Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts
Eric A.F. van TolLisa HoltFeng Ling LiFeng‐Ming KongRichard A. RippeMitsuo YamauchiJolanta B. PucilowskaP. Kay LundR. Balfour Sartor
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Normal luminal bacteria and bacterial cell wall polymers are implicated in the pathogenesis of chronic intestinal inflammation. To determine the direct involvement of bacteria and their products on intestinal fibrogenesis, the effects of purified bacterial cell wall polymers on collagen and cytokine synthesis were evaluated in intestinal myofibroblast cultures established from normal fetal and chronically inflamed cecal tissues. In this study, the intestines of Lewis rats were intramurally injected with peptidoglycan-polysaccharide polymers. Collagen and transforming growth factor (TGF)-β1 mRNA levels were measured and correlated with mesenchymal cell accumulation by immunohistochemistry. The direct effects of cell wall polymers on fibrogenic cytokine and collagen α1 (type I) expression were evaluated in intestinal myofibroblast cultures. We found that intramural injections of bacterial cell wall polymers induced chronic granulomatous enterocolitis with markedly increased collagen synthesis and concomitant increased TGF-β1 and interleukin (IL)-6 expression. Intestinal myofibroblast cultures were established, which both phenotypically and functionally resemble the mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell wall polymers directly stimulated collagen α1 (I), TGF-β1, IL-1β, and IL-6 mRNA expression in the intestinal myofibroblasts derived from both normal and inflamed cecum. Neutralization of endogenous TGF-β1 inhibited in vitro collagen gene expression. From our results, we conclude that increased exposure to luminal bacterial products can directly activate intestinal mesenchymal cells, which accumulate in areas of chronic intestinal inflammation, thus stimulating intestinal fibrosis in genetically susceptible hosts.Keywords:
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Scleroderma (SSc) is an autoimmune connective tissue disorder characterized by progressive fibrosis of the skin and internal organs. The leading cause of death in SSc patients is pulmonary dysfunction as a result of interstitial fibrosis and pulmonary vasculopathy. Our objective was to evaluate histopathological abnormalities associated with the development of pulmonary fibrosis in SSc.Postmortem SSc lung tissue from various stages of fibrosis and tissue from normal lung were analyzed by Masson's trichrome staining and immunohistochemistry. Monoclonal antibodies against smooth muscle-alpha actin (myofibroblast marker), von Willebrand Factor, platelet endothelial cell adhesion molecule-1 (endothelial cell markers), or caldesmon (smooth muscle cell marker) were employed.We found that in the early active stages of SSc lung fibrosis two major types of cellular abnormalities occur. One is the induction of a large number of smooth muscle alpha-actin-positive myofibroblasts in interstitia. The other is the excessive formation of alveolar capillaries (hypervascularity) accompanied by an increase in the number of microvascular endothelial cells. The vascular abnormality also involves the development of microvessels that are irregular in size and shape. However, the population of myofibroblasts and capillary endothelial cells decline as the fibrosis progresses to its most marked, later stages.We conclude that the induction of myofibroblasts and the overdevelopment of capillary microvessels characterize the progression of lung fibrosis in SSc. Using these histological alterations as criteria, therefore we have divided the fibrosis formed in the SSc lungs into four pathological stages. These results suggest that both fibroblast overproliferation and vascular abnormality play an important role in the pathogenesis of lung fibrosis in SSc.
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Abstract Background Fibrosis poses a substantial setback in regenerative medicine. Histopathologically, fibrosis is an excessive accumulation of collagen affected by myofibroblasts and this can occur in any tissue that is exposed to chronic injury or insult. Transforming growth factor (TGF)-β1, a crucial mediator of fibrosis, drives differentiation of fibroblasts into myofibroblasts. These cells exhibit α-smooth muscle actin (α-SMA) and synthesize high amounts of collagen I, the major extracellular matrix (ECM) component of fibrosis. While hormones stimulate cells in a pulsatile manner, little is known about cellular response kinetics upon growth factor impact. We therefore studied the effects of short TGF-β1 pulses in terms of the induction and maintenance of the myofibroblast phenotype. Results Twenty-four hours after a single 30 min TGF-β1 pulse, transcription of fibrogenic genes was upregulated, but subsided 7 days later. In parallel, collagen I secretion rate and α-SMA presence were elevated for 7 days. A second pulse 24 h later extended the duration of effects to 14 days. We could not establish epigenetic changes on fibrogenic target genes to explain the long-lasting effects. However, ECM deposited under singly pulsed TGF-β1 was able to induce myofibroblast features in previously untreated fibroblasts. Dependent on the age of the ECM (1 day versus 7 days’ formation time), this property was diminished. Vice versa , myofibroblasts were cultured on fibroblast ECM and cells observed to express reduced (in comparison with myofibroblasts) levels of collagen I. Conclusions We demonstrated that short TGF-β1 pulses can exert long-lasting effects on fibroblasts by changing their microenvironment, thus leaving an imprint and creating a reciprocal feed-back loop. Therefore, the ECM might act as mid-term memory for pathobiochemical events. We would expect this microenvironmental memory to be dependent on matrix turnover and, as such, to be erasable. Our findings contribute to the current understanding of fibroblast induction and maintenance, and have bearing on the development of antifibrotic drugs.
