Transforming growth factor-beta receptors in self-limited vs. chronic progressive nephritis in rats

Increases in transforming growth factor-beta (TGF-beta) expression and extracellular matrix accumulation are transient in acute self-limited mesangial proliferative glomerulonephritis induced by a single injection of anti-thymocyte serum (ATS), while these increases persist following repeated injections that produce chronic progressive sclerosing glomerulonephritis with tubulointerstitial lesions. However, little is known about the expression of TGF-beta receptors (TbetaRs) in cells involved in the proliferative and sclerosing renal lesions. A study of protein and mRNA expression for type I (TbetaRI), type II (TbetaRII), and type III (TbetaRIII) TbetaR in both forms of nephritis was therefore carried out by immunohistochemistry and in situ hybridization. Inhibition of cell proliferation and stimulation of matrix production by TGF-beta1 were assessed in isolated glomeruli using [(3)H]thymidine incorporation and [(3)H]proline metabolic labelling, respectively. In acute self-limited nephritis, expression of TbetaRI, TbetaRII, and TbetaRIII increased in the glomerular and Bowman's capsular epithelial cells comprising the glomerular tuft adhesions to Bowman's capsules. However, TbetaRII expression was not prominent in proliferating mesangial cells. Glomeruli isolated from rats with acute self-limited nephritis at day 7, when mesangial cell proliferation was maximal, were partially resistant to the mitoinhibitory effects of TGF-beta1. In contrast, expression of all three TbetaRs was elevated in glomerular and tubulointerstitial lesions in chronic progressive nephritis, and glomeruli isolated from rats with chronic progressive nephritis 7 days after the second ATS injection were sensitive to TGF-beta1. These data suggest that distinct cellular responses to TGF-beta1 resulting from differential expression of TbetaR underlie the difference between acute self-limited mesangial proliferative and chronic progressive sclerosing ATS nephritis in the development of proliferative and sclerotic renal lesions.     

Analysis of intestinal fibrosis in chronic colitis in mice induced by dextran sulfate sodium

Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in intestinal fibrosis, however, their precise role is largely unknown. To investigate their role in intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation with dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of proinflammatory cytokines are correlated with extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-β, and matrix metalloproteinases were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin(+), α-smooth muscle actin (α-SMA)(-)) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin(+), α-SMA(+)) were increased in mucosal but not in submucosal layers. Primary mouse subcutaneous fibroblast cultures experiments revealed that exogenously added TGF-β 1 substantially augmented the expressions of both vimentin and α-SMA proteins with increased production of collagen. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in this chronic DSS-induced colitis model.     

Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-β1.     

Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvβ6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvβ6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvβ6 using oral keratinocyte-derived cells genetically modified to express high αvβ6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvβ6 (OKF6/TERT-1). VB6 cells showed significant αvβ6-dependent activation of TGF-β1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-β1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvβ6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvβ6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvβ6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvβ6 integrin.     

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes

During pulmonary development, Sonic hedgehog (Shh) and transforming growth factor beta1 (TGF-beta1) signalling both contribute to branching morphogenesis. In interstitial lung disease, the complex alveolar structure of the lung is disrupted and remodelled, which leads to fibrosis, loss of respiratory surface, morbidity, and mortality. It is well documented that TGF-beta1 is involved in fibrosis. However, little is known about Shh signalling in damaged epithelia. This study examined whether or not components of the Shh signalling pathway, as well as TGF-beta1, are expressed in human fibrotic lung disease (cryptogenic fibrosing alveolitis and bronchiectasis) and in murine experimental models of fibrotic and non-fibrotic chronic pulmonary inflammation. Using immunohistochemistry, it was observed that Shh, like TGF-beta1, is up-regulated in epithelial cells at sites of fibrotic disease but not non-fibrotic inflammation. The Shh receptor patched was detected in infiltrating mononuclear cells and alveolar macrophages, as well as normal resting peripheral blood T lymphocytes. Neither Shh nor patched is expressed by hyperproliferative goblet cells in inflammatory epithelium. This study demonstrates that patched is present in human peripheral CD4 and CD8 lymphocytes at both protein and mRNA levels. Taken together, these results suggest that components of the highly conserved Shh signalling pathway may play a role in the remodelling of damaged pulmonary epithelium and that damaged epithelium and cells of the immune system may communicate via this pathway.     

