The mesenchymal context in inflammation, immunity and cancer.

Mesenchymal cells are mesoderm-derived stromal cells that are best known for providing structural support to organs, synthesizing and remodeling the extracellular matrix (ECM) and regulating development, homeostasis and repair of tissues. Recent detailed mechanistic insights into the biology of fibroblastic mesenchymal cells have revealed they are also significantly involved in immune regulation, stem cell maintenance and blood vessel function. It is now becoming evident that these functions, when defective, drive the development of complex diseases, such as various immunopathologies, chronic inflammatory disease, tissue fibrosis and cancer. Here, we provide a concise overview of the contextual contribution of fibroblastic mesenchymal cells in physiology and disease and bring into focus emerging evidence for both their heterogeneity at the single-cell level and their tissue-specific, spatiotemporal functional diversity.

Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities.

Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.

A20 controls RANK-dependent osteoclast formation and bone physiology.

The anti-inflammatory protein A20 serves as a critical brake on NF-κB signaling and NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-κB activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-κB signaling to control osteoclast differentiation, assuring proper bone development and turnover.

'SMASH' recommendations for standardised microscopic arthritis scoring of histological sections from inflammatory arthritis animal models.

Animal models for inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis are widely accepted and frequently used to identify pathological mechanisms and validate novel therapeutic strategies. Unfortunately, many publications reporting on these animal studies lack detailed description and appropriate assessment of the distinct histopathological features of arthritis: joint inflammation, cartilage damage and bone erosion. Therefore, the European consortium BeTheCure, consisting of 38 academic and industrial partners from 15 countries, set as goal to standardise the histological evaluation of joint sections from animal models of inflammatory arthritis. The consensual approach of a task force including 16 academic and industrial scientists as well as laboratory technicians has resulted in the development of the Standardised Microscopic Arthritis Scoring of Histological sections ('SMASH') recommendations for a standardised processing and microscopic scoring of the characteristic histopathological features of arthritis, exemplified by four different rodent models for arthritis: murine collagen-induced arthritis, collagen-antibody-induced arthritis, human tumour necrosis factor transgenic Tg197 mice and rat pristane-induced arthritis, applicable to any other inflammatory arthritis model. Through standardisation, the SMASH recommendations are designed to improve and maximise the information derived from in vivo arthritis experiments and to promote reproducibility and transparent reporting on such studies. In this manuscript, we will discuss and provide recommendations for analysis of histological joint sections: identification of the regions of interest, sample preparation, staining procedures and quantitative scoring methods. In conclusion, awareness of the different features of the arthritis pathology in animal models of inflammatory arthritis is of utmost importance for reliable research outcome, and the standardised histological processing and scoring methods in these SMASH recommendations will help increase uniformity and reproducibility in preclinical research on inflammatory arthritis.

Transmembrane TNF-α Reverse Signaling Inhibits Lipopolysaccharide-Induced Proinflammatory Cytokine Formation in Macrophages by Inducing TGF-ß: Therapeutic Implications.

TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α-bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti-TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-ß. Based on inhibitory experiments, the production of TGF-ß1 is regulated via Jun kinases, whereas that of other TGF-ßs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-ß by Abs prevented the mTNF-α-mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-ß. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-ß, we show that they do not use the mTNF-α signaling pathway. Because TGF-ß possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-ß synthesis by those TNF-α-targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn's disease, Wegener's granulomatosis, or sarcoidosis. Additionally, none of the TNF-α-targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.

Opposing role of tumor necrosis factor receptor 1 signaling in T cell-mediated hepatitis and bacterial infection in mice.

