Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

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.

Abstract The TNF superfamily member TWEAK acts through its receptor, Fn14, to mediate several key pathological processes known to be involved in lupus nephritis. To explore the potential for renal protection by targeting this pathway, we introduced the Fn14 null allele into the MRL/lpr lupus mouse strain. At 26 to 38 weeks of age, female MRL/lpr Fn14 knockout (KO) mice (backcross #8) had significantly lower levels of proteinuria, BUN, and creatinine as compared to Fn14 wild type (WT) mice. Furthermore, Fn14KO mice had significantly improved renal pathology, accompanied by attenuated glomerular and tubulointerstitial inflammation and less glomerular immune deposits. There were no detectable differences between the strains in either serum autoantibody levels or in splenic immune cell subsets, although less lymphadenopathy was observed in Fn14KO mice. Importantly, Fn14KO mice displayed substantial preservation of podocytes in kidney glomeruli. Moreover, we demonstrated that TWEAK signaling via Fn14 directly damaged the integrity of the glomerular filtration barrier through effects on both podocytes and glomerular endothelial cells. Inhibition of TWEAK signaling due to deficiency of the Fn14 receptor significantly improves renal disease in a spontaneous lupus nephritis model, likely through prevention of the direct injurious effects of TWEAK on the filtration barrier. Thus, blocking the TWEAK/Fn14 axis is a novel therapeutic target in immune-mediated proliferative glomerulonephritis.

TNF ligand superfamily member 12, also known as TNF-related weak inducer of apoptosis (TWEAK), acts through its receptor, fibroblast growth factor-inducible 14 (Fn14), to mediate several key pathologic processes involved in tissue injury relating to lupus nephritis. To explore the potential for renal protection in lupus nephritis by targeting this pathway, we introduced the Fn14 null allele into the MRL-lpr/lpr lupus mouse strain. At 26-38 weeks of age, female Fn14-knockout MRL-lpr/lpr mice had significantly lower levels of proteinuria compared with female wild-type MRL-lpr/lpr mice. Furthermore, Fn14-knockout mice had significantly improved renal histopathology accompanied by attenuated glomerular and tubulointerstitial inflammation. There was a significant reduction in glomerular Ig deposition in Fn14-knockout mice, despite no detectable differences in either serum levels of antibodies or splenic immune cell subsets. Notably, we found that the Fn14-knockout mice displayed substantial preservation of podocytes in glomeruli and that TWEAK signaling directly damaged barrier function and increased filtration through podocyte and glomerular endothelial cell monolayers. Our results show that deficiency of the Fn14 receptor significantly improves renal disease in a spontaneous lupus nephritis model through prevention of the direct injurious effects of TWEAK on the filtration barrier and/or modulation of cytokine production by resident kidney cells. Thus, blocking the TWEAK/Fn14 axis may be a novel therapeutic intervention in immune-mediated proliferative GN.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF ligand superfamily that is constitutively expressed by many immune cell types including monocytes/macrophages, DC, neutrophils, natural killer cells, and also T cells. The only known signaling receptor for TWEAK, FGF-inducible molecule 14 (Fn14), is a highly inducible molecule in epithelial cells (EC), endothelial cells, and other mesenchymal cell types wherein TWEAK signals through Fn14 to promote the canonical and noncanonical NF-κB, MAPK and potentially other pathways. We previously reported that TWEAK promoted colitis in mice by enhancing production of inflammatory mediators by EC. TWEAK also induced EC damage in the presence of IL-13, which is known as a pathogenic profibrotic T helper 2-type cytokine. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2-type immunity were significantly reduced in Fn14 knockout (KO) mice when compared with wild type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon EC than in WT EC. TWEAK/Fn14 was required for the induction by IL-13 of TSLP in colon explants. TWEAK and IL-13 synergistically promote embryonic fibroblast proliferation. Thus we propose an IL-13-TWEAK/ Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.

