Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic-spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial-gut axis in human disease.

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor-deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow-derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of nuclear factor kappaB activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL-1, dermal macrophage function, or expression of the toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF) 3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of up-regulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency.

Heme oxygenase-1 (HO-1) is a key cytoprotective, antioxidant, and antiinflammatory molecule. The pathophysiological functions of HO-1 have been associated with its enzymatic activities in heme catabolism. We have examined the immune functions of HO-1 by its conditional ablation in myeloid cells (HO-1(M-KO) mice). We demonstrate that myeloid HO-1 is required for the activation of interferon (IFN) regulatory factor (IRF) 3 after Toll-like receptor 3 or 4 stimulation, or viral infection. HO-1-deficient macrophages show reduced expression of IFN-beta and of primary IRF3 target genes encoding RANTES, IP-10 and MCP-1. In the presence of polyI:C, myeloid HO-1 knockout mice infected with Listeria monocytogenes, a model dependent on IFN-beta production, showed enhanced bacterial clearance and survival, whereas control mice succumbed to infection. Moreover, after induction of experimental autoimmune encephalomyelitis, mice with myeloid-specific HO-1 deficiency developed a higher incidence and an exacerbated, nonremitting clinical disease correlating with persistent activation of antigen-presenting cells, enhanced infiltration of Th17 cells, and a nonregressing myelin-specific T cell reactivity. Notably, these defects were rectified by exogenous administration of IFN-beta, confirming that HO-1 functions directly upstream of this critical immune pathway. These results uncover a novel direct function for myeloid HO-1 in the regulation of IFN-beta production, establishing HO-1 as a critical early mediator of the innate immune response.

The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)-specific T cells in Lewis rats, whereas transfer of S100beta- or myelin oligodendrocyte glycoprotein (MOG)-specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein(+) T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-kappaB (NF-kappaB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLD(ex7/8) mice), which is a deubiquitinating enzyme that is integral to NF-kappaB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-kappaB proteins p100 and RelB in CYLD(ex7/8) B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants.

Rheumatoid arthritis is a destructive arthropathy characterized by chronic synovial inflammation that imposes a substantial socioeconomic burden. Under the influence of the proinflammatory milieu, synovial fibroblasts (SFs), the main effector cells in disease pathogenesis, become activated and hyperplastic, releasing proinflammatory factors and tissue-remodeling enzymes. This study shows that activated arthritic SFs from human patients and animal models express significant quantities of autotaxin (ATX; ENPP2), a lysophospholipase D that catalyzes the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA). ATX expression from SFs was induced by TNF, and LPA induced SF activation and effector functions in synergy with TNF. Conditional genetic ablation of ATX in mesenchymal cells, including SFs, resulted in disease attenuation in animal models of arthritis, establishing the ATX/LPA axis as a novel player in chronic inflammation and the pathogenesis of arthritis and a promising therapeutic target.

Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved to modulate TNF function is the proteolytic cleavage of its cell surface receptors. In humans, mutations affecting shedding of the p55TNF receptor (R) have been linked with the development of the TNFR-associated periodic syndromes, disorders characterized by recurrent fever attacks and localized inflammation. Here we show that knock-in mice expressing a mutated nonsheddable p55TNFR develop Toll-like receptor-dependent innate immune hyperreactivity, which renders their immune system more efficient at controlling intracellular bacterial infections. Notably, gain of function for antibacterial host defenses ensues at the cost of disbalanced inflammatory reactions that lead to pathology. Mutant mice exhibit spontaneous hepatitis, enhanced susceptibility to endotoxic shock, exacerbated TNF-dependent arthritis, and experimental autoimmune encephalomyelitis. These results introduce a new concept for receptor shedding as a mechanism setting up thresholds of cytokine function to balance resistance and susceptibility to disease. Assessment of p55TNFR shedding may thus be of prognostic value in infectious, inflammatory, and autoimmune diseases.

Perivascular microglia activation is a hallmark of inflammatory demyelination in multiple sclerosis (MS), but the mechanisms underlying microglia activation and specific strategies to attenuate their activation remain elusive. Here, we identify fibrinogen as a novel regulator of microglia activation and show that targeting of the interaction of fibrinogen with the microglia integrin receptor Mac-1 (alpha(M)beta(2), CD11b/CD18) is sufficient to suppress experimental autoimmune encephalomyelitis in mice that retain full coagulation function. We show that fibrinogen, which is deposited perivascularly in MS plaques, signals through Mac-1 and induces the differentiation of microglia to phagocytes via activation of Akt and Rho. Genetic disruption of fibrinogen-Mac-1 interaction in fibrinogen-gamma(390-396A) knock-in mice or pharmacologically impeding fibrinogen-Mac-1 interaction through intranasal delivery of a fibrinogen-derived inhibitory peptide (gamma(377-395)) attenuates microglia activation and suppresses relapsing paralysis. Because blocking fibrinogen-Mac-1 interactions affects the proinflammatory but not the procoagulant properties of fibrinogen, targeting the gamma(377-395) fibrinogen epitope could represent a potential therapeutic strategy for MS and other neuroinflammatory diseases associated with blood-brain barrier disruption and microglia activation.

We describe new T cell receptor (TCR) transgenic mice (relapsing-remitting [RR] mice) carrying a TCR specific for myelin oligodendrocyte glycoprotein (MOG) peptide 92-106 in the context of I-A(s). Backcrossed to the SJL/J background, most RR mice spontaneously develop RR experimental autoimmune encephalomyelitis (EAE) with episodes often altering between different central nervous system tissues like the cerebellum, optic nerve, and spinal cord. Development of spontaneous EAE depends on the presence of an intact B cell compartment and on the expression of MOG autoantigen. There is no spontaneous EAE development in B cell-depleted mice or in transgenic mice lacking MOG. Transgenic T cells seem to expand MOG autoreactive B cells from the endogenous repertoire. The expanded autoreactive B cells produce autoantibodies binding to a conformational epitope on the native MOG protein while ignoring the T cell target peptide. The secreted autoantibodies are pathogenic, enhancing demyelinating EAE episodes. RR mice constitute the first spontaneous animal model for the most common form of multiple sclerosis (MS), RR MS.

The importance of mesenchymal cells in inflammation and/or neoplastic transformation is well recognized, but their role in the initiation of these processes, particularly in the intestine, remains elusive. Using mouse models of colorectal cancer, we show that IKKβ in intestinal mesenchymal cells (IMCs) is critically involved in colitis-associated, but not spontaneous tumorigenesis. We further demonstrate that IMC-specific IKKβ is involved in the initiation of colitis-associated cancer (CAC), as in its absence mice develop reduced immune cell infiltration, epithelial cell proliferation, and dysplasia at the early stages of the disease. At the molecular level, these effects are associated with decreased early production of proinflammatory and protumorigenic mediators, including IL-6, and reduced STAT3 activation. Ex vivo IKKβ-deficient IMCs show defective responses to innate immune stimuli such as LPS, as shown by decreased NF-κB signaling and reduced expression of important NF-κB target genes. Collectively, our results reveal a hitherto unknown role of mesenchymal IKKβ in driving inflammation and enabling carcinogenesis in the intestine.

TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

Innate mechanisms in the tumor stroma play a crucial role both in the initial rejection of tumors and in cancer promotion. Here, we provide a concise overview of the innate system in cancer and recent advances in the field, including the activation and functions of innate immune cells and the emerging innate properties and modulatory roles of the fibroblastic mesenchyme. Novel insights into the diverse identities and functions of the innate immune and mesenchymal cells in the microenvironment of tumors should lead to improved anticancer therapies.