Fibroblasts as a source of self-antigens for central immune tolerance.

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin ß-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.

Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

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

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

Lymphotoxin regulates commensal responses to enable diet-induced obesity.

Microbiota are essential for weight gain in mouse models of diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin, a key molecule in gut immunity, were resistant to DIO. Ltbr(-/-) mice had different microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr(-/-) mice with their obese siblings rescued weight gain in Ltbr(-/-) mice, demonstrating the communicability of the obese phenotype. Ltbr(-/-) mice lacked interleukin 23 (IL-23) and IL-22, which can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.

LIGHT signaling through LTßR and HVEM in keratinocytes promotes psoriasis and atopic dermatitis-like skin inflammation.

Psoriasis (PS) and atopic dermatitis (AD) are common skin inflammatory diseases characterized by hyper-responsive keratinocytes. Although, some cytokines have been suggested to be specific for each disease, other cytokines might be central to both diseases. Here, we show that Tumor necrosis factor superfamily member 14 (TNFSF14), known as LIGHT, is required for experimental PS, similar to its requirement in experimental AD. Mice devoid of LIGHT, or deletion of either of its receptors, lymphotoxin ß receptor (LTßR) and herpesvirus entry mediator (HVEM), in keratinocytes, were protected from developing imiquimod-induced psoriatic features, including epidermal thickening and hyperplasia, and expression of PS-related genes. Correspondingly, in single cell RNA-seq analysis of PS patient biopsies, LTßR transcripts were found strongly expressed with HVEM in keratinocytes, and LIGHT was upregulated in T cells. Similar transcript expression profiles were also seen in AD biopsies, and LTßR deletion in keratinocytes also protected mice from allergen-induced AD features. Moreover, in vitro, LIGHT upregulated a broad spectrum of genes in human keratinocytes that are clinical features of both PS and AD skin lesions. Our data suggest that agents blocking LIGHT activity might be useful for therapeutic intervention in PS as well as in AD.

A dendritic-cell-stromal axis maintains immune responses in lymph nodes.

Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTßR) ligands were critical mediators, and LTßR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTßR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.

Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin ß Receptors.

Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe(-/-) mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4(+) T cells, generated CD4(+), CD8(+), T regulatory (Treg) effector and central memory cells, converted naive CD4(+) T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin ß receptors (VSMC-LTßRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTßRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe(-/-)Ltbr(-/-) and to a similar extent in aged Apoe(-/-)Ltbr(fl/fl)Tagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTßRs participate in atherosclerosis protection via ATLOs.

Lymphotoxin beta-activated LTBR/NIK/RELB axis drives proliferation in cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the intrahepatic (iCCA) or extrahepatic (eCCA) bile ducts with poor prognosis and limited treatment options. Prior evidence highlighted a significant contribution of the non-canonical NF-κB signalling pathway in initiation and aggressiveness of different tumour types. Lymphotoxin-ß (LTß) stimulates the NF-κB-inducing kinase (NIK), resulting in the activation of the transcription factor RelB. However, the functional contribution of the non-canonical NF-κB signalling pathway via the LTß/NIK/RelB axis in CCA carcinogenesis and progression has not been established. METHODS: Human CCA-derived cell lines and organoids were examined to determine the expression of NF-κB pathway components upon activation or inhibition. Proliferation and cell death were analysed using real-time impedance measurement and flow cytometry. Immunoblot, qRT-PCR, RNA sequencing and in situ hybridization were employed to analyse gene and protein expression. Four in vivo models of iCCA were used to probe the activation and regulation of the non-canonical NF-κB pathway. RESULTS: Exposure to LTα1/ß2 activates the LTß/NIK/RelB axis and promotes proliferation in CCA. Inhibition of NIK with the small molecule inhibitor B022 efficiently suppresses RelB expression in patient-derived CCA organoids and nuclear co-translocation of RelB and p52 stimulated by LTα1/ß2 in CCA cell lines. In murine CCA, RelB expression is significantly increased and LTß is the predominant ligand of the non-canonical NF-κB signalling pathway. CONCLUSIONS: Our study confirms that the non-canonical NF-κB axis LTß/NIK/RelB drives cholangiocarcinogenesis and represents a candidate therapeutic target.

