Falk Weih

Lymphotoxin β 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 β receptor (LTβR) 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 LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Essential Role of RelB in Germinal Center and Marginal Zone Formation and Proper Expression of Homing Chemokines

High levels of the Rel/NF-κB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer’s patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB−/−) mice, in contrast to nfkb1−/−, but similar to nfkb2−/− mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB−/− splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB−/− spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.

Regulation of secondary lymphoid organ development by the nuclear factor-κB signal transduction pathway

Summary:  In primary lymphoid organs, such as thymus and bone marrow, B and T lymphocytes differentiate from lymphoid stem cells into mature albeit naïve effector cells. In contrast, secondary lymphoid organs, such as the spleen, lymph nodes, and Peyers patches (PPs), provide an environment that enable lymphocytes to interact with each other, with accessory cells, and with antigens, resulting in the initiation of antigen‐specific primary immune responses. Recently, the analysis of gene‐knockout mice has shed light on the signaling pathways, cellular requirements, and molecular mechanisms involved in secondary lymphoid organ development. In particular, signals that converge on the nuclear factor‐κB (NF‐κB) pathway have been demonstrated to play an important role in both early developmental steps as well as maintenance of secondary lymphoid organ structures. Analysis of the histopathological changes in secondary lymphoid tissues of mice lacking individual Rel/NF‐κB family members, upstream kinases, and receptors strongly indicates that activation of the recently described alternative NF‐κB pathway by membrane‐bound lymphotoxin, via p52–RelB heterodimers, plays a major role during initiation steps of secondary lymphoid organ development. Induction of the classical p50–RelA NF‐κB activity, as exemplified by tumor necrosis factor receptor signaling, clearly also contributes, but seems to be involved primarily in later developmental step, such as the proper cellular and structural organization of B‐cell follicles.

RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF.

Targeted disruption of the Rel/NF‐κB family members NF‐κB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyers patch (PP)‐organizing centers is impaired in both NF‐κB2‐ and RelB‐deficient animals. IL‐7‐induced expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP development, is not impaired in RelB‐deficient embryos. LTβ receptor (LTβR)‐deficient mice also lack PPs, and we demonstrate that LTβR signaling induces p52–RelB and classical p50–RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTβR‐induced binding of p52–RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF‐κB‐inducing kinase (NIK) and the IκB kinase (IKK) complex subunit IKKα, but not IKKβ or IKKγ. Activation of RelA requires all three IKK subunits, but is independent of NIK. Finally, we show that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA binding via the C‐terminus of p100. Our data indicate an important role of p52–RelB heterodimers in lymphoid organ development downstream of LTβR, NIK and IKKα.

Differential Requirement for Rel/Nuclear Factor κB Family Members in Natural Killer T Cell Development

Natural killer T (NKT) cells have been implicated in diverse immune responses ranging from suppression of autoimmunity to tumor rejection. Thymus-dependent NKT cells are positively selected by the major histocompatibility complex class I–like molecule CD1d, but the molecular events downstream of CD1d are still poorly understood. Here, we show that distinct members of the Rel/nuclear factor (NF)-κB family of transcription factors were required in both hematopoietic and nonhematopoietic cells for normal development of thymic NKT cells. Activation of NF-κB via the classical IκBα-regulated pathway was required in a cell autonomous manner for the transition of NK-1.1–negative precursors that express the TCR Vα14-Jα18 chain to mature NK-1.1–positive NKT cells. The Rel/NF-κB family member RelB, on the other hand, had to be expressed in radiation resistant thymic stromal cells for the generation of early NK-1.1–negative NKT precursors. Moreover, NF-κB–inducing kinase (NIK) was required for both constitutive thymic DNA binding of RelB and the specific induction of RelB complexes in vitro. Thus, distinct Rel/NF-κB family members in hematopoietic and nonhematopoietic cells regulate NKT cell development with a unique requirement for NIK-mediated activation of RelB in thymic stroma.

Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4

Abstract:  Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk‐B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)‐8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll‐like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA‐triggered activation of IL‐8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4‐specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP‐ and cytokine‐expression, in part in a TLR4‐dependent manner, providing new insights into the relationship between cancer and innate immunity.

