The Crucial Role of NLRP3 Inflammasome in Viral Infection-Associated Fibrosing Interstitial Lung Diseases.

Idiopathic pulmonary fibrosis (IPF), one of the most common fibrosing interstitial lung diseases (ILD), is a chronic-age-related respiratory disease that rises from repeated micro-injury of the alveolar epithelium. Environmental influences, intrinsic factors, genetic and epigenetic risk factors that lead to chronic inflammation might be implicated in the development of IPF. The exact triggers that initiate the fibrotic response in IPF remain enigmatic, but there is now increasing evidence supporting the role of chronic exposure of viral infection. During viral infection, activation of the NLRP3 inflammasome by integrating multiple cellular and molecular signaling implicates robust inflammation, fibroblast proliferation, activation of myofibroblast, matrix deposition, and aberrant epithelial-mesenchymal function. Overall, the crosstalk of the NLRP3 inflammasome and viruses can activate immune responses and inflammasome-associated molecules in the development, progression, and exacerbation of IPF.

Pinocembrin ameliorates post-infarct heart failure through activation of Nrf2/HO-1 signaling pathway.

BACKGROUND: Oxidative stress is an important factor involved in the progress of heart failure. The current study was performed to investigate whether pinocembrin was able to ameliorate post-infarct heart failure (PIHF) and the underlying mechanisms. METHODS: Rats were carried out left anterior descending artery ligation to induce myocardial infarction and subsequently raised for 6 weeks to produce chronic heart failure. Then pinocembrin was administrated every other day for 2 weeks. The effects were evaluated by echocardiography, western blot, Masson's staining, biochemical examinations, immunohistochemistry, and fluorescence. In vitro we also cultured H9c2 cardiomyocytes and cardiac myofibroblasts to further testify the mechanisms. RESULTS: We found that PIHF-induced deteriorations of cardiac functions were significantly ameliorated by administrating pinocembrin. In addition, the pinocembrin treatment also attenuated collagen deposition and augmented vascular endothelial growth factor receptor 2 in infarct border zone along with an attenuated apoptosis, which were related to an amelioration of oxidative stress evidenced by reduction of reactive oxygen species (ROS) in heart tissue and malondialdehyde (MDA) in serum, and increase of superoxide dismutase (SOD). This were accompanied by upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway. In vitro experiments we found that specific Nrf2 inhibitor significantly reversed the effects resulted from pinocembrin including antioxidant, anti-apoptosis, anti-fibrosis and neovascularization, which further indicated the amelioration of PIHF by pinocembrin was in a Nrf2/HO-1 pathway-dependent manner. CONCLUSION: Pinocembrin ameliorated cardiac functions and remodeling resulted from PIHF by ROS scavenging and Nrf2/HO-1 pathway activation which further attenuated collagen fibers deposition and apoptosis, and facilitated angiogenesis.

The Mechanism of CD8+ T Cells for Reducing Myofibroblasts Accumulation during Renal Fibrosis.

Renal fibrosis is a hallmark of chronic kidney disease (CKD) and a common manifestation of end-stage renal disease that is associated with multiple types of renal insults and functional loss of the kidney. Unresolved renal inflammation triggers fibrotic processes by promoting the activation and expansion of extracellular matrix-producing fibroblasts and myofibroblasts. Growing evidence now indicates that diverse T cells and macrophage subpopulations play central roles in the inflammatory microenvironment and fibrotic process. The present review aims to elucidate the role of CD8+ T cells in renal fibrosis, and identify its possible mechanisms in the inflammatory microenvironment.

Implant Fibrosis and the Underappreciated Role of Myofibroblasts in the Foreign Body Reaction.