Lymphotoxin beta receptor signaling directly controls airway smooth muscle deregulation and asthmatic lung dysfunction.

BACKGROUND: Dysregulation of airway smooth muscle cells (ASM) is central to the severity of asthma. Which molecules dominantly control ASM in asthma is unclear. High levels of the cytokine LIGHT (aka TNFSF14) have been linked to asthma severity and lower baseline predicted FEV1 percentage, implying that signals through its receptors might directly control ASM dysfunction. OBJECTIVE: Our study sought to determine whether signaling via lymphotoxin beta receptor (LTßR) or herpesvirus entry mediator from LIGHT dominantly drives ASM hyperreactivity induced by allergen. METHODS: Conditional knockout mice deficient for LTßR or herpesvirus entry mediator in smooth muscle cells were used to determine their role in ASM deregulation and airway hyperresponsiveness (AHR) in vivo. Human ASM were used to study signals induced by LTßR. RESULTS: LTßR was strongly expressed in ASM from normal and asthmatic subjects compared to several other receptors implicated in smooth muscle deregulation. Correspondingly, conditional deletion of LTßR only in smooth muscle cells in smMHCCreLTßRfl/fl mice minimized changes in their numbers and mass as well as AHR induced by house dust mite allergen in a model of severe asthma. Intratracheal LIGHT administration independently induced ASM hypertrophy and AHR in vivo dependent on direct LTßR signals to ASM. LIGHT promoted contractility, hypertrophy, and hyperplasia of human ASM in vitro. Distinguishing LTßR from the receptors for IL-13, TNF, and IL-17, which have also been implicated in smooth muscle dysregulation, LIGHT promoted NF-κB-inducing kinase-dependent noncanonical nuclear factor kappa-light-chain enhancer of activated B cells in ASM in vitro, leading to sustained accumulation of F-actin, phosphorylation of myosin light chain kinase, and contractile activity. CONCLUSIONS: LTßR signals directly and dominantly drive airway smooth muscle hyperresponsiveness relevant for pathogenesis of airway remodeling in severe asthma.

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

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

Cutting Edge: The RNA-Binding Protein Ewing Sarcoma Is a Novel Modulator of Lymphotoxin ß Receptor Signaling.

Lymphotoxin ß receptor (LTßR) signaling is crucial for lymphoid tissue organogenesis and immune homeostasis. To identify novel regulatory mechanisms for signaling, we implemented a two-step screen that uses coexpression analysis of human fibroblasts undergoing LTßR stimulation and affinity-purification mass spectrometry for the LTßR signaling protein TNFR-associated factor 3 (TRAF3). We identify Ewing sarcoma (EWS) protein as a novel LTßR signaling component that associates with TRAF3 but not with TNFR-associated factor 2 (TRAF2). The EWS:TRAF3 complex forms under unligated conditions that are disrupted following activation of the LTßR. We conclude that EWS limits expression of proinflammatory molecules, GM-CSF, and ERK-2, promoting immune homeostasis.

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

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

mTORC2 negatively controls the maturation process of medullary thymic epithelial cells by inhibiting the LTßR/RANK-NF-κB axis.

