Fibrogenic Cells Research Articles

Single-cell RNA transcriptomics in mice reveals embryonic origin of fibrosis due to maternal obesity

Over 40% of pregnant women in the USA are obese which negatively affects fetal development and offspring health. Maternal obesity (MO) leads to fibrotic infiltration in multiple tissues and organs of offspring during their adulthood although the origin and mechanisms are unclear. C57BL/6J female mice were fed a control and high-fat diet to mimic MO condition. Embryonic somatic tissues were obtained at E9.5, E11.5, and E13.5 (equivalent to 6 weeks of human pregnancy) from control (CON) and MO mice for single-cell RNA-sequencing (scRNA-seq). To explore the role of AMP-activated protein kinase (AMPK), AMPK was activated by metformin and A769662, and knocked out in embryonic mesenchymal cells (EMC) using AMPKα1 floxed mice. Using unsupervised clustering, we identified three major cell populations with fibrogenic capacity. Compared to CON, the population of fibrogenic cells increased dramatically (by ∼125%) due to MO, supporting an embryonic origin of fibrosis in the offspring. MO induced inflammatory response and elevated expression of transforming growth factor β (TGFβ) signalling and fibrogenic genes in embryos. MO inhibited AMPK and its activation by metformin and A769662 inhibited TGFβ signalling and fibrogenesis. MO profoundly enhances embryonic fibrogenesis, explaining the origin of fibrosis in the offspring of mothers living with obesity. Our data underscore the importance of early intervention, before 5-6 weeks of pregnancy, in improving embryonic development, and AMPK is an amiable target for suppressing excessive fibrogenesis in MO embryos to assist increasing populations of obese mothers having healthy children. This work was funded by National Institutes of Health Grant R01HD067449.

Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration.

Bone regeneration is mediated by skeletal stem/progenitor cells (SSPCs) that are mainly recruited from the periosteum after bone injury. The composition of the periosteum and the steps of SSPC activation and differentiation remain poorly understood. Here, we generated a single-nuclei atlas of the periosteum at steady-state and of the fracture site during early stages of bone repair ( https://fracture-repair-atlas.cells.ucsc.edu ). We identified periosteal SSPCs expressing stemness markers ( Pi16 and Ly6a /SCA1) and responding to fracture by adopting an injury-induced fibrogenic cell (IIFC) fate, prior to undergoing osteogenesis or chondrogenesis. We identified distinct gene cores associated with IIFCs and their engagement into osteogenesis and chondrogenesis involving Notch, Wnt and the circadian clock signaling respectively. Finally, we show that IIFCs are the main source of paracrine signals in the fracture environment, suggesting a crucial paracrine role of this transient IIFC population during fracture healing. Overall, our study provides a complete temporal topography of the early stages of fracture healing and the dynamic response of periosteal SSPCs to injury, redefining our knowledge of bone regeneration.

Cryptotanshinone alleviates liver fibrosis via inhibiting STAT3/CPT1A-dependent fatty acid oxidation in hepatic stellate cells

Hepatic stellate cells (HSCs) are a major source of fibrogenic cells and play a central role in liver fibrogenesis. HSC activation depends on metabolic activation, for which it is well established that fatty acid oxidation (FAO) sustains their rapid proliferative rate. Studies have indicated that tanshinones inhibit HSC activation, however, the anti-fibrosis mechanisms of tanshinones are remain unclear. Herein, we reported that cryptotanshinone (CTS), a lipid-soluble ingredient of Salvia miltiorrhiza Bunge, exhibited the strongest inhibitory effects on HSC-LX2 proliferation and activation. CTS could induce lipocyte phenotype in mouse primary HSC and HSC-LX2. Transcriptomic sequencing and qPCR revealed that CTS regulated fatty acid metabolism and inhibited CPT1A and CPT1B expression. Target prediction suggested CTS regulates lipid metabolism by targeting STAT3. Mechanistically, the level of ATP and acetyl-CoA were reduced by the treatment of CTS, indicating that CTS could inhibit the level of FAO. Furthermore, CTS could inhibit the phosphorylation and nuclear translocation of STAT3. Additionally, CPT1A overexpression reversed the efficacy of CTS. Finally, CTS (40 mg/kg/day) attenuated CCl4-induced liver fibrosis and inhibited collagen production and HSC activation. Moreover, the results of immunofluorescence showed that α-SMA and p-STAT3 were co-located, and CTS could reduce the levels of p-STAT3 and α-SMA. In summary, CTS alleviated liver fibrosis by inhibiting the p-STAT3/CPT1A-dependent FAO both in vitro and in vivo, making it a potential candidate drug for the treatment of liver fibrosis.

Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis.

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2 which drives an aberrant fibrotic cascade. Additional analyses by CUT&RUN-sequencing revealed loss of let-7afd hampers AT1 differentiation by eliciting aberrant histone EZH2 methylation which prevents the exit of AT2 transitional cells into terminal AT1s. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis.

Hepatic Fibrosis and Cancer: The Silent Threats of Metabolic Syndrome.

Metabolic dysfunction-associated steatotic (fatty) liver disease (MASLD), previously termed non-alcoholic fatty liver disease, is a worldwide epidemic that can lead to hepatic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The disease is typically a component of the metabolic syndrome that accompanies obesity, and is often overlooked because the liver manifestations are clinically silent until late-stage disease is present (i.e., cirrhosis). Moreover, Asian populations, including Koreans, have a higher fraction of patients who are lean, yet their illness has the same prognosis or worse than those who are obese. Nonetheless, ongoing injury can lead to hepatic inflammation and ballooning of hepatocytes as classic features. Over time, fibrosis develops following activation of hepatic stellate cells, the liver's main fibrogenic cell type. The disease is usually more advanced in patients with type 2 diabetes mellitus, indicating that all diabetic patients should be screened for liver disease. Although there has been substantial progress in clarifying pathways of injury and fibrosis, there no approved therapies yet, but current research seeks to uncover the pathways driving hepatic inflammation and fibrosis, in hopes of identifying new therapeutic targets. Emerging molecular methods, especially single cell sequencing technologies, are revolutionizing our ability to clarify mechanisms underlying MASLD-associated fibrosis and HCC.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

Novel Approaches in Chronic Renal Failure without Renal Replacement Therapy: A Review.

Chronic kidney disease (CKD) is characterized by renal parenchymal damage leading to a reduction in the glomerular filtration rate. The inflammatory response plays a pivotal role in the tissue damage contributing to renal failure. Current therapeutic options encompass dietary control, mineral salt regulation, and management of blood pressure, blood glucose, and fatty acid levels. However, they do not effectively halt the progression of renal damage. This review critically examines novel therapeutic avenues aimed at ameliorating inflammation, mitigating extracellular matrix accumulation, and fostering renal tissue regeneration in the context of CKD. Understanding the mechanisms sustaining a proinflammatory and profibrotic state may offer the potential for targeted pharmacological interventions. This, in turn, could pave the way for combination therapies capable of reversing renal damage in CKD. The non-replacement phase of CKD currently faces a dearth of efficacious therapeutic options. Future directions encompass exploring vaptans as diuretics to inhibit water absorption, investigating antifibrotic agents, antioxidants, and exploring regenerative treatment modalities, such as stem cell therapy and novel probiotics. Moreover, this review identifies pharmaceutical agents capable of mitigating renal parenchymal damage attributed to CKD, targeting molecular-level signaling pathways (TGF-β, Smad, and Nrf2) that predominate in the inflammatory processes of renal fibrogenic cells.

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction-associated steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells.

Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction-associated steatohepatitis (MASH). Transforming growth factor-β (TGFβ), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFβ antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction-associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFβ1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFβ signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.