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During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein-coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.
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Radiation fibrosis of human normal tissues is very common in radiotherapy. One of the main fundamental problems yet unsolved in fibrotic tissues is the origin of the chronic activation of myofibroblasts within these tissues. It has been postulated by some researchers that this chronic activation results from a continuous production of activating factors. So fibrosis could be defined as a wound where continuous signals for repair are emitted. Cytokines and growth factors probably play a vital role in this process. Among them transforming growth factor β1(TGF β1) is considered as a master switch for the fibrotic program. This review discusses recent evidence on the critical role played by TGF β1 in the initiation, development, and persistence of radiation fibrosis. It summarized the results concerning this factor after irradiation of various tissues and cells. All these researches show that the TGF β1 pathway may be a specific target for anti fibrotic agents. [
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Fibrosis refers to the connective tissue deposition and stiffness usually as a result of injury. Fibrosis tissue-resident mesenchymal cells, including fibroblasts, myofibroblast, smooth muscle cells, and mesenchymal stem/stromal cells (MSCs), are major players in fibrogenic processes under certain contexts. Acknowledging differentiation potential of MSCs to the aforementioned other types of mesenchymal cell lineages is essential for better understanding of MSCs' substantial contributions to progressive fibrogenesis. MSCs may represent a potential therapeutic option for fibrosis resolution owing to their unique pleiotropic functions and therapeutic properties. Currently, clinical trial efforts using MSCs and MSC-based products are underway but clinical data collected by the early phase trials are insufficient to offer better support for the MSC-based anti-fibrotic therapies. Given that MSCs are involved in the coagulation through releasing tissue factor, MSCs can retain procoagulant activity to be associated with fibrogenic disease development. Therefore, MSCs' functional benefits in translational applications need to be carefully balanced with their potential risks.
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The cytokine transforming growth factor β (TGFβ) is a major contributor to fibrogenic responses both in vitro and in vivo. TGFβ possesses many functions; thus, broadly targeting TGFβ signaling as an anti-fibrotic approach is anticipated to be problematic. Recent experiments, however, have begun to elucidate the signaling pathways through which TGFβ activates a fibrotic program. This review critically evaluates the evidence supporting TGFβ as a pro-fibrotic cytokine, with special attention to cardiac fibrosis, and suggests several possible points for selective drug intervention to combat chronic fibrotic disease.
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The relative abundance of fibroblasts and myofibroblasts in the portal tracts was assessed in liver biopsy specimens of baboons that developed fatty liver and perivenular fibrosis after alcohol (ethanol) consumption. Fourteen baboons pair-fed with diets containing alcohol, or isocaloric carbohydrate, for up to four years were studied. No fibrosis was observed in the portal tracts of these baboons. The number of mesenchymal cells per 1,000 sq microns of the portal tissue was not changed after alcohol consumption. Electron microscopy demonstrated no increase in the number of fibroblasts, myofibroblasts, or other mesenchymal cells. In view of an increased number of myofibroblasts and fibroblasts in association with the development of perivenular fibrosis as previously reported, the results herein suggest that mesenchymal cells in the portal tracts lack the proper stimuli for proliferation after alcohol consumption.
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Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-β1 (TGF-β1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-β1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-β1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.
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Idiopathic pulmonary fibrosis is a progressive and usually fatal lung disease characterized by fibroblast proliferation and extracellular matrix remodeling, which result in irreversible distortion of the lung's architecture. Although the pathogenetic mechanisms remain to be determined, the prevailing hypothesis holds that fibrosis is preceded and provoked by a chronic inflammatory process that injures the lung and modulates lung fibrogenesis, leading to the end-stage fibrotic scar. However, there is little evidence that inflammation is prominent in early disease, and it is unclear whether inflammation is relevant to the development of the fibrotic process. Evidence suggests that inflammation does not play a pivotal role. Inflammation is not a prominent histopathologic finding, and epithelial injury in the absence of ongoing inflammation is sufficient to stimulate the development of fibrosis. In addition, the inflammatory response to a lung fibrogenic insult is not necessarily related to the fibrotic response. Clinical measurements of inflammation fail to correlate with stage or outcome, and potent anti-inflammatory therapy does not improve outcome. This review presents a growing body of evidence suggesting that idiopathic pulmonary fibrosis involves abnormal wound healing in response to multiple, microscopic sites of ongoing alveolar epithelial injury and activation associated with the formation of patchy fibroblast–myofibroblast foci, which evolve to fibrosis. Progress in understanding the fibrogenic mechanisms in the lung is likely to yield more effective therapies.
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