Body implants and implantable medical devices have dramatically improved and prolonged the life of countless patients. However, our body repair mechanisms have evolved to isolate, reject, or destroy any object that is recognized as foreign to the organism and inevitably mounts a foreign body reaction (FBR). Depending on its severity and chronicity, the FBR can impair implant performance or create severe clinical complications that will require surgical removal and/or replacement of the faulty device. The number of review articles discussing the FBR seems to be proportional to the number of different implant materials and clinical applications and one wonders, what else is there to tell? We will here take the position of a fibrosis researcher (which, coincidentally, we are) to elaborate similarities and differences between the FBR, normal wound healing, and chronic healing conditions that result in the development of peri-implant fibrosis. After giving credit to macrophages in the inflammatory phase of the FBR, we will mainly focus on the activation of fibroblastic cells into matrix-producing and highly contractile myofibroblasts. While fibrosis has been discussed to be a consequence of the disturbed and chronic inflammatory milieu in the FBR, direct activation of myofibroblasts at the implant surface is less commonly considered. Thus, we will provide a perspective how physical properties of the implant surface control myofibroblast actions and accumulation of stiff scar tissue. Because formation of scar tissue at the surface and around implant materials is a major reason for device failure and extraction surgeries, providing implant surfaces with myofibroblast-suppressing features is a first step to enhance implant acceptance and functional lifetime. Alternative therapeutic targets are elements of the myofibroblast mechanotransduction and contractile machinery and we will end with a brief overview on such targets that are considered for the treatment of other organ fibroses.

Natural killer T cell/IL-4 signaling promotes bone marrow-derived fibroblast activation and M2 macrophage-to-myofibroblast transition in renal fibrosis.

Renal fibrosis is a histological manifestation of chronic kidney disease. Natural killer T (NKT) cells have a critical role in the pathogenesis of fibrotic disorder. However, the role of NKT cells in regulating kidney fibrosis remains largely unknown. In the current study, we showed that the percentages of NKT+ cells and NKT+-IL-4+ cells were notably increased in folic acid (FA) and obstructive nephropathy. CD1d deficiency protected mice from renal fibrosis induced by FA and obstructive injury. Specifically, Loss of CD1d reduced bone marrow-derived myofibroblasts and CD206+/α-smooth muscle actin+ cells in the kidneys of injured mice. But mice treated with α-galactosylceramide (α-GC, a specific activator of NKT cells) developed more severe fibrosis, accumulated more myeloid myofibroblasts and M2 macrophages-myofibroblasts transition (M2MMT) cells in FA injured kidneys. Furthermore, IL-4 expression was markedly reduced in CD1d deficiency mice but increased in α-GC-treated mice. Administration of IL-4 abrogates the inhibiting effect of CD1d deficiency on renal fibrosis, bone marrow-derived fibroblasts activation, and M2MMT in FA injured kidneys. Conversely, pharmacological inhibition of IL-4 attenuated the development of renal fibrosis, decreased bone marrow-derived myofibroblasts, and suppressed M2MMT. Thus, this study revealed a novel role of NKT cells in the bone marrow-derived fibroblasts activation and M2MMT during renal fibrosis. Targeting NKT cell/IL-4 signaling may be an effective treatment for renal fibrosis.

Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice.

BACKGROUND AND AIMS: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing. APPROACH AND RESULTS: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers. CONCLUSIONS: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.

Human cardiac fibroblasts produce pro-inflammatory cytokines upon TLRs and RLRs stimulation.

Heart inflammation is one of the major causes of heart damage that leads to dilated cardiomyopathy and often progresses to end-stage heart failure. In the present study, we aimed to assess whether human cardiac cells could release immune mediators upon stimulation of Toll-like receptors (TLRs) and Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs).Commercially available human cardiac fibroblasts and an immortalized human cardiomyocyte cell line were stimulated in vitro with TLR2, TLR3, and TLR4 agonists. In addition, cytosolic RLRs were activated in cardiac cells after transfection of polyinosinic-polycytidylic acid (PolyIC). Upon stimulation of TLR3, TLR4, MDA5, and RIG-I, but not upon stimulation of TLR2, human cardiac fibroblasts produced high amounts of the pro-inflammatory cytokines IL-6 and IL-8. On the contrary, the immortalized human cardiomyocyte cell line was unresponsive to the tested TLRs agonists. Upon RLRs stimulation, cardiac fibroblasts, and to a lesser extent the cardiomyocyte cell line, induced anti-viral IFN-ß expression.These data demonstrate that human cardiac fibroblasts and an immortalized human cardiomyocyte cell line differently respond to various TLRs and RLRs ligands. In particular, human cardiac fibroblasts were able to induce pro-inflammatory and anti-viral cytokines on their own. These aspects will contribute to better understand the immunological function of the different cell populations that make up the cardiac tissue.

Echinochrome A Treatment Alleviates Fibrosis and Inflammation in Bleomycin-Induced Scleroderma.

