (WT)→LTα−/− mice) or PPs alone (WT→LTβ−/− mice) remained refractory to disease, demonstrating an important role for the PPs. Although early scrapie agent accumulation also occurs within the mesenteric lymph nodes, their presence in WT→LTβ−/− mice did not restore disease susceptibility. We have also shown that isolated lymphoid follicles (ILFs) are important novel sites of TSE agent accumulation in the intestine. Mice that lacked PPs but contained numerous FDC-containing mature ILFs succumbed to scrapie at similar times to control mice. Because the formation and maturation status of ILFs is inducible and influenced by the gut flora, our data suggest that such factors could dramatically affect susceptibility to orally acquired TSE agents. In conclusion, these data demonstrate that following oral exposure TSE agent accumulation upon FDCs within lymphoid tissue within the intestine itself is critically required for efficient neuroinvasion.

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.

Intermittent administration of parathyroid hormone (PTH) promotes oral osseous wound healing and protects against ligature-induced alveolar bone loss. However, its therapeutic value on periapical periodontitis is unknown. The goal of this study was to determine the effect of intermittent PTH administration on the progression of periapical periodontitis., Seven lymphotoxin alpha-deficient mice received pulp exposures of mandibular first and second molars. Exposed pulp in the right mandible was covered with plaque-contaminated fibrin, whereas exposed pulp in the left mandible was left open. After 4 weeks, the periapical tissues were examined to determine the effect of plaque-contaminated fibrin to induce periapical lesions. Fourteen mice received pulp exposure covered with plaque-contaminated fibrin. PTH (40 μg/kg/d) was administered intermittently to half of the mice for 3 weeks beginning 1 week after pulp exposure. The remaining half received saline injections as the vehicle control. At sacrifice, mandibles and tibiae were harvested and processed for histologic examination. Evaluation of neutrophils and blood vessels was performed after staining with immunofluorescence, and periradicular bone was histomorphometrically analyzed., The exposed pulp covered with plaque-contaminated fibrin resulted in significantly larger periapical lesions compared with the control. Intermittent PTH administration reduced the size of periapical lesions significantly. Significantly less neutrophil infiltration around the root apex was found in PTH-treated animals compared with the control., PTH treatment suppressed periapical inflammation by reducing neutrophil infiltration and protected against tissue destruction by periapical periodontitis.

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish ( Danio rerio ) to investigate how furin regulates host responses against mycobacteria. In steady-state furinA td204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum -infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinA td204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a ( tnfa ), lymphotoxin alpha ( lta ) and interleukin 17a/f3 ( il17a/f3 ) expression levels. The enhanced innate immune response in the furinA td204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.

Contains fulltext : 154818.pdf (Publisher’s version ) (Closed access) Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

