Redefining the Role of Lymphotoxin Beta Receptor in the Maintenance of Lymphoid Organs and Immune Cell Homeostasis in Adulthood.

Lymphotoxin beta receptor (LTßR) is a promising therapeutic target in autoimmune and infectious diseases as well as cancer. Mice with genetic inactivation of LTßR display multiple defects in development and organization of lymphoid organs, mucosal immune responses, IgA production and an autoimmune phenotype. As these defects are imprinted in embryogenesis and neonate stages, the impact of LTßR signaling in adulthood remains unclear. Here, to overcome developmental defects, we generated mice with inducible ubiquitous genetic inactivation of LTßR in adult mice (iLTßRΔ/Δ mice) and redefined the role of LTßR signaling in organization of lymphoid organs, immune response to mucosal bacterial pathogen, IgA production and autoimmunity. In spleen, postnatal LTßR signaling is required for development of B cell follicles, follicular dendritic cells (FDCs), recruitment of neutrophils and maintenance of the marginal zone. Lymph nodes of iLTßRΔ/Δ mice were reduced in size, lacked FDCs, and had disorganized subcapsular sinus macrophages. Peyer`s patches were smaller in size and numbers, and displayed reduced FDCs. The number of isolated lymphoid follicles in small intestine and colon were also reduced. In contrast to LTßR-/- mice, iLTßRΔ/Δ mice displayed normal thymus structure and did not develop signs of systemic inflammation and autoimmunity. Further, our results suggest that LTßR signaling in adulthood is required for homeostasis of neutrophils, NK, and iNKT cells, but is dispensable for the maintenance of polyclonal IgA production. However, iLTßRΔ/Δ mice exhibited an increased sensitivity to C. rodentium infection and failed to develop pathogen-specific IgA responses. Collectively, our study uncovers new insights of LTßR signaling in adulthood for the maintenance of lymphoid organs, neutrophils, NK and iNKT cells, and IgA production in response to mucosal bacterial pathogen.

mTORC1 activation in B cells confers impairment of marginal zone microarchitecture by exaggerating cathepsin activity.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.

Developmental expression of LTßR and differential expression in Escherichia coli F18 resistant/sensitive piglets.

We analyzed LTßR mRNA expression in piglets from birth to weaning and compared the differential expression between Escherichia coli F18-resistant and sensitive populations to determine whether this gene could be used as a genetic marker for E. coli F18 resistance. Sutai piglets of different age groups (8, 18, 30, and 35 days; N = 4 each) and piglets demonstrating resistance/sensitivity to E. coli F18 were used. LTßR expression levels were determined by real-time PCR. The LTßR expression levels in the lymph node, duodenum, and jejunum were significantly higher in 8-day-old piglets than in the other age groups (P < 0.01), and the expression levels were significantly higher in the lungs of 8-day-old piglets than in 35-day-old piglets (P < 0.01) and 30 day-old piglets (P < 0.05). In liver tissue, the expression level was significantly higher in the 35-day-old piglets than in other age groups (P < 0.01). In the stomach tissue, the expression level was significantly higher in 35-day-old piglets than in 18-day-old piglets (P < 0.05). LTßR expression in the lymph nodes was significantly higher in the resistant group than in the sensitive group (P < 0.01), but there was no significant difference in the other tissues (P > 0.05). These results indicate that 8 days after birth is a crucial stage in the formation of mesentery lymph nodes and immune barriers in pigs, and increased expression of LTßR may be beneficial for developing resistance to E. coli F18.

TNF superfamily members play distinct roles in shaping the thymic stromal microenvironment.

The differentiation and proper function of thymic epithelial cells (TECs) depend on various tumor necrosis factor superfamily (TNFSF) signals that are needed to maintain the thymic stromal microenvironment. Nevertheless, the direct transcriptional effects of these signals on TECs remain unclear. To address this issue, we stimulated murine embryonic thymus tissue with selected TNFSF ligands and performed a gene expression profiling study. We show that Aire expression is a direct and specific effect of RANKL stimulation, whereas LTß and TNFα are major inducers of chemokines in the thymic stroma and we propose differential NF-κB binding as one possible cause of these gene expression patterns. Our work provides further insight into the complex molecular pathways that shape the thymic microenvironment and maintain central tolerance.