Scleroderma is an autoimmune disease caused by the abnormal regulation of extracellular matrix synthesis and is activated by non-regulated inflammatory cells and cytokines. Echinochrome A (EchA), a natural pigment isolated from sea urchins, has been demonstrated to have antioxidant activities and beneficial effects in various disease models. The present study demonstrates for the first time that EchA treatment alleviates bleomycin-induced scleroderma by normalizing dermal thickness and suppressing collagen deposition in vivo. EchA treatment reduces the number of activated myofibroblasts expressing α-SMA, vimentin, and phosphorylated Smad3 in bleomycin-induced scleroderma. In addition, it decreased the number of macrophages, including M1 and M2 types in the affected skin, suggesting the induction of an anti-inflammatory effect. Furthermore, EchA treatment markedly attenuated serum levels of inflammatory cytokines, such as tumor necrosis factor-α and interferon-γ, in a murine scleroderma model. Taken together, these results suggest that EchA is highly useful for the treatment of scleroderma, exerting anti-fibrosis and anti-inflammatory effects.

Wnt Inhibition Sensitizes PD-L1 Blockade Therapy by Overcoming Bone Marrow-Derived Myofibroblasts-Mediated Immune Resistance in Tumors.

Cancer-associated fibroblasts (CAFs) has been recognized as one cause of tumor resistance to immune checkpoint blockade therapy, but the underlying mechanisms still remain elusive. In the present study, a bone marrow-derived CAF (BMF) -rich tumor model is successfully established by subcutaneously mixed inoculation of BMFs and tumor cells into mice and the BMF-mixed tumor xenografts are demonstrated to be resistant to anti-PD-L1 antibody immunotherapy compared to the mere tumor xenografts. In vitro assays via the co-culture system of BMFs and tumor cells indicate that the co-cultured BMFs are induced to overexpress PD-L1, while there is no such a phenomenon in the co-cultured cancer cells. The further knock-out of PD-L1 in BMFs rescues the sensitivity of BMF-mixed tumor xenografts to PD-L1 blockade therapy. Mechanistically, via the microarray assay, we identify that the upregulation of PD-L1 in BMFs stimulated by cancer cells is medicated by the activation of the Wnt/ß-catenin signaling pathway in BMFs. Moreover, the administration of Wnt/ß-catenin signaling inhibitors, including XAV-939 and Wnt-C59, distinctly inhibits the upregulation of PD-L1 expression in the co-cultured BMFs. The further combination administration of XAV-939 significantly potentiates the therapeutic outcome of PD-L1 blockade therapy in BMF-mixed tumors. In summary, our study demonstrates that Wnt inhibition augments PD-L1 blockade efficacy by overcoming BMF-mediated immunotherapy resistance.

UUO induces lung fibrosis with macrophage-myofibroblast transition in rats.

Progression of chronic kidney disease (CKD) to uremia is often accompanied by varying degrees of lung damage and this is also an important cause of death. Although there are many studies on the mechanism of lung injury, it is not clearly understood. Inflammatory macrophages may associated with fibrosis in the lungs. Here, we investigated the role of macrophage-myofibroblast transition (MMT) in lung fibrosis with unilateral ureteral obstruction (UUO) rats. We found that cells undergoing MMT accounted for an important part of the myofibroblast population, and correlated with lung fibrosis, MMT cells in lungs have a predominant M2 phenotype, and this process was attenuated after treatment with eplerenone. In conclusion, our studies provide a possible mechanism for UUO-induced kidney damage and lung injury, indicating the possibility of using eplerenone, a mineralocorticoid receptor blocker, to treat UUO to reduce kidney damage and protect lung function.

Plasminogen activator inhibitor-1 reduces cardiac fibrosis and promotes M2 macrophage polarization in inflammatory cardiomyopathy.