MicroRNA‐214 exerts a Cardio‐protective effect by inhibition of fibrosis

The miRNAs play important roles in regulating myocardial fibrosis. The purpose of this study was to determine the potential roles of microRNA‐214 (miR‐214) in cardiac fibrosis in vitro and in vivo. In vitro experiment, Ang II‐induced cardiac fibroblasts (CFBs) are transfected with pre‐miR‐214, anti‐miR‐214 and their oligo controls. Gene expression was checked by Quantitative realtime‐PCR (qRT‐PCR) and western blotting. In the present experiment, compared with controls, expressions of collagen type I (COL I), collagen type III (COL III), transforming growth factor (TGF)‐β1, and tissue inhibitors of metalloproteinase (TIMP)‐1 were all increased, but matrix metalloproteinase (MMP)‐1 was reduced in CFB by Ang II treatment at both mRNA and protein levels, and these alterations were found reversed by miR‐214 transfection. In vivo, an anterior transmural acute myocardial infarction (AMI) was created by occlusion of the left anterior descending coronary artery after Ad‐pre‐miR‐214, Ad‐anti‐miR‐214 or Ad‐GFP was delivered separately. Four weeks after AMI, protein contents of COL I, COL III and TGF‐β1 in tissue from border area were found increased after AMI, but impaired by overexpression of miR‐214. While the expression of MMP‐1 was increased by miR‐214 stimulation but decreased by miR‐214 inhibition. These results suggested that miR‐214 exerts cardio‐protective effects by inhibition of fibrosis and the inhibitory effect involves TGF‐β1 suppression and MMP‐1/TIMP‐1 regulation. Anat Rec, 299:1348–1357, 2016. © 2016 Wiley Periodicals, Inc.     

Reduced efficacy of treatment of strongyloidiasis in HTLV-I carriers related to enhanced expression of IFN-gamma and TGF-beta1

Strongyloidiasis, a human intestinal infection caused by Strongyloides stercoralis (S. stercoralis), is difficult to cure with drugs. In particular, a decrease of the efficacy of treatment has been reported in patients dually infected with S. stercoralis and human T-cell leukaemia virus type I (HTLV-I), both of which are endemic in Okinawa, Japan. However, the factors influencing this resistance remain unclear. In the present study, patients infected with S. stercoralis, with or without HTLV-I infection, were treated with albendazole, followed up for one year and separated into two groups, cured and non-cured. The cure rate of S. stercoralis was lower in HTLV-I carriers (P < 0.05). Serum levels of S. stercoralis-specific IgA, IgE, IgG, IgG1 and IgG4 antibodies were estimated, and a decrease of IgE (P < 0.05) and an increase of IgG4 (P < 0.05) were observed in the non-cured group, especially in HTLV-I carriers. RT-PCR of cytokines using peripheral blood mononuclear cells revealed that S. stercoralis patients with HTLV-I showed a high frequency of expression of IFN-gamma and TGF-beta1, whereas those without HTLV-I showed no expression of these cytokines. IFN-gamma- and TGF-beta1-positive HTLV-I carriers showed a decrease of IgE (P < 0.05), an increase of IgG4 (P < 0.01) and a lower cure rate (P < 0.01) compared with those who were negative for both cytokines. These results suggest that persistent infection with HTLV-I affected S. stercoralis-specific immunity and reduced therapeutic efficacy.     

