TNF superfamily control of tissue remodeling and fibrosis.

Fibrosis is the result of extracellular matrix protein deposition and remains a leading cause of death in USA. Despite major advances in recent years, there remains an unmet need to develop therapeutic options that can effectively degrade or reverse fibrosis. The tumor necrosis super family (TNFSF) members, previously studied for their roles in inflammation and cell death, now represent attractive therapeutic targets for fibrotic diseases. In this review, we will summarize select TNFSF and their involvement in fibrosis of the lungs, the heart, the skin, the gastrointestinal tract, the kidney, and the liver. We will emphasize their direct activity on epithelial cells, fibroblasts, and smooth muscle cells. We will further report on major clinical trials targeting these ligands. Whether in isolation or in combination with other anti-TNFSF member or treatment, targeting this superfamily remains key to improve efficacy and selectivity of currently available therapies for fibrosis.

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.

Bone Marrow Transplantation Rescues Monocyte Recruitment Defect and Improves Cystic Fibrosis in Mice.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders.

Mucus secretion is an important feature of asthma that highly correlates with morbidity. Current therapies, including administration of mucolytics and anti-inflammatory drugs, show limited effectiveness and durability, underscoring the need for novel effective and longer lasting therapeutic approaches. Here we show that mucus production in the lungs is regulated by the TNF superfamily member 15 (TL1A) acting through the mucus-inducing cytokine IL-13. TL1A induces IL13 expression by innate lymphoid cells leading to mucus production, in addition to promoting airway inflammation and fibrosis. Reciprocally, neutralization of IL13 signaling through its receptor (IL4Rα), completely reverses TL1A-induced mucus secretion, while maintaining airway inflammation and fibrosis. Importance of TL1A is further demonstrated using a preclinical asthma model induced by chronic house dust mite exposure where TL1A neutralization by genetic deletion or antagonistic blockade of its receptor DR3 protected against mucus production and fibrosis. Thus, TL1A presents a promising therapeutic target that out benefits IL13 in reversing mucus production, airway inflammation and fibrosis, cardinal features of severe asthma in humans.

TL1A Promotes Lung Tissue Fibrosis and Airway Remodeling.

Lung fibrosis and tissue remodeling are features of chronic diseases such as severe asthma, idiopathic pulmonary fibrosis, and systemic sclerosis. However, fibrosis-targeted therapies are currently limited. We demonstrate in mouse models of allergen- and bleomycin-driven airway inflammation that neutralization of the TNF family cytokine TL1A through Ab blocking or genetic deletion of its receptor DR3 restricted increases in peribronchial smooth muscle mass and accumulation of lung collagen, primary features of remodeling. TL1A was found as a soluble molecule in the airways and expressed on the surface of alveolar macrophages, dendritic cells, innate lymphoid type 2 cells, and subpopulations of lung structural cells. DR3 was found on CD4 T cells, innate lymphoid type 2 cells, macrophages, fibroblasts, and some epithelial cells. Suggesting in part a direct activity on lung structural cells, administration of recombinant TL1A into the naive mouse airways drove remodeling in the absence of other inflammatory stimuli, innate lymphoid cells, and adaptive immunity. Correspondingly, human lung fibroblasts and bronchial epithelial cells were found to express DR3 and responded to TL1A by proliferating and/or producing fibrotic molecules such as collagen and periostin. Reagents that disrupt the interaction of TL1A with DR3 then have the potential to prevent deregulated tissue cell activity in lung diseases that involve fibrosis and remodeling.

Increased Production of LIGHT by T Cells in Eosinophilic Esophagitis Promotes Differentiation of Esophageal Fibroblasts Toward an Inflammatory Phenotype.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFß1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE. METHODS: We analyzed publicly available esophageal CD3+ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFß1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry. RESULTS: LIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFß1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFß1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1. CONCLUSIONS: T cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFß1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV.

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.

LIGHT-HVEM signaling in keratinocytes controls development of dermatitis.

Dermatitis is often associated with an allergic reaction characterized by excessive type 2 responses leading to epidermal acanthosis, hyperkeratosis, and dermal inflammation. Although factors like IL-4, IL-13, and thymic stromal lymphopoietin (TSLP) are thought to be instrumental for the development of this type of skin disorder, other cytokines may be critical. Here, we show that the tumor necrosis factor (TNF) superfamily protein LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes) is required for experimental atopic dermatitis, and LIGHT directly controls keratinocyte hyperplasia, and production of periostin, a matricellular protein that contributes to the clinical features of atopic dermatitis as well as other skin diseases such as scleroderma. Mice with a conditional deletion of the LIGHT receptor HVEM (herpesvirus entry mediator) in keratinocytes phenocopied LIGHT-deficient mice in exhibiting reduced epidermal thickening and dermal collagen deposition in a model of atopic dermatitis driven by house dust mite allergen. LIGHT signaling through HVEM in human epidermal keratinocytes directly induced proliferation and periostin expression, and both keratinocyte-specific deletion of HVEM or antibody blocking of LIGHT-HVEM interactions after disease onset prevented expression of periostin and limited atopic dermatitis symptoms. Developing reagents that neutralize LIGHT-HVEM signaling might be useful for therapeutic intervention in skin diseases where periostin is a central feature.

The TWEAK/Fn14 pathway is required for calcineurin inhibitor toxicity of the kidneys.

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis.

TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis.