UNLABELLED: Death receptor (DR) ligands such as tumor necrosis factor (TNF) have been identified as fundamental mediators of liver damage both in mouse models and in humans. While the essential site of function of DR signaling is conceivably the hepatocyte, a systematic analysis is missing. Using mice with conditional gene ablation, we analyzed the tissue-specific function of DR signaling in T cell-dependent (concanavalin A) and independent (lipopolysaccharide/galactosamine) hepatitis and in models of bacterial infection (Listeria monocytogenes, lipopolysaccharide). We report that lipopolysaccharide/galactosamine-induced liver injury depends on hepatocyte-intrinsic TNF receptor 1 (p55, TNFR1). In contrast, we show that T cell-induced hepatitis was independent of TNFR1 signaling in hepatocytes, T cells, or endothelial cells. Moreover, T cell-induced hepatitis was independent of hepatocyte-intrinsic Fas-associated protein with death domain, TNF-related apoptosis-inducing ligand receptor, or Fas signaling. Instead, concanavalin A-induced hepatitis was completely prevented in mice with myeloid-derived cell (MDC)-specific deletion of TNFR1. Significantly, however, mice lacking TNFR1 in MDCs succumbed to listeria infection, although they displayed similar sensitivity toward endotoxin-induced septic shock when compared to control mice. These results suggest that TNFR1 signaling in MDCs is a critical mediator of both the detrimental and the protective functions of TNF in T cell-induced hepatitis and bacterial infection, respectively. CONCLUSION: The critical site of action of DRs is completely dependent on the nature of hepatitis; the data specify MDCs as the essential cell type of TNFR1 function in T cell-mediated hepatitis and in the response to listeria, thereby identifying the opposing role of MDC TNFR1 in autoimmunity and bacterial infection. (Hepatology 2016;64:508-521).

RANKL expressed on synovial fibroblasts is primarily responsible for bone erosions during joint inflammation.

OBJECTIVE: RANKL is mainly expressed by synovial fibroblasts and T cells within the joints of rheumatoid arthritis patients. The relative importance of RANKL expression by these cell types for the formation of bone erosions is unclear. We therefore aimed to quantify the contribution of RANKL by each cell type to osteoclast differentiation and bone destruction during inflammatory arthritis. METHODS: RANKL was specifically deleted in T cells (Tnfsf11(flox/Δ) Lck-Cre), in collagen VI expressing cells including synovial fibroblasts (Tnfsf11(flox/Δ) Col6a1-Cre) and in collagen II expressing cells including articular chondrocytes (Tnfsf11(flox/Δ) Col2a1-Cre). Erosive disease was induced using the collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA) models. Osteoclasts and cartilage degradation were assessed by histology and bone erosions were assessed by micro-CT. RESULTS: The inflammatory joint score during CAIA was equivalent in all mice regardless of cell-targeted deletion of RANKL. Significant increases in osteoclast numbers and bone erosions were observed in both the Tnfsf11(flox/Δ) and the Tnfsf11(flox/Δ) Lck-Cre groups during CAIA; however, the Tnfsf11(flox/Δ) Col6a1-Cre mice showed significant protection against osteoclast formation and bone erosions. Similar results on osteoclast formation and bone erosions were obtained in CIA mice. The deletion of RANKL on any cell type did not prevent articular cartilage loss in either model of arthritis used. CONCLUSIONS: The expression of RANKL on synovial fibroblasts rather than T cells is predominantly responsible for the formation of osteoclasts and erosions during inflammatory arthritis. Synovial fibroblasts would be the best direct target in RANKL inhibition therapies.

Cyld restrains the hyperactivation of synovial fibroblasts in inflammatory arthritis by regulating the TAK1/IKK2 signaling axis.

TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Herein, we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NF-κB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis.

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

Single-cell multimodal analysis identifies common regulatory programs in synovial fibroblasts of rheumatoid arthritis patients and modeled TNF-driven arthritis.

BACKGROUND: Synovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined. METHODS: We applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas. RESULTS: By comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1- SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks. CONCLUSIONS: We revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.

Ectopic bone formation and systemic bone loss in a transmembrane TNF-driven model of human spondyloarthritis.