Lymphotoxin alpha (LTα), tumor necrosis factor (TNF), and LTβ are related cytokines whose genes are clustered within 12 kb of genomic DNA inside of the major histocompatibility complex (10, 34). Their products have important but often overlapping functions in peripheral lymphoid organ development, microstructure maintenance, and immune response (2, 16, 50). LTα can exist in two main forms: the soluble homotrimeric form (sLTα), which shares receptors with TNF, and the transmembrane LTα1β2 heterotrimeric form, which interacts with a separate LTβ receptor (10, 18, 20, 50). Because of the close proximity of LT/TNF genes, one can envision that any manipulation with one gene, involving deletions or insertions, may influence the expression of the others. Indeed, defective TNF production by conventionally engineered LTα knockout (KO) mice was previously reported (3). Additionally, another report showed that Mycobacterium bovis BCG-infected LTα KO mice displayed a 10-fold decrease in TNF production at an early stage of infection and a twofold reduction at a later time point (9). Besides, low or high copy TNF transgenes improved T/B-cell zone segregation in the LTα−/− mice via a TNF receptor I (TNFRI)-dependent mechanism, and partially restored the organization of splenic B-cell follicles, suggesting that defective TNF expression might contribute to the phenotype of LTα-deficient mice (3). Such a role of TNF was further supported by the phenotype of the double LTβ/TNF KO mice, whose splenic architecture was disrupted similarly to that in “conventional” LTα KO mice (26). Thus, downregulation of TNF expression in conventional LTα KO mice might be due to targeting artifacts. An alternative hypothesis is that the LTα and TNF genes are involved in mutual regulation, perhaps through TNFRI, TNFRII, LTβ receptor (LTβR), and NF-κB. The essential role of TNF in control of several infections was documented (15, 35). At the same time, several studies utilizing LTα-deficient mice provided evidence for the TNF-independent role of soluble LTα in various physiological processes, including resistance to pathogens such as Mycobacterium tuberculosis, Listeria monocytogenes, and Toxoplasma gondii (41, 42, 44). Interestingly, a significant decrease in TNF mRNA expression after T. gondii infection was noted in the brains of neo cassette-containing LTα KO mice. Primed spleen cells from these mice displayed significantly reduced TNF synthesis upon restimulation with Toxoplasma antigen while peritoneal macrophages produced severely reduced amounts of TNF in response to different activating stimuli (44). Thus, defective TNF production by conventional LTα knockout animals could potentially lead to misinterpretation of the role of LTα in some pathophysiological models. The relative TNF deficiency in conventional LTα KO mice has been perplexing for many years but there was no tool available to directly address this problem. In this report, we describe a novel strain of LTα-deficient mice which allow investigation of these important issues. We used LoxP-Cre technology to create neo-free LTα KO (LTαΔ/Δ) mice capable of producing normal amounts of TNF both in vitro and in vivo. Our data strongly suggest that the decreased level of TNF production in LTα deficiency is not due to mutual regulation of LT and TNF but rather correlates with the presence of the neo cassette in the genetically manipulated LTα gene locus. On the other hand, our data support the main conclusions regarding the critical role of the LTα/LTβ-LTβR axis in development and maintenance of peripheral lymphoid tissues. Overall, the new LTαΔ/Δ mice will help to better define distinct and overlapping functions of TNF and LTα.

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

Although the essential role of tumor necrosis factor (TNF) in resistance toListeria monocytogenesinfection is well established, the roles of the related cytokines lymphotoxin alpha (LTα) and lymphotoxin beta (LTβ) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTα, and LTβ in the host response toListeria. To overcome the lack of peripheral lymph nodes in LTα−/−and LTβ−/−mice, bone marrow chimeras were constructed. TNF−/−and LTα−/−chimeras that lacked both secreted LTα3and membrane-bound LTα1β2and LTα2β1were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTβ−/−chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. TheListeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTα−/−and LTβ−/−chimeras, WT chimeras, and TNF−/−and WT mice). TNF−/−mice and LTα−/−chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF−/−and LTα−/−chimeras. Therefore, in addition to TNF, soluble LTα3plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor κB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE–RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-α in the presence of cofactors such as TGF-β. We provide direct evidence against the current paradigm that the TRANCE–RANK–TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Interactions between lymphotoxin (LT)alpha(1)beta(2) on inducer cells and the lymphotoxin beta receptor (LTbetaR) on stromal cells initiate development of lymph nodes and Peyer's patches. In this study, we assessed the contributions of LTalpha and LTbetaR to the development of cryptopatches (CP), aggregates of T cell precursors in the mouse small intestine. Mice genetically deficient in LTalpha or LTbetaR lacked CP. Bone marrow from LTalpha-deficient mice was unable to initiate development of CP or isolated lymphoid follicles (ILF) after transfer to CD132-null mice lacking CP and ILF. However, LTalpha-deficient bone marrow-derived cells contributed to CP formed in CD132-null mice receiving a mixture of wild-type and LTalpha-deficient bone marrow cells. Transfer of wild-type bone marrow into irradiated LTalpha-deficient mice resulted in reconstitution of both CP and ILF. However, the LT-dependent formation of CP was distinguished from the LT-dependent formation of ILF and Peyer's patches by not requiring the presence of an intact NF-kappaB-inducing kinase gene. CP but not ILF were present in the small intestine from NF-kappaB-inducing kinase-deficient alymphoplasia mice, indicating that the alternate NF-kappaB activation pathway required for other types of LTbetaR-dependent lymphoid organogenesis is dispensable for CP development. In addition, we identified VCAM-1(+) cells within both CP and ILF that are candidates for the stromal cells involved in receiving LT-dependent signals from the hemopoietic precursors recruited to CP. These findings demonstrate that interactions between cells expressing LTalpha(1)beta(2) and LTbetaR are a shared feature in the development of all small intestinal lymphoid aggregates.