Atopic dermatitis (AD) and psoriasis are driven by alternate type 2 and type 17 immune responses, but some proteins might be critical to both diseases. Here we show that a deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in defective maintenance of AD-specific T helper type 2 (Th2) and psoriasis-specific Th17 cells in the skin, and impaired expression of disease-characteristic chemokines and cytokines, such as CCL17 and TSLP in AD, and CCL20 and IL-19 in psoriasis. The TWEAK receptor, Fn14, is upregulated in keratinocytes and dermal fibroblasts, and TWEAK induces these cytokines and chemokines alone and in synergy with the signature T helper cytokines of either disease, IL-13 and IL-17. Furthermore, subcutaneous injection of recombinant TWEAK into naive mice induces cutaneous inflammation with histological and molecular signs of both diseases. TWEAK is therefore a critical contributor to skin inflammation and a possible therapeutic target in AD and psoriasis.

The control of tissue fibrosis by the inflammatory molecule LIGHT (TNF Superfamily member 14).

The TNF Superfamily member LIGHT (TNFSF14) has recently emerged as a potential target for therapeutic interventions aiming to halt tissue fibrosis. In this perspective, we discuss how LIGHT may influence the inflammatory and remodeling steps that characterize fibrosis, relevant for many human diseases presenting with scarring such as asthma, idiopathic pulmonary fibrosis, systemic sclerosis, and atopic dermatitis. LIGHT acts through two receptors in the TNF receptor superfamily, HVEM (TNFRSF14) and LTßR (TNFRSF3), which are broadly expressed on hematopoietic and non-hematopoietic cells. LIGHT can regulate infiltrating T cells, macrophages, and eosinophils, controlling their trafficking or retention in the inflamed tissue, their proliferation, and their ability to produce cytokines that amplify fibrotic processes. More interestingly, LIGHT can act on structural cells, namely epithelial cells, fibroblasts, smooth muscle cells, adipocytes, and endothelial cells. By signaling through either HVEM or LTßR expressed on these cells, LIGHT can contribute to their proliferation and expression of chemokines, growth factors, and metalloproteinases. This will lead to hyperplasia of epithelial cells, fibroblasts, and smooth muscle cells, deposition of extracellular matrix proteins, vascular damage, and further immune alterations that in concert constitute fibrosis. Because of its early expression by T cells, LIGHT may be an initiator of fibrotic diseases, but other sources in the immune system could also signify a role for LIGHT in maintaining or perpetuating fibrotic activity. LIGHT may then be an attractive prognostic marker as well as an appealing target for fibrosis therapies relevant to humans.

The Tumor Necrosis Factor Superfamily Molecule LIGHT Promotes Keratinocyte Activity and Skin Fibrosis.

Several inflammatory diseases including scleroderma and atopic dermatitis display dermal thickening, epidermal hypertrophy, or excessive accumulation of collagen. Factors that might promote these features are of interest for clinical therapy. We previously reported that LIGHT, a TNF superfamily molecule, mediated collagen deposition in the lungs in response to allergen. We therefore tested whether LIGHT might similarly promote collagen accumulation and features of skin fibrosis. Strikingly, injection of recombinant soluble LIGHT into naive mice, either subcutaneously or systemically, promoted collagen deposition in the skin and dermal and epidermal thickening. This replicated the activity of bleomycin, an antibiotic that has been previously used in models of scleroderma in mice. Moreover skin fibrosis induced by bleomycin was dependent on endogenous LIGHT activity. The action of LIGHT in vivo was mediated via both of its receptors, HVEM and LTßR, and was dependent on the innate cytokine TSLP and TGF-ß. Furthermore, we found that HVEM and LTßR were expressed on human epidermal keratinocytes and that LIGHT could directly promote TSLP expression in these cells. We reveal an unappreciated activity of LIGHT on keratinocytes and suggest that LIGHT may be an important mediator of skin inflammation and fibrosis in diseases such as scleroderma or atopic dermatitis.

Tumor necrosis factor superfamily 14 (LIGHT) controls thymic stromal lymphopoietin to drive pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis is characterized by excessive accumulation of collagen and α-smooth muscle actin in the lung. The key molecules that promote these phenotypes are of clinical interest. OBJECTIVES: Thymic stromal lymphopoietin (TSLP) has been found at high levels in patients with asthma and idiopathic pulmonary fibrosis, and TSLP has been proposed as a primary driver of lung fibrotic disease. We asked whether tumor necrosis factor superfamily protein 14 (TNFSF14) (aka LIGHT) controls TSLP production to initiate fibrosis. METHODS: Expression of TSLP and initiation of pulmonary fibrosis induced by bleomycin were assessed in mice deficient in LIGHT. The ability of recombinant LIGHT, given intratracheally to naive mice, to promote TSLP and fibrosis was also determined. RESULTS: Genetic deletion of LIGHT abolished lung TSLP expression driven by bleomycin, accompanied by near-complete absence of accumulation of lung collagen and α-smooth muscle actin. Furthermore, recombinant LIGHT administered in vivo induced lung expression of TSLP in the absence of other inflammatory stimuli, and strikingly reproduced the primary features of bleomycin-driven disease in a TSLP-dependent manner. Blockade of LIGHT binding to either of its receptors, herpes virus entry mediator and lymphotoxin beta receptor, inhibited clinical symptoms of pulmonary fibrosis, and correspondingly both receptors were found on human bronchial epithelial cells, a primary source of TSLP. Moreover, LIGHT induced TSLP directly in human bronchial epithelial cells and synergized with IL-13 and TGF-ß in vivo to promote TSLP in the lungs and drive fibrosis. CONCLUSIONS: These results show that LIGHT is a profibrogenic cytokine that may be a key driver of TSLP production during the initiation and development of lung fibrotic disease.