Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

Cholesterol restricts lymphotoxin ß receptor-triggered NF-κB signaling.

BACKGROUND: Lymphotoxin ß receptor (LTßR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTßR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTßR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTßR-induced NF-κB signaling. METHODS: To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-ß-cyclodextrin and simvastatin were applied. LTßR localization was studied by confocal microscopy. The activity of LTßR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTßR with its protein partners were investigated by immunoprecipitation. RESULTS: We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-ß-cyclodextrin impaired LTßR internalization and potentiated LTßR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTßR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS: Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTßR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTßR-based therapies. Video abstract.

Lymphotoxin ß receptor signalling executes Helicobacter pylori-driven gastric inflammation in a T4SS-dependent manner.

OBJECTIVE: Lymphotoxin ß receptor (LTßR) signalling has been implicated in inflammation-associated tumour development in different tissues. We have analysed the role of LTßR and alternative NF-κB signalling in Helicobacter pylori-mediated gastric inflammation and pathology. DESIGN: We analysed several ligands and receptors of the alternative NF-κB pathway, RelB, p52 nuclear translocation and target genes in tissue samples of H. pylori-infected patients with different degrees of gastritis or early gastric tumours by in situ hybridisation, immunohistochemistry, Western blot and real-time PCR analyses. Molecular mechanisms involved in LTßR activation by H. pylori were assessed in vitro using human gastric cancer cell lines and distinct H. pylori isolates. The effects of blocking or agonistically activating LTßR on gastric pathology during challenge with a human pathogenic H. pylori strain were studied in a mouse model. RESULTS: Among the tested candidates, LT was significantly increased and activated alternative NF-κB signalling was observed in the gastric mucosa of H. pylori-infected patients. H. pyloriinduced LTßR-ligand expression in a type IV secretion system-dependent but CagA-independent manner, resulting in activation of the alternative NF-κB pathway, which was further enhanced by blocking canonical NF-κB during infection. Blocking LTßR signalling in vivo suppressed H. pylori-driven gastritis, whereas LTßR activation in gastric epithelial cells of infected mice induced a broadened pro-inflammatory chemokine milieu, resulting in exacerbated pathology. CONCLUSIONS: LTßR-triggered activation of alternative NF-κB signalling in gastric epithelial cells executes H. pylori-induced chronic gastritis, representing a novel target to restrict gastric inflammation and pathology elicited by H. pylori, while exclusively targeting canonical NF-κB may aggravate pathology by enhancing the alternative pathway.

The lymphotoxin ß receptor is a potential therapeutic target in renal inflammation.

Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin ß receptor (LTßR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTßR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTßR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTß ligands, as well as LTßR. The LTßR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTß was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTß mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTßR signaling. Importantly, in a murine lupus model, LTßR blockade improved renal function without the reduction of serum autoantibody titers or glomerular immune complex deposition. Thus, a preclinical mouse model and human studies strongly suggest that LTßR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons, respectively.

A receptor-ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type-specific post-receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death-triggering factor in motoneurons through LT-ßR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT-induced axonal elongation and branching require ERK and caspase-9 pathways. This distinct response involves a compartment-specific activation of LIGHT signals, with somatic activation-inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.

LIGHT/HVEM/LTßR interaction as a target for the modulation of the allogeneic immune response in transplantation.

The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC-cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell-mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin ß receptor (LTßR) engagement. LIGHT-deficient mice, or WT mice treated with LIGHT-targeting decoy receptors HVEM-Ig, LTßR-Ig or sDcR3-Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTßR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor-specific tolerance.

Lymphotoxin-beta receptor blockade induces inflammation and fibrosis in tolerized cardiac allografts.