Plasminogen activator inhibitor-1 (PAI-1) has a cardioprotective function in mice by repressing cardiac fibrosis through TGF-ß and plasminogen-mediated pathways. In addition it is known to be involved in the recruitment and polarization of monocytes/macrophages towards a M2 phenotype in cancer. Here, we investigated the expression of PAI-1 in human dilated cardiomyopathy (DCM) and inflammatory dilated cardiomyopathy (DCMi) and its effect on cardiac fibrosis and macrophage polarization. We retrospectively analyzed endomyocardial biopsies (EMBs) of patients with DCM or DCMi for PAI-1 expression by immunohistochemistry. Furthermore, EMBs were evaluated for the content of fibrotic tissue, number of activated myofibroblasts, TGF-ß expression, as well as for M1 and M2 macrophages. Patients with high-grade DCMi (DCMi-high, CD3+ lymphocytes > 30 cells/mm2) had significantly increased PAI-1 levels compared to DCM and low-grade DCMi patients (DCMi-low, CD3+ lymphocytes = 14-30 cells/mm2) (15.5 ± 0.4% vs. 1.0 ± 0.1% and 4.0 ± 0.1%, p ≤ 0.001). Elevated PAI-1 expression in DCMi-high subjects was associated with a diminished degree of cardiac fibrosis, decreased levels of TGF-ß and reduced number of myofibroblasts. In addition, DCMi-high patients revealed an increased proportion of non-classical M2 macrophages towards classical M1 macrophages, indicating M2 macrophage-favoring properties of PAI-1 in inflammatory cardiomyopathies. Our findings give evidence that elevated expression of cardiac PAI-1 in subjects with high-grade DCMi suppresses fibrosis by inhibiting TGF-ß and myofibroblast activation. Moreover, our data indicate that PAI-1 is involved in the polarization of M2 macrophages in the heart. Thus, PAI-1 could serve as a potential prognostic biomarker and as a possible therapeutic target in inflammatory cardiomyopathies.

NLRP3 inflammasome activation in alveolar epithelial cells promotes myofibroblast differentiation of lung-resident mesenchymal stem cells during pulmonary fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is a lethal and agnogenic interstitial lung disease, which has limited therapeutic options. Recently, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated as an important contributor to various fibrotic diseases following its persistent activation. However, the role of NLRP3 inflammasome in pulmonary fibrogenesis still needs to be further clarified. Here, we found that the activation of the NLRP3 inflammasome was raised in fibrotic lungs. In addition, the NLRP3 inflammasome was found to be activated in alveolar epithelial cells (AECs) in the lung tissue of both IPF patients and pulmonary fibrosis mouse models. Further research revealed that epithelial cells, following activation of the NLRP3 inflammasome, could induce the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs). In addition, inhibiting the activation of the NLRP3 inflammasome in epithelial cells promoted the expression of dickkopf-1 (DKK1), a secreted Wnt antagonist. DKK1 was capable of suppressing the profibrogenic differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis. In conclusion, this study not only provides a further in-depth understanding of the pathogenesis of pulmonary fibrosis, but also reveals a potential therapeutic strategy for disorders associated with pulmonary fibrosis.

Elimination of NF-κB signaling in Vimentin+ stromal cells attenuates tumorigenesis in a mouse model of Barrett's Esophagus.

Chronic inflammation induces Barrett's Esophagus (BE) which can advance to esophageal adenocarcinoma. Elevated levels of interleukin (IL)-1b, IL-6 and IL-8 together with activated nuclear factor-kappaB (NF-κB), have been identified as important mediators of tumorigenesis. The inflammatory milieu apart from cancer cells and infiltrating immune cells contains myofibroblasts (MFs) that express aSMA and Vimentin. As we observed that increased NF-κB activation and inflammation correlates with increased MF recruitment and an accelerated phenotype we here analyze the role of NF-κB in MF during esophageal carcinogenesis in our L2-IL-1B mouse model. To analyze the effect of NF-κB signaling in MFs, we crossed L2-IL-1B mice to tamoxifen inducible Vim-Cre (Vim-CreTm) mice and floxed RelA (p65fl/fl) mice to specifically eliminate NF-κB signaling in MF (IL-1b.Vim-CreTm.p65fl/fl). The interaction of epithelial cells and stromal cells was further analyzed in mouse BE organoids and patient-derived human organoids. Histological scoring of IL-1b.Vim-CreTm.p65fl/fl mice showed a significantly attenuated phenotype compared with L2-IL-1B mice, with mild inflammation, decreased metaplasia and no dysplasia. This correlated with decreased proliferation and increased differentiation in cardia tissue of IL-1b.Vim-CreTm.p65fl/fl compared with L2-IL-1B mice. Distinct changes of cytokines and chemokines within the local microenvironment in IL-1b.Vim-CreTm.p65fl/fl mice reflected the histopathological abrogated phenotype. Co-cultured NF-κB inhibitor treated MF with mouse BE organoids demonstrated NF-κB-dependent growth and migration. MFs are essential to form an inflammatory and procarcinogenic microenvironment and NF-κB signaling in stromal cells emerges as an important driver of esophageal carcinogenesis. Our data suggest anti-inflammatory approaches as preventive strategies during surveillance of BE patients.