Cooperative role of lymphotoxin ß receptor and tumor necrosis factor receptor p55 in murine liver regeneration.

BACKGROUND & AIMS: The liver exhibits a unique capacity for regeneration in response to injury. Lymphotoxin-ß receptor (LTßR), a core member of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily is known to play an important role in this process. However, the function of LTßR during pathophysiological alterations and its molecular mechanisms during liver regeneration are so far ill-characterized. METHODS: LTßR(-/-) mice were subjected to 70% hepatectomy and liver regeneration capacity, bile acid profiles, and transcriptome analysis were performed. RESULTS: LTßR(-/-) deficient mice suffered from increased and prolonged liver tissue damage after 70% hepatectomy, accompanied by deregulated bile acid homeostasis. Pronounced differences in the expression patterns of genes relevant for bile acid synthesis and recirculation were observed. LTßR and TNFRp55 share downstream signalling elements. Therefore, LTßR(-/-) mice were treated with etanercept to create mice functionally deficient in both signalling pathways. Strikingly, the combined blockade of TNFRp55 and LTßR signalling leads to complete failure of liver regeneration resulting in death within 24 to 48h after PHx. Transcriptome analysis revealed a marked disparity in gene expression programs in livers of LTßR(-/-) and etanercept-treated LTßR(-/-) vs. wild-type animals after PHx. Murinoglobulin 2 was identified as a significantly differentially regulated gene. CONCLUSIONS: LTßR is essential for efficient liver regeneration and cooperates with TNFRp55 in this process. Differences in survival kinetics strongly suggest distinct functions for these two cytokine receptors in liver regeneration. Failure of TNFR and LTßR signalling renders liver regeneration impossible.

LTßR expression on hematopoietic cells regulates acute inflammation and influences maturation of myeloid subpopulations.

Lymphotoxin beta-receptor (LTßR) is involved in the formation and maintenance of secondary lymphoid structures, as well as in the regulation of inflammatory responses. Because LTßR lymphoid structure formation continues to develop in infants, we compared two different chimera models: one using adult mice and the other using a transplantation model of neonatal mice. To elucidate the function of LTßR on lymphoid and non-lymphoid cells, we generated bone marrow chimeras on the wild type C57Bl/6 and the LTßR-deficient (LTßR(-/-)) background, and reconstituted the mice with bone marrow cells reciprocally. These chimeric mice were analyzed in the experimental model of acute dextran sulfate sodium-induced colitis. Interestingly, both models revealed not only equal reconstitution levels but also similar immunological responses: LTßR expression on stromal cells is essential for lymph node formation, whereas LTBR on hematopoietic cells is crucial for a decrease in inflammation. In addition, mice lacking LTßR on hematopoietic cells revealed (a) an increase of immature granulocytic cells in the spleen and (b) a reduced proportion of myeloid cells in peripheral blood and spleen expressing CD11b(+)Ly6C(+)Ly6G(-) (myeloid-derived suppressor cells expression profile). In conclusion, LTßR expression on hematopoietic cells seems to be involved in the down-regulation of acute inflammatory reactions paralleled by the appearance of immature myeloid cells.

Lymphotoxin-beta receptor expression and its related signaling pathways govern dendritic cell homeostasis and function.

Dendritic cells (DCs) play a fundamental function, either positive or detrimental, in regulating immune responses. Numerous specialized DC subsets exist in different organs. However, the trophic factors regulating their origin, location, homeostasis and functions remains to be fully understood. Recent evidence indicates that signaling via the lymphotoxin ß receptor (LTßR) can function as a trophic signaling system for specific DCs. LTßR is part of a complex signaling network that provides both positive and inhibitory signals to DC subsets. In this review, we focus on the role of LTßR expressed in DC subsets and its associated signaling pathways that regulate DC homeostasis and function. Therapeutically targeting the LTßR signaling pathway could support the development of a beneficial immune response for the host.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) beta-receptor-dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTalpha(1)beta(2)-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A-independent, LT- and B cell-dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.

Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells.

Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.

LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms.

BACKGROUND & AIMS: LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. METHODS: The effects of LIGHT and interferon-gamma on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers. RESULTS: LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-gamma, which induced lymphotoxin beta receptor (LT beta R) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LT beta R knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis. CONCLUSIONS: T cell-derived LIGHT activates intestinal epithelial LT beta R to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.