The lymphotoxin system (LT) regulates interactions between lymphocytes and stromal cells to maintain lymphoid microenvironmental homeostasis. Soluble LT beta-receptor-Ig (LTßRIg) blocks lymphocyte LTα1ß2-stromal cell LTßR signaling. In a murine cardiac allograft model, LTbRIg treatment reversed the tolerance induced by anti-CD40L antibody leading to graft inflammation and fibrosis. LTßRIg treatment decreased PD-L1 expression by blood endothelial cells, and decreased VCAM-1 while increasing CXCL1, CXCL2, CXCL12, CCL5, CCL21 and IL-6 expression in fibroblastic reticular cells. In secondary lymphoid organs these effects caused T- and B cell zone disruption, loss of CD35(+) follicular dendritic cells and abnormal recruitment of CD11b(+) Ly6G(+) neutrophils. These disruptions correlated with increased numbers of CD8(+) T cells and CD11b(+) Ly6G(+) neutrophils, and decreased numbers of CD4(+) T cells and Foxp3(+) regulatory T cells in the grafts. Depleting neutrophils or blocking neutrophil-attracting chemokines restored normal histology in lymph node, spleen and grafts. Taken together, LTßRIg treatment altered stromal subset, particularly fibroblastic reticular cell, production of cytokines and chemokines, resulting in changes in neutrophil recruitment in spleen, lymph node and grafts, and inflammation and fibrosis associated with decreased Foxp3(+) regulatory T cells and increased CD8(+) T cell infiltration of grafts.

Adiponectin inhibits lymphotoxin-ß receptor-mediated NF-κB signaling in human umbilical vein endothelial cells.

Adiponectin exerts anti-diabetic and anti-atherogenesis properties through its 2 receptors (AdipoR1 and AdipoR2). However, the signaling pathways responsible for the anti-inflammatory effects of adiponectin are largely unknown. In this study, we identified the lymphotoxin (LT)-ß receptor (LTBR) as an interacting partner of human AdipoR1 by using a yeast two-hybrid screening. The interaction between LTBR and AdipoR1 was confirmed by co-immunoprecipitation and co-localization analysis. Furthermore, adiponectin incubation inhibited lymphotoxin-induced NF-κB activation and the expression of adhesion molecules in human umbilical vein endothelial cells. These results indicated that AdipoR1 interacted with LTBR and mediated the inhibition of LTBR-activated NF-κB pathway.

Blocking lymphotoxin beta receptor signalling exacerbates acute DSS-induced intestinal inflammation--opposite functions for surface lymphotoxin expressed by T and B lymphocytes.

The lymphotoxin beta receptor (LTbetaR) signalling pathway is involved in the development of secondary lymphoid organs and the maintenance of organized lymphoid tissues. Additionally, previous studies clearly demonstrated the involvement of the LTbetaR interaction with its ligands in promoting intestinal inflammation. In order to dissect the role of LTbetaR activation in the mouse model of acute DSS-induced colitis we treated mice with a functional inhibitor of LTbetaR activation (LTbetaR:Ig) and compared it to disease in LTbetaR-deficient and LTalphabeta-deficient mice. All these modes of LTbetaR signalling ablation resulted in significant aggravation of the disease and in release of inflammatory cytokines such as TNF, IL-6, and IFNgamma. Finally, using mice with conditionally ablated expression of membrane bound LTbeta on T or B cells, respectively, distinct and opposite contributions of surface LTbeta expressed on T or B cells was found. Thus, activation of LTbetaR by LTalphabeta mainly expressed on T lymphocytes is crucial for the down regulation of the inflammatory response in this experimental model.

Lymphotoxin beta receptor (Lt betaR): dual roles in demyelination and remyelination and successful therapeutic intervention using Lt betaR-Ig protein.

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin beta receptor (Lt betaR) during the demyelination phase of this model, and Lt betaR is found in areas enriched with microglial and astroglial cells. Deletion of the Lt betaR gene (Lt betaR-/-) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of Lt betaR signaling by an Lt betaR-Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this Lt betaR-Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of Lt betaR-Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems of Lt betaR-/- mice. These findings bode well for the use of an inhibitory Lt betaR-Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling.

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

Effects of lymphotoxin beta receptor blockade on intestinal mucosal immunity.