Imaging of fibrogenesis in the liver by [18F]TZ-Z09591, an Affibody molecule targeting platelet derived growth factor receptor β

BackgroundPlatelet-derived growth factor receptor beta (PDGFRβ) is a receptor overexpressed on activated hepatic stellate cells (aHSCs). Positron emission tomography (PET) imaging of PDGFRβ could potentially allow the quantification of fibrogenesis in fibrotic livers. This study aims to evaluate a fluorine-18 radiolabeled Affibody molecule ([18F]TZ-Z09591) as a PET tracer for imaging liver fibrogenesis.ResultsIn vitro specificity studies demonstrated that the trans-Cyclooctenes (TCO) conjugated Z09591 Affibody molecule had a picomolar affinity for human PDGFRβ. Biodistribution performed on healthy rats showed rapid clearance of [18F]TZ-Z09591 through the kidneys and low liver background uptake. Autoradiography (ARG) studies on fibrotic livers from mice or humans correlated with histopathology results. Ex vivo biodistribution and ARG revealed that [18F]TZ-Z09591 binding in the liver was increased in fibrotic livers (p = 0.02) and corresponded to binding in fibrotic scars.ConclusionsOur study highlights [18F]TZ-Z09591 as a specific tracer for fibrogenic cells in the fibrotic liver, thus offering the potential to assess fibrogenesis clearly.Graphical abstract

THE MORPHOLOGICAL AND FUNCTIONAL ASSESSMENT OF FAP+ AND α-SMA+-CELLS IN DIFFERENT TIMES OF RAT TOXIC LIVER FIBROSIS

Qualitative study of the source of the fibro-genic cell population in relation to the etiology and stage of fibrosis, as well as an understanding of the molecular mechanisms that regulate changes in the phenotype of hepatic fibroblasts, are of paramount importance in the development of pharmacological drugs. The purpose of the study was a morphological and functional assessment of activated portal fibroblasts (FAP+) and fat-accumulating cells (α-SMA+) of the liver at various stages of toxic liver fibrosis in rats. Liver fibrosis and cirrhosis in male Wistar rats were induced with thioacetamide solution for 17 weeks. Morphological examination of the liver was carried out on paraffin sections stained with hematoxylin and eosin using the Mallory method; immunohistochemical examination was carried out using polyclonal rabbit antibodies to the portal fibroblast antigen FAP and using monoclonal mouse antibodies to the α-SMA+ cell antigen. Before the onset of liver fibrosis stage F3/F4, from weeks 3 to 7, the number of FAP+ and α-SMA+ cells increased alternately. During the stages of transformation of fibrosis into cirrhosis from 7 to 11 weeks, their number increased slightly. At the stage of incomplete (F5) and before the onset of significant cirrhosis (F6) from weeks 11 to 15, the number of FAP+ and α-SMA+ cells were inconsistent and there was an alternating increase and decrease in their number. α-SMA+ cells before the start of the process of transformation of fibrosis into cirrhosis (F4/F5) were observed in sinusoids and foci of necrosis. Then they were detected both in sinusoids and in connective tissue trabeculae. FAP+ cells at the stage of portal fibrosis (F1) were localized near the interlobular vessels and interlobular bile ducts of the portal zones, and from the F2/F3 period they were detected in connective tissue trabeculae and sinusoids. In quantitative terms, α-SMA+ cells predominated at all stages of fibrosis. Based on the results obtained, it can be assumed that FAP+ cells make a major contribution to the development of the portal and initial stages of bridging fibrosis. They should be considered as one of the myofibroblast populations in thioacetamide-induced liver fibrogenesis.

The Role of Supporting Cell Populations in Satellite Cell Mediated Muscle Repair.

Skeletal muscle has a high capacity to repair and remodel in response to damage, largely through the action of resident muscle stem cells, termed satellite cells. Satellite cells are required for the proper repair of skeletal muscle through a process known as myogenesis. Recent investigations have observed relationships between satellite cells and other cell types and structures within the muscle microenvironment. These findings suggest that the crosstalk between inflammatory cells, fibrogenic cells, bone-marrow-derived cells, satellite cells, and the vasculature is essential for the restoration of muscle homeostasis. This review will discuss the influence of the cells and structures within the muscle microenvironment on satellite cell function and muscle repair.

