Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.

Extensive phenotypic characterization of a new transgenic mouse reveals pleiotropic perturbations in physiology due to mesenchymal hGH minigene expression.

The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues.

Epigenetically-driven anatomical diversity of synovial fibroblasts guides joint-specific fibroblast functions.

A number of human diseases, such as arthritis and atherosclerosis, include characteristic pathology in specific anatomical locations. Here we show transcriptomic differences in synovial fibroblasts from different joint locations and that HOX gene signatures reflect the joint-specific origins of mouse and human synovial fibroblasts and synovial tissues. Alongside DNA methylation and histone modifications, bromodomain and extra-terminal reader proteins regulate joint-specific HOX gene expression. Anatomical transcriptional diversity translates into joint-specific synovial fibroblast phenotypes with distinct adhesive, proliferative, chemotactic and matrix-degrading characteristics and differential responsiveness to TNF, creating a unique microenvironment in each joint. These findings indicate that local stroma might control positional disease patterns not only in arthritis but in any disease with a prominent stromal component.

Proof of concept: enthesitis and new bone formation in spondyloarthritis are driven by mechanical strain and stromal cells.

OBJECTIVES: Spondyloarthritides (SpA) are characterised by both peripheral and axial arthritis. The hallmarks of peripheral SpA are the development of enthesitis, most typically of the Achilles tendon and plantar fascia, and new bone formation. This study was undertaken to unravel the mechanisms leading towards enthesitis and new bone formation in preclinical models of SpA. RESULTS: First, we demonstrated that TNF(ΔARE) mice show typical inflammatory features highly reminiscent of SpA. The first signs of inflammation were found at the entheses. Importantly, enthesitis occurred equally in the presence or absence of mature T and B cells, underscoring the importance of stromal cells. Hind limb unloading in TNF(ΔARE) mice significantly suppressed inflammation of the Achilles tendon compared with weight bearing controls. Erk1/2 signalling plays a crucial role in mechanotransduction-associated inflammation. Furthermore, new bone formation is strongly promoted at entheseal sites by biomechanical stress and correlates with the degree of inflammation. CONCLUSIONS: These findings provide a formal proof of the concept that mechanical strain drives both entheseal inflammation and new bone formation in SpA.

Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis.

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.

Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology.

TNF plays a crucial role in the pathogenesis of Crohn disease. Dysregulated TNF production in mice that bear the genetic deletion of the TNF AU-rich regulatory elements (ARE) (Tnf(ΔARE/+) mice) results in TNF receptor I (TNFRI)-dependent spontaneous Crohn-like pathology. Current concepts consider intestinal epithelial cell (IEC) responses to TNF to be critical for intestinal pathology, but the potential contribution of IEC-derived TNF in disease pathogenesis has not been addressed. In this study we examined whether IEC are sufficient as cellular targets or sources of TNF in the development of intestinal pathology. Using IEC-specific reactivation of a hypomorphic Tnf(ΔAREneo) allele in mice, we show that selective chronic overproduction of TNF by IEC suffices to cause full development of Crohn-like pathology. Epithelial TNF overexpression leads to early activation of the underlying intestinal myofibroblast, a cell type previously identified as a sufficient target of TNF for disease development in the Tnf(ΔARE) model. By contrast, restricted TNFRI expression on IEC although sufficient to confer IEC apoptosis after acute exogenous TNF administration, fails to induce pathology following chronic specific targeting of IEC by endogenous TNF in Tnf(ΔARE/+) mice. Our results argue against IEC being early and sufficient responders to chronic TNF-mediated pathogenic signals and suggest that proinflammatory aberrations leading to chronic TNF production by IEC may initiate pathology in Crohn disease.

Cellular mechanisms of TNF function in models of inflammation and autoimmunity.

The TNF/TNF receptor (TNFR) system has a prominent role in the pathogenesis of chronic inflammatory and autoimmune disorders. Extensive research in animal models with deregulated TNF expression has documented that TNF may initiate or sustain inflammatory pathology, while at the same time may exert immunomodulatory or disease-suppressive activities. The TNF/TNFR system encompassing both the soluble and the transmembrane form of TNF with differential biological activities, as well as the differential usage of its receptors, mediating distinct functions, appears to confer complexity but also specificity in the action of TNF. The inherent complexity in TNF-mediated pathophysiology highlights the requirement to address the role of TNF taking into account both proinflammatory tissue-damaging and immunomodulatory functions in a cellular and receptor-specific manner. In this review, we discuss our current understanding of the involvement of TNF in chronic inflammation and autoimmunity, focusing on TNF-mediated cellular pathways leading to the pathogenesis or progression of joint and intestinal inflammatory pathology. Knowledge of the mechanisms by which TNF either initiates or contributes to disease pathology is fundamentally required for the design of safe and effective anti-TNF/TNFR therapies for human inflammatory and autoimmune disorders.

Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases.

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.

Generation and characterization of p38beta (MAPK11) gene-targeted mice.

p38 mitogen-activated protein kinases (MAPKs) are activated primarily in response to inflammatory cytokines and cellular stress, and inhibitors which target the p38alpha and p38beta MAPKs have shown potential for the treatment of inflammatory disease. Here we report the generation and initial characterization of a knockout of the p38beta (MAPK11) gene. p38beta-/- mice were viable and exhibited no apparent health problems. The expression and activation of p38alpha, ERK1/2, and JNK in response to cellular stress was normal in embryonic fibroblasts from p38beta-/- mice, as was the activation of p38-activated kinases MAPKAP-K2 and MSK1. The transcription of p38-dependent immediate-early genes was also not affected by the knockout of p38beta, suggesting that p38alpha is the predominant isoform involved in these processes. The p38beta-/- mice also showed normal T-cell development. Lipopolysaccharide-induced cytokine production was also normal in the p38beta-/- mice. As p38 is activated by tumor necrosis factor, the p38beta-/- mice were crossed onto a TNFDeltaARE mouse line. These mice overexpress tumor necrosis factor, which results in development symptoms similar to rheumatoid arthritis and inflammatory bowel disease. The progression of these diseases was not however moderated by knockout of p38beta. Together these results suggest that p38alpha, and not p38beta, is the major p38 isoform involved in the immune response and that it would not be necessary to retain activity against p38beta during the development of p38 inhibitors.

Cytoskeletal rearrangements in synovial fibroblasts as a novel pathophysiological determinant of modeled rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology-assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.

Functional analysis of an arthritogenic synovial fibroblast.

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellular matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.

Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn's-like inflammatory bowel 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.