Water intake accelerates ATP release from myofibroblast cells in rats: ATP-mediated podoplanin-dependent control for physiological function and immunity.

We previously demonstrated that water intake increased mesenteric lymph flow and the total flux of IL-22 in rat jejunum. The drained water and the higher permeability of albumin in the jejunal microcirculation contributed to increase the lymph flow and IL-22 transport via the activation of great bulk flow in the jejunal villi. To address the effects of water intake-mediated great bulk flow-dependent mechanical force on jejunal physiological function and immunological regulation of innate lymphoid cells (ILC)-3, we examined the effects of shear stress stimulation on cultured rat myofibroblast cells. Next, we investigated the effects of water intake on podoplanin and IL-22 expressions in cultured human intestinal epithelial cells and rat in vivo jejunal preparations, respectively. Shear stress stimulation of the myofibroblast cells induced ATP release via an activation of cell surface F1/F0 ATP synthase. ATP produced podoplanin expression in the intestinal epithelial cells. Water intake accelerated immunohistochemical expressions of podoplanin and IL-22 in the interepithelial layers and lamina propria of the jejunum. ATP dose-dependently increased IL-22 mRNA expression in ILC-3, which are housed in the lamina propria. Water intake also increased immunohistochemical and mRNA expressions of ecto-nucleoside triphosphate diphosphohydrolases 2 and 5 in jejunal villi. In conclusion, water intake-mediated shear stress stimulation-dependent ATP release from myofibroblast cells maintains higher tissue colloid osmotic pressure in the jejunal microcirculation through podoplanin upregulation in the interepithelial layers. ATP induces IL-22 mRNA expression in ILC-3 in jejunal villi, which may contribute to regulation of mucosal immunity in small intestine.NEW & NOTEWORTHY We investigated effects of shear stress stimulation on cultured myofibroblast cells and water intake on podoplanin and IL-22 expressions in rat jejunal villi. The stimulation induced ATP release from the cells. Water intake accelerated podoplanin and IL-22 expression levels. ATP increased IL-22 mRNA expression in innate lymphoid cells (ILC)-3. Hence, water intake maintains higher osmotic pressure in the jejunal villi through ATP release and podoplanin upregulation. Water intake may regulate the mucosal immunity.

Macrophage to myofibroblast transition contributes to subretinal fibrosis secondary to neovascular age-related macular degeneration.

BACKGROUND: Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis. METHODS: Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-ß for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo. RESULTS: Approximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-ß and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions. CONCLUSIONS: MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-ß and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-ß1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch's membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor ß. a-SMA = alpha smooth muscle actin. C3a = complement C3a.

Contribution of Autophagy-Notch1-Mediated NLRP3 Inflammasome Activation to Chronic Inflammation and Fibrosis in Keloid Fibroblasts.

Keloid is a representative chronic fibroproliferative condition that occurs after tissue injury. Emerging evidence showed that activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in the pro-inflammatory response in injured tissues. However, the role of NLRP3 inflammasome in keloid progression remains unclear. Notch signaling, which activates NLRP3 inflammasome, is known to contribute to scar formation in keloid, but the cause of enhanced Notch signaling in keloid is not clear. We sought to investigate whether autophagy regulates Notch1 signaling in keloid fibroblasts and determine whether Notch1 signaling might regulate NLRP3 inflammasomes and myofibroblast differentiation. An in vitro model of keloid was established by culturing primary keloid fibroblasts from patients. Expression levels of Notch1, NLRP3 inflammasome proteins, pro-inflammatory cytokines, and myofibroblast markers in keloid fibroblasts were examined and compared with those in normal fibroblasts. Autophagy known to mediate Notch1 degradation was also monitored in fibroblasts. Small interfering RNA (siRNA) targeting Notch1 was used to transfect keloid fibroblasts to further examine the role of Notch signaling in NLRP3 inflammasome activation. Expression levels of Notch1 and NLRP3 inflammasome in keloid fibroblasts increased compared to those in normal fibroblasts. Such increases were accompanied by increased LC3 levels and reduced autophagic flux. Notch1 silencing in keloid fibroblasts by siRNA transfection significantly suppressed increased levels of overall NLRP3 inflammasome complex proteins, NF-kB, and α-smooth muscle actin. Autophagy induction by rapamycin treatment in keloid fibroblasts effectively suppressed expression levels of Notch1 and NLRP3 inflammasome proteins. Decreased autophagy activity in keloid can result in Notch1-mediated myofibroblast activation and NLRP3 inflammasome signaling activation which is critical for chronic inflammation. Collectively, these results identify Notch1 as a novel activator of NLRP3 inflammasome signaling leading to chronic tissue damage and myofibroblast differentiation in keloid progression.