Previously it was shown that the TNF superfamily member TWEAK (TNFSF12) acts through its receptor, Fn14, to promote proinflammatory responses in kidney cells, including the production of MCP-1, RANTES, IP-10 and KC. In addition, the TWEAK/Fn14 pathway promotes mesangial cell proliferation, vascular cell activation, and renal cell death. To study the relevance of the TWEAK/Fn14 pathway in the pathogenesis of antibody-induced nephritis using the mouse model of nephrotoxic serum nephritis (NTN), we induced NTN by passive transfer of rabbit anti-glomerular antibodies into Fn14 knockout (KO) and wild type (WT) mice. Severe proteinuria as well as renal histopathology were induced in WT but not in Fn14 KO mice. Similarly, a pharmacologic approach of anti-TWEAK mAb administration into WT mice in the NTN model significantly ameliorated proteinuria and improved kidney histology. Anti-TWEAK treatment did not affect the generation of mouse anti-rabbit antibodies; however, within the kidney there was a significant decrease in glomerular immunoglobulin deposition, as well as macrophage infiltrates and tubulointerstitial fibrosis. The mechanism of action is most likely due to reductions in downstream targets of TWEAK/Fn14 signaling, including reduced renal expression of MCP-1, VCAM-1, IP-10, RANTES as well as Fn14 itself, and other molecular pathways associated with fibrosis in anti-TWEAK treated mice. Thus, TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the NTN model, apparently mediating a cascade of pathologic events locally in the kidney rather than by impacting the systemic immune response. Disrupting TWEAK/Fn14 interactions may be an innovative kidney-protective approach for the treatment of lupus nephritis and other antibody-induced renal diseases.

BACKGROUND & AIMS:TWEAK, a member of the tumor necrosis factor (TNF) superfamily, promotes intestinal epithelial cell injury and signals through the receptor Fn14 following irradiation-induced tissue damage and during development of colitis in mice. Interleukin (IL)-13, an effector of tissue damage in similar models, has been associated with the pathogenesis of ulcerative colitis (UC). We investigated interactions between TWEAK and IL-13 following mucosal damage in mice.METHODS:We compared patterns of gene expression in intestinal tissues from wild-type and TWEAK knockout mice following γ-irradiation. Intestinal explants from these mice were used to detect cell damage induced by IL-13 and TNF-α. Levels of messenger RNA for IL-13, TWEAK, and Fn14 were measured in mucosal samples from patients with UC.RESULTS:Based on gene expression analysis, TWEAK mediates γ-irradiation-induced epithelial cell cycle arrest and apoptosis. However, TWEAK alone did not induce damage or apoptosis of primary intestinal epithelial cells. On the other hand, exogenous IL-13 activated caspase-3 in naïve intestinal explants; this process required TWEAK, Fn14, and secretion of endogenous TNF-α which was mediated by ADAM17. Conversely, activation of caspase by exogenous TNF-α required IL-13, TWEAK, and Fn14. In mucosa from patients with UC, messenger RNA levels of IL-13, TWEAK, and Fn14 increased with level of disease severity.CONCLUSIONS:IL-13-induced damage of intestinal epithelial cells requires TWEAK, its receptor (Fn14), and TNF-α. IL-13, TNF-α, TWEAK, and Fn14 could perpetuate and aggravate intestinal inflammation in patients with UC.

TWEAK (TNF-like weak inducer of apoptosis) is a TNF family cytokine that mediates pleiotropic effects, including proinflammatory and pro-angiogenetic roles, through its receptor, FGF-inducible molecule 14 (Fn14).Fn14 expression is highly upregulated in contexts of tissue injury and regeneration, and chronic inflammatory disease.We previously investigated colitis in mouse models and found that TWEAK deficiency, Fn14 deficiency and treatment with TWEAK blocking monoclonal antibody significantly ameliorated TNBS colitis, with less deformity and branching in the regenerating crypt.This result prompted us to investigate role of TWEAK/Fn14 in epithelial cell regeneration.Whole body γ-irradiation induced upregulation of Fn14 mRNA in the both small and large intestine.Messenger RNA level of TWEAK was also elevated 24 hours after γ-irradiation, at the time point when many of intestinal epithelial cells underwent apoptosis.TWEAK or Fn14 deficient mice in both BALB/c and C57BL/6 background obviously had less numbers of apoptotic cells in the small and large intestine after γ-irradiation when compared with wild type mice.Treatment of WT mice with anti-TWEAK antibody also increased the number of regenerating crypts.Thus, TWEAK/Fn14 pathway enhances epithelial cell death, and blocking TWEAK may prevent epithelial cell damage and mucositis in irradiation therapy.

Objective— TWEAK is a multifunctional cytokine belonging to the tumor necrosis factor superfamily and binds to the receptor Fn14. TWEAK and Fn14 are expressed in atherosclerotic plaques in areas rich in macrophages and foam cells. We investigated the role of TWEAK/Fn14 interactions in ApoE −/− mice and bone marrow–derived macrophages in vitro. Methods and Results— ApoE −/− mice were treated with TWEAK-inhibiting fusion protein, Fn14-Fc, in an early (5 to 17 weeks of age) or delayed (17 to 29 weeks of age) setting. In the aortic arch, Fn14-Fc as compared to control treatment resulted in advanced plaques which were smaller (early treatment), fewer (delayed treatment), lower in fibrotic content (early and delayed treatment), and exhibited an increased macrophage content and smaller macrophage size (delayed treatment). There were no differences in apoptosis in atherosclerotic plaques after Fn14-Fc versus control Ab treatment. However, blocking TWEAK resulted in less macrophage uptake of modified lipids in vitro. Conclusions— Fn14-Fc fusion protein treatment did not prevent lesion initiation but inhibited some features of plaque progression and induced a unique advanced plaque phenotype with increased macrophage content and smaller macrophage size, which may be attributable to reduced lipid uptake. These findings indicate that TWEAK/Fn14 interactions regulate atherosclerosis and mediate lipid uptake in macrophages.