Seoul virus enhances regulatory and reduces proinflammatory responses in male Norway rats

Zoonotic pathogens, including hantaviruses, are maintained in the environment by causing persistent infection in the absence of disease in their reservoir hosts. Spillover of hantaviruses to humans can cause severe disease that is mediated by excessive proinflammatory responses. The mechanisms mediating hantaviral persistence in rodent reservoirs remain largely unknown. Male Norway rats were inoculated with their species-specific hantavirus, Seoul virus (SEOV), and viral RNA, cytokine, and chemokine responses were evaluated in spleen and lung tissue. More viral RNA was detectable in the lungs than spleen, with copies of SEOV peaking 15-30 days post-inoculation (p.i.) and persisting for 60 days p.i. In the lungs, the expression and production of proinflammatory mediators (i.e., IL-1beta, IL-6, TNF-alpha, IFN-gamma, CCL5, CCL2, CX3CL1, CXCL10, VCAM, VEGF, and NOS2) remained at or below baseline throughout SEOV infection; whereas, regulatory factors, including TGF-beta and FoxP3 were elevated. Conversely, in the spleen, proinflammatory responses were induced while regulatory responses remained unchanged during infection. To determine whether reduced proinflammatory responses mediate hantavirus persistence in the lungs, male rats were administered rIL-1beta or vehicle for 30 days during SEOV infection. SEOV persistence and shedding were not affected by IL-1beta treatment. Proinflammatory responses were elevated in rIL-1beta-treated rats, but remained within physiological levels, suggesting that supra-physiological concentrations may be necessary for viral clearance at the cost of causing disease. Elevated regulatory responses may suppress excessively high proinflammatory responses at a site of elevated SEOV replication to contribute to viral persistence and prevent proinflammatory-mediated disease in reservoir hosts.     

Transforming growth factor-beta expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis

Retinochoroiditis caused by Toxoplasma gondii infection results in inflammation and necrosis of the retina. We have used human retinal pigment epithelial cultures (HRPE) as an in vitro model to investigate the role of TGF-beta in T. gondii-induced retinochoroiditis. RT-PCR analyses showed enhanced steady state levels of TGF-beta1 and TGF-beta2 mRNA in T. gondii-infected HRPE. Uninfected HRPE secrete TGF-beta1 in a latent form while 10-30% of the secreted TGF-beta2 was in the active form. T. gondii infection induced a significant increase (P < 0.01) in total TGF-beta1 and TGF-beta2 secretion by HRPE. In addition, soluble extracts of T. gondii (ST) stimulated secretion of both TGF-beta1 and TGF-beta2 significantly (P < 0.01). Interestingly, T. gondii infection as well as ST of the parasites completely inhibited secretion of the active form of TGF-beta2. Studies evaluating the effect of TGF-beta on T. gondii replication in HRPE revealed that TGF-beta enhanced parasite replication. The interactions between host retinal cells and T. gondii may play an active role in the pathogenesis of retinochoroiditis.     

Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma

Accumulative data have demonstrated that plasminogen activator inhibitor-1 (PAI-1) plays an important role in the extracellular matrix metabolism; however, the involvement of PAI-1 in scleroderma has not been fully elucidated. In this study, we investigated the role of PAI-1 in bleomycin-induced murine scleroderma. 100 microg of bleomycin was injected subcutaneously to the back skin of C3H/HeJ mice on alternate day for 4 weeks. Histopathological findings revealed that PAI-1 was positive in macrophage-like cells and fibroblastic cells in the dermis, in parallel with the induction of dermal sclerosis. PAI-1 mRNA expression in the whole skin was up-regulated at 1 and 4 weeks. The production of active PAI-1 protein in the lesional skin was significantly increased 3 and 4 weeks after bleomycin treatment. Next, we examined whether dermal sclerosis is induced by bleomycin in PAI-1-deficient (PAI-1-/-) mice. 10 microg of bleomycin was subcutaneously injected to PAI-1-/- and wild type (WT) mice 5 days per week for 4 weeks. Histological examination revealed that dermal sclerosis was similarly induced even in PAI-1-/- as well as WT mice. Dermal thickness and collagen contents in the skin were significantly increased by bleomycin injection in both PAI-1-/- and WT mice, and the rate of increase was similar. These data suggest that PAI-1 plays an important role, possibly via TGF-beta pathway activation. However, the fact that PAI-1 deficiency did not ameliorate skin sclerosis suggest that PAI-1 is not the essential factor in the development of bleomycin-induced scleroderma, and more complex biochemical effects other than PA/plasmin system are greatly suspected.     