BACKGROUND: The transmembrane-TNF transgenic mouse, TgA86, has been shown to develop spontaneously peripheral arthritis with signs of axial involvement. To assess similarity to human spondyloarthritis, we performed detailed characterization of the axial, peripheral, and comorbid pathologies of this model. METHODS: TgA86 bone pathologies were assessed at different ages using CT imaging of the spine, tail vertebrae, and hind limbs and characterized in detail by histopathological and immunohistochemical analysis. Cardiac function was examined by echocardiography and electrocardiography and bone structural parameters by µCT analysis. The response of TgA86 mice to either early or late anti-TNF treatment was evaluated clinically, histopathologically, and by µCT analysis. RESULTS: TgA86 mice developed with 100% penetrance spontaneous axial and peripheral pathology which progressed with time and manifested as reduced body weight and body length, kyphosis, tail bendings, as well as swollen and distorted hind joints. Whole-body CT analysis at advanced ages revealed bone erosions of sacral and caudal vertebrae as well as of sacroiliac joints and hind limbs and, also, new ectopic bone formation and eventually vertebral fusion. The pathology of these mice highly resembled that of SpA patients, as it evolved through an early inflammatory phase, evident as enthesitis and synovitis in the affected joints, characterized by mesenchymal cell accumulation, and neutrophilic infiltration. Subsequently, regression of inflammation was accompanied by ectopic bone formation, leading to ankylosis. In addition, both systemic bone loss and comorbid heart valve pathology were evident. Importantly, early anti-TNF treatment, similar to clinical treatment protocols, significantly reduced the inflammatory phase of both the axial and peripheral pathology of TgA86 mice. CONCLUSIONS: The TgA86 mice develop a spontaneous peripheral and axial biphasic pathology accompanied by comorbid heart valvular dysfunction and osteoporosis, overall reproducing the progression of pathognomonic features of human spondyloarthritis. Therefore, the TgA86 mouse represents a valuable model for deciphering the role of transmembrane TNF in the pathogenic mechanisms of spondyloarthritis and for assessing the efficacy of human therapeutics targeting different phases of the disease.

Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis.

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.

RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.

Hepatocyte apoptosis is intrinsically linked to chronic liver disease and hepatocarcinogenesis. Conversely, necroptosis of hepatocytes and other liver cell types and its relevance for liver disease is debated. Using liver parenchymal cell (LPC)-specific TGF-beta-activated kinase 1 (TAK1)-deficient (TAK1LPC-KO) mice, which exhibit spontaneous hepatocellular and biliary damage, hepatitis, and early hepatocarcinogenesis, we have investigated the contribution of apoptosis and necroptosis in hepatocyte and cholangiocyte death and their impact on liver disease progression. Here, we provide in vivo evidence showing that TAK1-deficient cholangiocytes undergo spontaneous necroptosis induced primarily by TNFR1 and dependent on RIPK1 kinase activity, RIPK3, and NEMO. In contrast, TAK1-deficient hepatocytes die by FADD-dependent apoptosis, which is not significantly inhibited by LPC-specific RIPK1 deficiency, inhibition of RIPK1 kinase activity, RIPK3 deficiency or combined LPC-specific deletion of TNFR1, TRAILR, and Fas. Accordingly, normal mouse cholangiocytes can undergo necroptosis, while primary hepatocytes are resistant to it and die exclusively by apoptosis upon treatment with cell death-inducing stimuli in vitro, likely due to the differential expression of RIPK3. Interestingly, the genetic modifications that conferred protection from biliary damage also prevented the spontaneous lethality that was often observed in TAK1LPC-KO mice. In the presence of chronic hepatocyte apoptosis, preventing biliary damage delayed but did not avert hepatocarcinogenesis. On the contrary, inhibition of hepatocyte apoptosis fully prevented liver tumorigenesis even in mice with extensive biliary damage. Altogether, our results suggest that using RIPK1 kinase activity inhibitors could be therapeutically useful for cholestatic liver disease patients.

A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain.

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1ß release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.

The p55TNFR-IKK2-Ripk3 axis orchestrates arthritis by regulating death and inflammatory pathways in synovial fibroblasts.

NFκB activation and regulated cell death are important in tissue homeostasis, inflammation and pathogenesis. Here we show the role of the p55TNFR-IKK2l-Ripk3 axis in the regulation of synovial fibroblast homeostasis and pathogenesis in TNF-mediated mouse models of arthritis. Mesenchymal-specific p55TNFR triggering is indispensable for arthritis in acute and chronic TNF-dependent models. IKK2 in joint mesenchymal cells is necessary for the development of cartilage destruction and bone erosion; however, in its absence synovitis still develops. IKK2 deletion affects arthritic and antiapoptotic gene expression leading to hypersensitization of synovial fibroblasts to TNF/Ripk1-mediated death via district mechanisms, depending on acute or chronic TNF signals. Moreover, Ripk3 is dispensable for TNF-mediated arthritis, yet it is required for synovitis in mice with mesenchymal-specific IKK2 deletion. These results demonstrate that p55TNFR-IKK2-Ripk3 signalling orchestrates arthritogenic and death responses in synovial fibroblasts, suggesting that therapeutic manipulation of this pathway in arthritis may require combinatorial blockade of both IKK2 and Ripk3 signals.

Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium.

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.