Innate Immunity Effector Cells as Inflammatory Drivers of Cardiac Fibrosis.

Despite relevant advances made in therapies for cardiovascular diseases (CVDs), they still represent the first cause of death worldwide. Cardiac fibrosis and excessive extracellular matrix (ECM) remodeling are common end-organ features in diseased hearts, leading to tissue stiffness, impaired myocardial functional, and progression to heart failure. Although fibrosis has been largely recognized to accompany and complicate various CVDs, events and mechanisms driving and governing fibrosis are still not entirely elucidated, and clinical interventions targeting cardiac fibrosis are not yet available. Immune cell types, both from innate and adaptive immunity, are involved not just in the classical response to pathogens, but they take an active part in "sterile" inflammation, in response to ischemia and other forms of injury. In this context, different cell types infiltrate the injured heart and release distinct pro-inflammatory cytokines that initiate the fibrotic response by triggering myofibroblast activation. The complex interplay between immune cells, fibroblasts, and other non-immune/host-derived cells is now considered as the major driving force of cardiac fibrosis. Here, we review and discuss the contribution of inflammatory cells of innate immunity, including neutrophils, macrophages, natural killer cells, eosinophils and mast cells, in modulating the myocardial microenvironment, by orchestrating the fibrogenic process in response to tissue injury. A better understanding of the time frame, sequences of events during immune cells infiltration, and their action in the injured inflammatory heart environment, may provide a rationale to design new and more efficacious therapeutic interventions to reduce cardiac fibrosis.

Adaptive and innate immune mechanisms in cardiac fibrosis complicating pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a syndrome diagnosed by increased mean pulmonary artery (PA) pressure and resistance and normal pulmonary capillary wedge pressure. PAH is characterized pathologically by distal pulmonary artery remodeling, increased pulmonary vascular resistance, and plexiform lesions (PLs). Right ventricular fibrosis and hypertrophy, leading to right ventricular failure, are the main determinants of mortality in PAH. Recent work suggests that right ventricular fibrosis results from resident cardiac fibroblast activation and conversion to myofibroblasts, leading to replacement of contractile cardiomyocytes with nondistensible tissue incapable of conductivity or contractility. However, the origins, triggers, and consequences of myofibroblast expansion and its pathophysiological relationship with PAH are unclear. Recent advances indicate that signals generated by adaptive and innate immune cells may play a role in right ventricular fibrosis and remodeling. This review summarizes recent insights into the mechanisms by which adaptive and innate immune signals participate in the transition of cardiac fibroblasts to activated myofibroblasts and highlights the existing gaps of knowledge as relates to the development of right ventricular fibrosis.

Bi-directional communication: Conversations between fibroblasts and immune cells in systemic sclerosis.

Systemic Sclerosis (SSc) is an autoimmune idiopathic connective tissue disease, characterized by aberrant fibro-proliferative and inflammatory responses, causing fibrosis of multiple organs. In recent years the interactions between innate and adaptive immune cells with resident fibroblasts have been uncovered. Cross-talk between immune and stromal cells mediates activation of stromal cells to myofibroblasts; key cells in the pathophysiology of fibrosis. These cells and their cytokines appear to mediate their effects in both a paracrine and autocrine fashion. This review examines the role of innate and adaptive immune cells in SSc, focusing on recent advances that have illuminated our understanding of ongoing bi-directional communication between immune and stromal cells. Finally, we appraise current and future therapies and how these may be useful in a disease that currently has no specific disease modifying treatment.