Epicutaneous immunization induces alphabeta T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-beta

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen-non-specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by alphabeta-T-cell receptor CD4(+) CD8(+) double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex-restricted nor antigen specific. Both CD25(+) and CD25(-) CD4(+) CD8(+) T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity. In vivo treatment with monoclonal antibodies showed that the cytokines interleukin (IL)-4, IL-10 and transforming growth factor-beta (TGF-beta) are involved in the induction of the Ts cells. Additionally, using IL-10(-/-) mice we found that IL-10 is involved in skin induced tolerance. Further in vitro experiments showed that lymph node cells of skin tolerized mice non-specifically suppress [(3)H]thymidine incorporation by antigen-stimulated immune cells and this effect can be abolished by adding anti-TGF-beta, but not anti-IL-4 nor anti-IL-10 antibodies. These studies indicate the crucial role of TGF-beta in skin induced tolerance due to non-antigen-specific Ts cells and also show that IL-4, IL-10 and TGF-beta play an important role in the induction of epicutaneously induced Ts cell suppression.     

Expression of TGF-betas in the embryonic nervous system: analysis of interbalance between isoforms.

Transforming growth factor-beta (TGF-beta) is a family of growth factors with essential and multiple roles during embryonic development. In mammals, three isoforms (TGF-beta1, TGF-beta2, TGF-beta3) have been described. In the nervous system, the presence of TGF-beta1 has remained undetectable in other structures than meninges and choroids plexus, while TGF-beta2 and TGF-beta3 were considered as the neural members of the family. In the present study, we have analysed the expression pattern of the three isoforms in the neural tube, brain, and spinal cord during development in both mouse and chicken. The data reveal specific patterns for each isoform. This work also shows that both TGF-beta1 and TGF-beta3 are expressed in neural crest cells. In addition, we demonstrate the existence of interbalance between TGF-beta1 and TGF-beta3 with possible functional implications, which, together with the expression of TGF-beta1 in the CNS, represents one of the most important contributions of this work.     

Influence of human T lymphotrophic virus type I on diffuse pan-bronchiolitis

Human T lymphotrophic virus type-I (HTLV-I), a human retrovirus, infects CD4(+) lymphocytes and is thought to modify their function and a possible association with pulmonary diseases has also been suggested. However, little is known about the influence of HTLV-I on diffuse pan-bronchiolitis (DPB), a chronic inflammatory lung disease with infiltration of lymphocytes and hyperplasia of the bronchus-associated lymphoid tissue. In this study, 35 DPB patients with and without HTLV-I infection were examined. HTLV-I positive DPB patients were likely to have a larger affected area with lower FEV(1). The CD3(+)/CD25(+) lymphocyte percentage was significantly higher in the BALF of HTLV-I positive patients than in negative patients. MIP-1 alpha, IP-10 and levels in BALF were also significantly higher in HTLV-I positive patients than in negative patients. The levels of MCP-1 and IL-8 were not significantly different. In HTLV-I positive patients, the MIP-1 alpha and IP-10 levels showed a significant positive correlation with the percentage of CD3(+)/CD25 lymphocytes. BALF cells of all HTLV-I positive DPB patients showed expression of p40(tax) mRNA. We suggest that HTLV-I infection may modify DPB pathogenesis via activation of T cells. We also found that the frequency of ATL development in HTLV-I positive DPB patients was significantly higher than in all HTLV-I positive patients (OR = 8.22, 95% CI = 2.61-25.9, P < 0.01). The levels of TGF-beta in patients who developed ATL were significantly lower than in patients who did not develop ATL. Sensitivity and specificity were 80% and 85.7%, respectively (cut-off = 20 pg/ml). We also propose that these features should be taken into consideration in the treatment of DPB in HTLV-I infected individuals.