BACKGROUND: Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) directs lymphocyte migration into gut-associated lymphoid tissue (GALT) through Peyer's patches (PPs). Parenteral nutrition (PN) impairs mucosal immunity by reducing PPs MAdCAM-1 expression, T and B cells in GALT, and intestinal and respiratory immunoglobulin (Ig) A levels. We previously showed that PN reduces lymphotoxin beta receptor blockade (LTbetaR) in PPs and intestine, and that stimulation with LTbetaR agonist antibodies reverses these defects. To confirm that LTbetaR regulates transcription of MAdCAM-1 message and more fully understand the effects of LTbetaR on MAdCAM-1 function within the mucosal immune system, we studied the effect of LTbetaR blockade with a chimeric LTbetaR Ig-fusion protein on MAdCAM-1 mRNA levels, PP lymphocyte mass and IgA levels in the intestinal and respiratory tracts. METHODS: Mice were cannulated and killed 3 days after receiving chow + control Ig, chow + LTbetaR-Ig fusion protein (100 microg IV), or PN + control Ig. The PPs of half of the animals were processed for lymphocyte count, and the other half were processed for complementary DNA and subsequent polymerase chain reaction (PCR). mRNA levels of MAdCAM-1 were determined by real-time PCR; intestinal and respiratory IgA levels were measured by ELISA. RESULTS: PN significantly reduced PP lymphocyte mass, MAdCAM-1 mRNA, and intestinal IgA. As anticipated, LTbetaR blockade significantly decreased PP cells and MAdCAM-1 mRNA, but not intestinal IgA because chow feeding was maintained. Both LTbetaR blockade and PN decreased nasal IgA, but not significantly. CONCLUSIONS: LTbetaR blockade in chow animals significantly reduces transcription of MAdCAM-1 gene and PPs lymphocyte mass. These data implicate inadequate LTbetaR signaling as a major mechanism for decreased GALT cells with lack of enteral stimulation, and further establish the role of LTbetaR in the mucosal immune system.

The Lymphotoxin Network: orchestrating a type I interferon response to optimize adaptive immunity.

The Lymphotoxin (LT) pathway is best known for its role in orchestrating the development and homeostasis of lymph nodes and Peyer's patches through the regulation of homeostatic chemokines. More recently an appreciation of the LTßR pathway in the production of Type I interferons (IFN-I) during homeostasis and infection has emerged. LTßR signaling is essential in differentiating stromal cells and macrophages in lymphoid organs to rapidly produce IFN-I in response to virus infections independently of the conventional TLR signaling systems. In addition, LTßR signaling is required to produce homeostatic levels of IFN-I from dendritic cells in order to effectively cross-prime a CD8+ T cell response to protein antigen. Importantly, pharmacological inhibition of LTßR signaling in mice has a profound positive impact on a number of autoimmune disease models, although it remains unclear if this efficacy is linked to IFN-I production during chronic inflammation. In this review, we will provide a brief overview of how the "Lymphotoxin Network" is linked to the IFN-I response and its impact on the immune system.

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-ß receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-ß receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases-for example, by lymphotoxin-ß receptor activation-allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

Lymphotoxin-beta receptor signalling regulates cytokine expression via TRIM30α in a TRAF3-dependent manner.

Our earlier studies indicated that activation of the lymphotoxin beta receptor (LTßR) by T cell derived LTα(1)ß(2) regulates inflammatory cytokine expression. While characterizing the cellular and molecular mechanisms responsible for the down regulation of the inflammatory reaction after LTßR stimulation we were able to identify the specific induction of TRIM30α expression as a result of LTßR signalling in mouse macrophages. Furthermore, we could demonstrate that LTßR activation in these cells results in the down regulation of pro-inflammatory cytokine (e.g. TNF and IL-6) and mediator expression upon TLR4 and TLR9 re-stimulation, demonstrating that LTßR activation on mouse macrophages dampens pro-inflammatory cytokine and mediator expression. Thus, LTßR signalling renders macrophages hypo-responsive to subsequent stimulation with TLR ligands. The observation of an LTßR-mediated TLR-tolerance in the human monocyte cell line THP-1 suggests that similar signalling mechanisms seem to exist in human cells. Signalling pathway analysis clearly demonstrated that LTßR-induced TRIM30α expression is mediated by an IκBα-dependent signalling pathway. Furthermore, the LTßR-induced TRIM30α expression seems to be TRAF3 dependent. Our data suggest that LTßR activation on mouse macrophages is involved in the control of pro-inflammatory cytokine and mediator expression by activation of a signalling pathway that controls exacerbating inflammatory cytokine production.