internalization via the GRK5/c-Src pathway.Methods.Activation of c-Src and tyrosine phosphorylation of soluble guanylyl cyclase (sGC) in response to CB 1 agonist anandamide were determined in the presence or absence of CB 1 antagonist AM251, or the c-Src inhibitor PP2, and in cells expressing dominant negative GRK5 (K215R) that blocks CB 1 receptor internalization.sGC activity, cGMP formation and muscle relaxation in response to the NOdonor S-nitrosoglutathione (GSNO) were measured in the presence or absence of anandamide.Results.Anandamide stimulated c-Src activity and induced phosphorylation of sGC, which were blocked by AM251 and PP2, and in cells expressing GRK5(K215R), suggesting activation of c-Src upon CB 1 receptor internalization results in phosphorylation of sGC.GSNO stimulated sGC activity, cGMP formation and caused muscle relaxation.Pretreatment of cells with anandamide inhibited GSNO-stimulated sGC activity, cGMP formation and muscle relaxation; the inhibitory effects of anandamide were blocked by AM251 and PP2.Anandamide also attenuated sGC activity and cGMP formation in response to NO-independent activator of sGC, YC-1.The pattern implied that phosphorylation of sGC by c-Src kinase inhibits sGC activity and the inhibition was not due to decrease in the binding of NO, but probably due to decrease in catalytic activity.Anandamide also inhibited forskolin-stimulated cAMP formation and muscle relaxation.Treatment of cells with pertussis toxin (PTx) blocked the effect of anandamide on forskolin-induced cAMP formation and muscle relaxation, but had no effect on GSNO-induced cGMP formation and muscle relaxation.The results imply that inhibition of adenylyl cyclase is mediated via activation of PTx-sensitive Gα i2 , whereas inhibition of sGC is mediated via G protein-independent activation of c-Src involving CB 1 receptor internalization.Conclusion.cGMP levels are regulated by CB 1 receptors following their internalization via GRK5: the mechanism involves c-Src-dependent phosphorylation of sGC leading to inhibition of sGC activity and cGMP formation.The study identifies a novel mechanism for attenuation of NO/sGC/cGMP signal by G i -coupled receptor agonists that is dependent on activation of c-Src.

BACKGROUND AND AIMS:Tumor necrosis factor (TNF) superfamily members have attracted attention as new therapeutic targets for treating inflammatory disease. TNF-like weak inducer of apoptosis (TWEAK) is a unique, multifunctional TNF family cytokine that signals through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). The role of this pathway in the intestine has not been previously reported.METHODS:The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model was conducted in TWEAK- or Fn14-deficient mice or in normal mice treated with a TWEAK-blocking monoclonal antibody, and clinical severity, histopathology, immunohistochemistry for cell infiltrates, TWEAK and Fn14, gene expression profiling in the colon, and systemic adaptive immunity were assessed. The effect of TWEAK on colon epithelial cell production of inflammatory mediators was analyzed in vitro. The gamma-irradiation injury model was conducted in TWEAK- or Fn14-deficient mice, and crypt epithelial death was assessed.RESULTS:Colitis severity and histologic scores were significantly reduced by TWEAK pathway deficiency or TWEAK-blocking monoclonal antibody. Neutrophil and macrophage infiltrates, chemokines, cytokines, and matrix metalloproteinase expression were reduced in the TWEAK-deficient colon after TNBS administration; however, systemic adaptive immune responses to trinitrophenyl were not altered. Fn14 is expressed on colon epithelial cells in TNBS colitis, and TWEAK induces epithelial production of pathogenic mediators. TWEAK also regulates intestinal epithelial turnover, as evidenced by reduced epithelial cell death after gamma-irradiation injury in TWEAK and Fn14 knockout mice.CONCLUSIONS:Our studies elucidate a nonredundant TWEAK-intestinal epithelial cell axis and suggest that blocking TWEAK may dampen chronic intestinal inflammation and allow normal epithelial repair.