Mice lacking the transcription factor RelB develop T cell-dependent skin lesions similar to human atopic dermatitis.

Mice with a targeted disruption of the Rel / NF‐κB family member RelB develop a complex inflammatory phenotype and hematopoietic abnormalities. RelB‐deficient (relB– / –) mice were clinically normal until 4 – 10 weeks after birth when thickening of the skin and hair loss developed. Histological and immunohistochemical evaluation of relB– / – skin lesions revealed hyperkeratosis and marked epidermal hyperplasia. Many CD4+ T cells and eosinophils mixed with lesser numbers of CD8+ T cells and neutrophils were present in the dermis. There was a moderate increase of MHC class II‐positive dermal dendritic cells and dermal mast cells. Increased expression of Th2 cytokines correlated with increased mRNA levels of eotaxin and CCR3 in relB– / – skin. The dermatitis did not develop in the offspring of relB– / – mice crossed with transgenic mice that lack peripheral T cells, demonstrating that the skin lesions were T cell dependent. The dermatitis observed in RelB‐deficient mice had many similarities with atopic dermatitis in human patients including infiltrating CD4+ T cells and eosinophils in the skin, increased number of eosinophils in the blood and increased serum IgE. Thus, the relB– / – mouse should be a useful model to study the pathogenesis of this common allergic human disease.

MHC class II and CD40 play opposing roles in dendritic cell survival

In contrast to very immature dendritic cells (DC), mature DC are largely resistant to death by CD95 (CD95/APO‐1) ligation. Investigation of other potential death‐inducing ligands showed that mature DC were instead highly susceptible to apoptosis induced by cross‐linking of MHC class II. Thus, increasing DC maturity correlates with increased resistance to CD95 killing, but an increased susceptibility to class II‐mediated killing. Anti‐I‐A/I‐E monoclonal antibodies (mAb) induced rapid (<2 h) apoptotic cell death in mature epidermal, spleen and bone marrow‐derived DC, as determined by annexin/propidium iodide staining, morphological changes, decreased diploidy and loss in mitochondrial membrane potential. Although full class II‐mediated killing required DC cytoskeletal motion, divalent cations and phosphatase activity, neither caspase activation, respiration, RNA or protein synthesis, NO production, nor CD95:CD95L interactions were required. Strikingly, DC pretreated by CD40 mAb cross‐linking, but not by lipopolysaccharide or TNF‐α , were completely resistant to class II‐mediated killing. CD40‐mediated protection was reduced in the presence of the SB202190 inhibitor of the mitogen‐activated protein kinase p38 pathway, but appeared to be independent of p42/44 extracellular signal‐related kinase or NF‐K B activation. Our findings show that in addition to its role as an activator of antigen‐presenting cell function, CD40 provides an important counter‐signal against class II‐induced apoptosis. Thus, these data point to an important role of the T cell in regulating DC survival.

Mouse Aorta Smooth Muscle Cells Differentiate Into Lymphoid Tissue Organizer-Like Cells on Combined Tumor Necrosis Factor Receptor-1/Lymphotoxin β-Receptor NF-κB Signaling

Objective—Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin &bgr;-receptor (LT&bgr;R). Circumstantial evidence has linked the SMC LT&bgr;R to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LT&bgr;R signaling in cultured SMC. Methods and Results—TNFR-1 signaling activated the classical RelA NF-&kgr;B pathway, whereas LT&bgr;R signaling activated the classical RelA and alternative RelB NF-&kgr;B pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-&kgr;B. Microarrays showed that simultaneous TNFR-1/LT&bgr;R activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LT&bgr;R cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LT&bgr;R–activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr−/− SMC indicated that LT&bgr;R-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype. Conclusion—SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.

Artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell Lymphotoxin β receptors

Summary 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 β receptors (VSMC-LTβRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTβRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe−/−Ltbr−/− and to a similar extent in aged Apoe−/−Ltbrfl/flTagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTβRs participate in atherosclerosis protection via ATLOs.