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome.

INTRODUCTION: In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome. METHODS: Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. RESULTS: LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). CONCLUSIONS: Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.

Blockade of lymphotoxin-beta receptor signaling reduces aspects of Sjögren's syndrome in salivary glands of non-obese diabetic mice.

INTRODUCTION: The lymphotoxin-beta receptor (LTbetaR) pathway is important in the development and maintenance of lymphoid structures. Blocking this pathway has proven beneficial in murine models of autoimmune diseases such as diabetes and rheumatoid arthritis. The aim of this study was to determine the effects of LTbetaR pathway blockade on Sjögren syndrome (SS)-like salivary gland disease in non-obese diabetic (NOD) mice. METHODS: The course of SS-like disease was followed in NOD mice that were given lymphotoxin-beta receptor-immunoglobulin fusion protein (LTbetaR-Ig) starting at 9 weeks of age. Treatment was given as a single weekly dose for 3, 7, or 10 weeks. Age-matched NOD mice treated with mouse monoclonal IgG1, or not treated at all, were used as controls. The severity of inflammation, cellular composition, and lymphoid neogenesis in the submandibular glands were determined by immunohistochemistry. Mandibular lymph nodes were also studied. Saliva flow rates were measured, and saliva was analyzed by a multiplex cytokine assay. The salivary glands were analyzed for CXCL13, CCL19, and CCL21 gene expression by quantitative polymerase chain reaction. RESULTS: Treatment with LTbetaR-Ig prevented the increase in size and number of focal infiltrates normally observed in this SS-like disease. Compared with the controls, the submandibular glands of LTbetaR-Ig-treated mice had fewer and smaller T- and B-cell zones and fewer high endothelial venules per given salivary gland area. Follicular dendritic cell networks were lost in LTbetaR-Ig-treated mice. CCL19 expression was also dramatically inhibited in the salivary gland infiltrates. Draining lymph nodes showed more gradual changes after LTbetaR-Ig treatment. Saliva flow was partially restored in mice treated with 10 LTbetaR-Ig weekly injections, and the saliva cytokine profile of these mice resembled that of mice in the pre-disease state. CONCLUSIONS: Our findings show that blocking the LTbetaR pathway results in ablation of the lymphoid organization in the NOD salivary glands and thus an improvement in salivary gland function.

Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice.

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Isolated lymphoid follicle maturation induces the development of follicular dendritic cells.

Isolated lymphoid follicles (ILFs) are recently identified lymphoid structures in the small intestine with features similar to Peyer's patches (PPs). Using immunohistochemistry we characterized the composition of ILFs in the small intestines of immunocompetent mice and of mice that lacked PPs as a result of either genetic deficiency of lymphotoxin or temporary in utero lymphotoxin-beta receptor-signalling blockade. We showed that although both immature and mature ILFs were present in the intestines of immunocompetent mice, PP-deficiency induced a significantly greater number of mature ILFs. We found that in addition to B-lymphocyte-containing germinal centres, mature ILFs also possessed large networks of follicular dendritic cells (FDCs). These features were not detected within immature ILFs. Indeed, the presence of FDCs could be used to reliably distinguish ILF maturity. Further analysis revealed that the area occupied by the FDCs within mature ILFs was substantial. The total area occupied by FDCs in all the mature ILFs in mice lacking PPs was equivalent to the total area occupied by FDCs in all the PPs and the few mature ILFs in immunocompetent mice. Based on these data we reasoned that in the absence of PPs, mature ILFs are important inductive sites for intestinal immune responses. Indeed, in mice that lacked PPs, ILF maturation coincided with a restoration of faecal immunoglobulin A levels to values that were comparable to those found in immunocompetent mice. Taken together, these data imply that the induction of germinal centres and FDC networks within mature ILFs in response to PP deficiency provides an important compensatory mechanism.

LIGHT Is critical for IL-12 production by dendritic cells, optimal CD4+ Th1 cell response, and resistance to Leishmania major.

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.