THE (PRO)RENIN RECEPTOR IS INVOLVED IN KIDNEY ORGANOID DEVELOPMENT

Objective: The (pro)renin receptor [(P)RR], a receptor for prorenin and renin, is widely distributed in the body, including the kidneys. Studies in (P)RR knockout mouse models indicate that (P)RR ablation can be detrimental to renal development and function. However, the lack of human models hinders investigation into the role of (P)RR in human kidney development. The advent of human induced pluripotent stem cell (iPSC) – derived kidney organoids provides a tool to study the role of (P)RR in human kidney development. Design and method: To investigate the effects of (P)RR knockdown on kidney organoid development, human iPSC – derived kidney organoids were generated according to a previously described protocol. We silenced the (P)RR by introducing (P)RR antisense oligonucleotides (ASOs) by electroporation at the stage of iPSCs and nephron progenitor cells induction, respectively. The development of kidney organoids was monitored morphologically and through protein expression analysis of nephron markers by immunohistochemistry staining which was quantified by Image J. Results: After silencing the (P)RR at the initial stage of differentiation, the size of iPSCs-derived organoids was substantially smaller, but the size of tubular structures was bigger. We observed a decrease by 50% in both WT1 (glomerular cell marker) and PDGFR↑ (stromal cell marker) expressions in (P)RR-knockdown organoids, a 4-fold and 2-fold increase in expressions of fibrogenic cell markers ↑-SMA and COL1A1 respectively, and no change in Villin1 (proximal tubular cell marker) and Cadherin-1 (distal tubular cell marker) expression. Additionally, (P)RR knockdown at the stage of nephron progenitor cells induction decreased the expressions of Villin1 and Cadherin-1 in organoids by 50% and 60% separately, while it induced a 4-, 3-, 2- and 2-fold increase in the expressions of CD31 (endothelial cell marker), PDGFR↑, ↑-SMA and COL1A1 respectively. Conclusions: This study shows that (P)RR silencing at the initial stage of differentiation or at the stage of nephron progenitor cells induction impaired normal development of kidney organoids. Its absence caused a shift into the direction of fibrogenic cells. This study provides new insights into the understanding the role of (P)RR in human kidney development.

Identifying fibrogenic cells following salivary gland obstructive injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen significantly increased at 14days post-ligation. Macrophages, which participate in extracellular matrix remodeling, and Gli1+ and PDGFRα+ stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, Gli1 + cells were not found in discrete clusters at embryonic day 16 but were found in clusters expressing the stromal genes Pdgfra and/or Pdgfrb. In adult mice, Gli1+ cells were similarly heterogeneous but more cells co-expressed PDGFRα and PDGFRβ. Using Gli1-CreERT2; ROSA26tdTomato lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato+ cells expressed vimentin and PDGFRβ following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRβ, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1+ cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Some Pdgfra + /Pdgfrb + stromal cell subpopulations expanded in response to ligation, with two stromal cell subpopulations showing increased expression of Col1a1 and a greater diversity of matrisome genes, consistent with these cells being fibrogenic. However, only a few cells in these subpopulations expressed Gli1, consistent with a minor contribution of these cells to extracellular matrix production. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

An inducible model for genetic manipulation and fate-tracing of PDGFRβ-expressing fibrogenic cells in the liver

Myofibroblasts are the source of extracellular matrix protein during liver fibrogenesis. Fibroblasts, hepatic stellate cells (HSCs) and vascular smooth muscle cells are mesenchymal subpopulations in the liver that are characterized by the expression of PDGFRβ and contribute to the pool of these myofibroblasts. Conditional knockout models are important to better understand the function of specific liver cell populations including mesenchymal cells. While there is a limited number of constitutive mouse models for liver mesenchymal cell specific transgene expression, there is no established model for inducible gene targeting in HSCs or PDGFRβ-expressing mesenchymal cell populations in the liver. To address this, we investigated whether the tamoxifen inducible PDGFRβ-P2A-CreERT2 mouse can be used as a reliable tool to specifically express transgens in liver mesenchymal cells. Our data demonstrate, that PDGFRβ-P2A-CreERT2 specifically and efficiently marks over 90% of retinoid positive HSCs in healthy and fibrotic liver in mice upon tamoxifen injection, and that those cells give rise to Col1a1-expressing myofibroblasts in different models of liver fibrosis. Together with a negligible background recombination of only about 0.33%, this confirms that the PDGFRβ-P2A-CreERT2 mouse is nearly as efficient as established constitutive LratCre and PDGFRβ-Cre mouse models for recombination in HSCs, and that it is a powerful model for mesenchymal liver cell studies that require an inducible Cre approach.

Stellate cell in hepatic inflammation and acute injury.