The differentiation of mature medullary thymic epithelial cells (mTECs) is critical for the induction of central immune tolerance. Although the critical effect of mechanistic target of rapamycin complex 1 (mTORC1) in shaping mTEC differentiation has been studied, the regulatory role of mTORC2 in the differentiation and maturation of mTECs is poorly understood. We herein reported that TEC-specific ablation of a rapamycin-insensitive companion of mTOR (RICTOR), a key component of mTORC2, significantly decreased the thymus size and weight, the total cell number of TECs, and the cell number of mTECs with a smaller degree of reduced cortical thymic epithelial cells. Interestingly, RICTOR deficiency significantly accelerated the mTEC maturation process, as indicated by the increased ratios of mature mTECs (MHCIIhi , CD80+ , and Aire+ ) to immature mTECs (MHCIIlo , CD80- , and Aire- ) in Rictor-deficient mice. The RNA-sequencing assays showed that the upregulated nuclear factor-κB (NF-κB) signaling pathway in Rictor-deficient mTECs was one of the obviously altered pathways compared with wild-type mTECs. Our studies further showed that Rictor-deficient mTECs exhibited upregulated expression of receptor activator of NF-κB (RANK) and lymphotoxin ß receptor (LTßR), as well as increased activity of canonical and noncanonical NF-κB signaling pathways as determined by ImageStream and Simple Western. Finally, our results showed that inhibition of NF-κB signaling pathways could partially reverse the accelerated maturation of mTECs in Rictor conditional KO mice. Thus, mTORC2 negatively controls the kinetics of the mTEC maturation process by inhibiting the LTßR/RANK-NF-κB signal axis.

Langerhans Cells Control Lymphatic Vessel Function during Inflammation via LIGHT-LTßR Signaling.

The lymphatic vasculature is an important route for dendritic cell (DC) or tumor cell migration from peripheral tissues to draining lymph nodes (DLNs). However, the underlying molecular and cellular mechanisms remain poorly understood. In this study, using conventional bone marrow chimeric mice and additional UVB radiation, we found that deficiency of LIGHT but not lymphotoxin (LT) α1ß2, likely on radioresistant Langerhans cells (LCs), resulted in impaired skin DC migration to DLNs during LPS-induced inflammation. In addition, LT ß receptor (LTßR), but not herpes virus entry mediator, was found to be the receptor of LIGHT controlling DC migration. Furthermore, conditional deficiency of LTßR in Tie2 cre or Lyve1 cre mice, but not in LTßR-deficient bone marrow chimeric mice, impaired DC migration, suggesting an important role of LTßR in radioresistant lymphatic endothelial cells (LECs), although the role of LTßR in blood endothelial cells remains intriguing. Mechanistically, the gene expression of both CCL21 and CCL19 was found to be reduced in skin LECs isolated from LC-LIGHT-conditionally deficient or Lyve1 cre Ltbr fl/fl mice compared with their controls upon LPS stimulation. Soluble recombinant LIGHT was able to upregulate CCL21 and CCL19 gene expression on SVEC4-10 endothelial cells. Doxycycline, an inhibitor of soluble LIGHT release in the inflamed skin, impaired skin CCL21 and CCL19 expression and DC migration. In addition, melanoma cell metastasis to DLNs was also inhibited in LC-LIGHT-conditionally deficient or Lyve1 cre Ltbr fl/fl mice. Together, our data suggest, to our knowledge, a previously unrecognized scenario in which LCs activate LECs via the LIGHT-LTßR signaling axis to promote DC migration or tumor cell metastasis.

Lymphotoxin-ß receptor-NIK signaling induces alternative RELB/NF-κB2 activation to promote metastatic gene expression and cell migration in head and neck cancer.

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

Increased expression of the TNF superfamily member LIGHT/TNFSF14 and its receptors (HVEM and LTßR) in patients with systemic sclerosis.