Lymph node development depends on well-orchestrated interactions between lymphoid tissue inducer cells and stromal cells. In this issue of Immunity, Pikor and colleagues (2015) find that signals from IL-17-producing T helper cells can alter the stromal microenvironment of the inflamed brain to generate a neo-lymphoid organ that drives further inflammation.

Studies in mouse infection models clearly demonstrate tumour necrosis factor (TNF) to be a critical component of both the antibacterially protective and the inflammatory immune response to Mycobacterium tuberculosis. It is therefore not surprising that treatment of patients—for example, those with rheumatoid arthritis—with biological agents interfering with TNF activity have shown an increased risk of reactivating tuberculosis. However, conceivably, TNF targeting biological agents can be developed that because of their particular mode of action and their specific pharmacodynamics may be less likely to have this side effect.

In order to evaluate the role and mode of action of TNF in bleomycin-induced lung fibrosis, mice deficient for TNF and LT alpha (delta TNF/LT alpha) were examined at 2 months of age and after 3 weekly i.v. injections of bleomycin. The body weight of the delta TNF/LT alpha mice was 88 +/- 11% of that of the wild type littermates. Lung collagen, evaluated by its hydroxyproline content, was also lower (81 +/- 9%) in mutant than in wild type littermates. Bleomycin induced a diffuse alveolitis with focal areas of alveolar remodelling in wild type but not in delta TNF/LT alpha, mice. Lymphoid infiltration was also prominent in wild type, but absent from delta TNF/LT alpha, mice. Bleomycin injections increased collagen deposition, as evaluated by the lung hydroxyproline content, more markedly in wild type, than in delta TNF/LT alpha, mice. Cell trapping in the alveolar capillaries was evaluated by semi-quantitative electron microscopy. Bleomycin markedly increased platelet trapping in the alveolar capillaries of wild type, but not of delta TNF/LT alpha, mice. This study indicates that the expression of TNF/LT alpha genes increases the deposition of collagen in both untreated and inflamed lung and that these genes may act, at least in part, by promoting platelet trapping.

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cells express high levels of lymphotoxin and use this molecule as an autocrine growth factor. We hypothesized that the EBV-derived latent membrane protein 1 (LMP1) mediates lymphotoxin production by inducing NF-kappaB binding to the lymphotoxin promoter. We assessed lymphotoxin production, LMP1 expression, and NF-kappaB activation in Z-43 (EBV-positive lymphoblastoid cells), Daudi (EBV-positive Burkitt's cells), and 3A4 (EBV-negative Burkitt's cells containing a stably transfected tetracycline-inducible LMP1 construct). Z-43 cells expressed high levels of LMP1 (immunoblot) and lymphotoxin (ELISA); the EBV-positive Burkitt's lymphoma line Daudi expressed neither LMP1 nor lymphotoxin. Similarly, induction of LMP1 in the 3A4 cells (exposed to tetracycline) was accompanied by a 13-fold increase in lymphotoxin levels (ELISA) as compared to uninduced (LMP1-negative) cells. EMSAs demonstrated high levels of NF-kappaB activation in Z-43 and tetracycline-induced 3A4 cells, but much lower levels in the uninduced 3A4 cells. Exposure of these cells to Bay 11-7082 (an inhibitor of IkappaB phosphorylation and, therefore, NF-kappaB activation) abrogated NF-kappaB binding and lymphotoxin production in a dose-dependent manner in both Z-43 and 3A4 cells. Therefore, in our model system, autocrine lymphotoxin production is largely driven by NF-kappaB activation, which is in turn mediated by EBV-derived LMP1 signaling.