The perisinusoidal hepatic stellate cells (HSCs) have been investigated extensively for their role as the major fibrogenic cells during chronic liver injury. HSCs also produce numerous cytokines, chemokines, and growth mediators, and express cell adhesion molecules constitutively and in response to stimulants such as endotoxin (lipopolysaccharide). With this property and by interacting with resident and recruited immune and inflammatory cells, HSCs regulate hepatic immune homeostasis, inflammation, and acute injury. Indeed, experiments with HSC-depleted animal models and cocultures have provided evidence for the prominent role of HSCs in the initiation and progression of inflammation and acute liver damage due to various toxic agents. Thus HSCs and/or mediators derived thereof during acute liver damage may be considered as potential therapeutic targets.

CD14 is not required for carbon tetrachloride-induced hepatic inflammation and fibrosis with or without lipopolysaccharide challenge.

Binding of lipopolysaccharide (LPS) to CD14 is required for its cellular effects via TLR4. A role of LPS/TLR4-mediated signaling in activated hepatic stellate cells (aHSCs), the major fibrogenic cells, in liver fibrosis has been reported. We investigated effects of LPS on carbon tetrachloride (CCl4)-induced fibrosis in CD14-knockout (KO) mice in vivo, and culture-activated HSCs in vitro. CCl4(biweekly; 4 weeks)-treated wild type (WT) and CD14-KO mice were challenged with single LPS administration for 24 h. Liver injury, inflammation and fibrosis were determined. Culture-activated HSCs from WT or CD14-KO mice were stimulated with LPS. Parameters of fibrogenic activity (expression of collagen1a1 [Col1a1], α-smooth muscle actin [αSMA] and TGFβ1) and inflammatory cytokines/chemokines were measured. CCl4 treatment caused similar liver injury and fibrosis in WT and CD14-KO mice. LPS increased liver injury and inflammation similarly in CCl4-treated WT and CD14-KO mice, but downregulated Timp1 and upregulated Mmp13. LPS elicited similar NFκB activation and inflammatory response in WT and CD14-KO aHSCs. LPS similarly downregulated Acta2 (encodes αSMA), Pdgfrb, Col1a1 and Mmp13 expression but did not affect Timp1 expression in WT and CD14-KO aHSCs. LPS did not alter Tgfb1 but increased expression of decorin (Dcn) (inhibitor of TGFβ1) expression in WT and CD14-KO aHSCs. The results indicate that the effects of LPS on HSCs are CD14-independent, and CD14 is not required for hepatic fibrosis. LPS-induced down-modulation of fibrogenic markers in aHSCs is also CD14-independent.

IL-8 concurrently promotes idiopathic pulmonary fibrosis mesenchymal progenitor cell senescence and PD-L1 expression enabling escape from immune cell surveillance.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We discovered fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that display cell-autonomous fibrogenicity and drive fibrotic progression. In a study of the IPF MPC nuclear proteome, we identified DNA damage as one of the most altered functions in IPF MPCs. In prior work we found that IL-8 drives IPF MPC self-renewal. IL-8 can promote replicative stress and DNA damage and induce senescence through the CXCR2 receptor. We hypothesized that IL-8 promotes DNA damage-mediated senescence in IPF MPCs. We show that IL-8 induces DNA damage and promotes IPF MPC senescence. We discovered that IL-8 concurrently promotes senescence and upregulation of the programmed death ligand 1 (PD-L1) in a CXCR2-dependent manner. Disruption of programmed cell death protein-1 (PD-1)-PD-L1 interaction promotes natural killer (NK) cell killing of IPF MPCs in vitro and arrests IPF MPC-mediated experimental lung fibrosis in vivo. Immunohistochemical (IHC) analysis of IPF lung tissue identified PD-L1-expressing IPF MPCs codistributing with NK cells and β-galactosidase-positive cells. Our data indicate that IL-8 simultaneously promotes IPF MPC DNA damage-induced senescence and high PD-L1 expression, enabling IPF MPCs to elude immune cell-targeted removal. Disruption of PD-1-PD-L1 interaction may limit IPF MPC-mediated fibrotic progression.NEW & NOTEWORTHY Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway.

Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.

Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update.

In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.