OBJECTIVES: This study aimed to assess the potential role of the TNF superfamily member lymphocyte T-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) in SSc through evaluation of: skin expression of LIGHT and its receptors, herpesvirus entry mediator and lymphotoxin ß-related receptor, and serum concentration of LIGHT in SSc patients. METHODS: Expression of LIGHT and its receptors was investigated by immunohistochemistry and evaluated semi-quantitatively in skin biopsies from 19 SSc patients and 9 healthy controls. Serum levels of LIGHT were measured using ELISA in 329 patients with SSc and 50 control subjects. RESULTS: Expression of LIGHT and both receptors was higher in SSc patients compared with controls (P < 0.05 for all comparisons). Patients with early SSc (⩽ 3 years from the first non-Raynaud's phenomenon symptom) showed higher expression of LIGHT and herpesvirus entry mediator compared with patients with longer disease duration (P < 0.05 for both comparisons). The mean serum concentration of LIGHT was significantly higher in SSc patients compared with the controls (P < 0.05). High serum concentration of LIGHT was associated with male sex, presence of digital ulcers, muscle involvement (defined by elevated serum creatine kinase levels), steroid treatment and lack of ACA. However, in multivariate regression analysis only presence of digital ulcers and creatine kinase elevation were independently associated with serum concentration of LIGHT. CONCLUSION: These data provide the first evidence of overexpression of LIGHT and its receptors in SSc and suggest that the LIGHT axis might contribute to the pathogenesis of SSc. Increased serum concentrations of LIGHT seem to reflect vascular injury in SSc.

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.

MKK4 activates non-canonical NFκB signaling by promoting NFκB2-p100 processing.

The NFκB family of transcription factors is crucial for innate or adaptive immunity, inflammation, and diseases including cancer. The two NFκB signaling pathways (canonical and non-canonical) differ from each other in extracellular signals, membrane receptors, signaling adaptors, and dimer subunits. The p52 (NFκB2) subunit, which participates in the non-canonical pathway, is generated by ubiquitin-mediated processing of the p100 precursor. Here, we found that NFκB2 processing and activation were mediated by mitogen-activated protein kinase kinase-4 (MKK4) and its substrate c-Jun N-terminal kinase (JNK). In MKK4-null mouse embryonic fibroblasts (MEFs), serum- and lymphotoxin ß receptor (LTßR) antibody-induced processing of p100 and nuclear translocation of p52 were found to be defective. Serum and LTßR antibody activated the MKK4-JNK signaling pathway, and SP600125, a JNK inhibitor, blocked p100 processing. Cellular senescence, one of the responses regulated by the non-canonical NFκB pathway, was observed more frequently in MKK4-null MEFs than in wildtype cells. These results suggest that the MKK4/JNK-dependent pathway regulates NFκB2 processing/activation and, through this mechanism, MKK4 and NFκB2 control cellular growth and senescence.

Deficiency in lymphotoxin ß receptor protects from atherosclerosis in apoE-deficient mice.

RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

A Lymphotoxin/Type I IFN Axis Programs CD8+ T Cells To Infiltrate a Self-Tissue and Propagate Immunopathology.

Type I IFNs (IFN-I) are cytokines that can mediate both immune suppression and activation. Dendritic cells (DC) are significant producers of IFN-I, and depending on the context (nature of Ag, duration of exposure to Ag), DC-derived IFN-I can have varying effects on CD8(+) T cell responses. In this study, we report that in the context of a CD8(+) T cell response to a self-Ag, DC-intrinsic expression of IFN regulatory factor 3 is required to induce optimal proliferation and migration of autoreactive CD8(+) T cells, ultimately determining their ability to infiltrate a target tissue (pancreas), and the development of glucose intolerance in rat insulin promoter-glycoprotein (RIP-GP) mice. Moreover, we show that signals through the lymphotoxin-ß receptor (LTßR) in DC are also required for the proliferation of autoreactive CD8(+) T cells, the upregulation of VLA4/LFA1 on activated CD8(+) T cells, and their subsequent infiltration into the pancreas both in vitro and in vivo. Importantly, the defects in autoreactive CD8(+) T cell proliferation, accumulation of CD8(+) T cells in the pancreas, and consequent glucose intolerance observed in the context of priming by LTßR(-/-) DC could be rescued by exogenous addition of IFN-I. Collectively, our data demonstrate that the LTßR/IFN-I axis is essential for programming of CD8(+) T cells to mediate immunopathology in a self-tissue. A further understanding of the IFN-I/LTßR axis will provide valuable therapeutic insights for treatment of CD8(+) T cell-mediated autoimmune diseases.