Infusion of Kupffer Cells Expanded in Vitro Ameliorated Liver Fibrosis in a Murine Model of Liver Injury.
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ABSTRACT:
SUBMITTER: Li W
PROVIDER: S-EPMC8020097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Activation of myofibroblasts (MF) and extracellular matrix (ECM) deposition predispose the expansion and differentiation of liver progenitor cells (LPC) during chronic liver injury. Because Kupffer cells (KC) are active modulators of tissue response and fibrosis, we analyzed their role in a model of LPC proliferation. A choline-deficient diet, supplemented by ethionine (CDE) was administrated to C57Bl/6J mice that were depleted of KC by repeated injections of clodronate (CLO) and compared to PBS-injected mice. On CDE, massive KC activation was observed in the PBS group, but this was blunted in CLO-treated mice. The depletion of KC did not influence LPC proliferation but reduced their invasive behavior. Instead of being found far into the parenchyma, as was found in the PBS group (mean distance from portal vein: 209 ?m), LPC of CLO mice remained closer to the portal area (138 ?m), forming aggregates and phenotypically resembling cells of biliary lineage. Notably, removal of KC was also associated with a significant decrease in amount of MF and ECM and in the expression of profibrotic factors. Thus, besides ECM and MF, KC are also a significant component of the microenvironmental changes preceding LPC expansion. Depletion of KC may limit the LPC parenchymal invasion through a deficiency in chemoattracting factors, reduced activation of MF, and/or a paucity of the ECM framework necessary for cell motility.
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Project description:Chronic liver injury can be caused by viral hepatitis, alcohol, obesity, and metabolic disorders resulting in fibrosis, hepatic scarring, and cirrhosis. Novel therapies are urgently required and previous work has demonstrated that treatment with bone marrow derived macrophages can improve liver regeneration and reduce fibrosis in a murine model of hepatic injury and fibrosis. Here, we describe a protocol whereby pure populations of therapeutic macrophages can be produced in vitro from murine embryonic stem cells on a large scale. Embryonic stem cell derived macrophages display comparable morphology and cell surface markers to bone marrow derived macrophages but our novel imaging technique revealed that their phagocytic index was significantly lower. Differences were also observed in their response to classical induction protocols with embryonic stem cell derived macrophages having a reduced response to lipopolysaccharide and interferon gamma and an enhanced response to IL4 compared to bone marrow derived macrophages. When their therapeutic potential was assessed in a murine, carbon tetrachloride-induced injury and fibrosis model, embryonic stem cell derived macrophages significantly reduced the amount of hepatic fibrosis to 50% of controls, down-regulated the number of fibrogenic myofibroblasts and activated liver progenitor cells. To our knowledge, this is the first study that demonstrates a therapeutic effect of macrophages derived in vitro from pluripotent stem cells in a model of liver injury. We also found that embryonic stem cell derived macrophages repopulated the Kupffer cell compartment of clodronate-treated mice more efficiently than bone marrow derived macrophages, and expressed comparatively lower levels of Myb and Ccr2, indicating that their phenotype is more comparable to tissue-resident rather than monocyte-derived macrophages.
| S-EPMC5677947 | biostudies-literature
Project description:Kupffer cells (KCs) contribute to liver fibrosis resolution by production of a large spectrum of matrix metalloproteinases (MMPs). MMP9 is a major MMP expressed by KCs. However, its role in liver fibrosis resolution remains unclear. In this study, rodent liver fibrosis was induced by intraperitoneal thioacetamide (TAA) and the resolution process was initiated by TAA withdrawal. The role of KC-derived MMP9 in fibrolysis was investigated by adoptive transfer of KCs with or without MMP9 following their depletion. The levels of serum alanine aminotransferase (ALT) and hepatic cytokines were measured during fibrosis regression. The mRNA levels of MMPs and tissue inhibitor of metalloproteinases (TIMPs) were analyzed as well. It was found that removing KCs delayed fibrosis resolution. Adoptive transfer of KCs from WT animals promoted liver fibrosis resolution, compared with transfer of KCs from MMP9-/- mice. Depletion of KCs also resulted in prolonged liver wound healing, which was reversed partially by transferred KCs from either WT or MMP9-/- mice. Likewise, the absence of KCs led to reduction in MMPs mRNA levels and elevation in TIMPs mRNA levels. The expression patterns of MMPs or TIMPs were restored by adoptive transfer of the wild-type but not MMP9-/- KCs. In addition, liver fibrosis resolution was accelerated in MMP9-/- mice by adoptive transferred KCs from WT animals, compared to the KCs from MMP9-/- mice. Overall, KC-derived MMP9 plays a critical role in fibrosis resolution, which might serve as the foundation for developing anti-fibrosis therapy.
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Project description:The fate of resolution of liver fibrosis after withdrawal of liver injury is still incompletely elucidated. Toll-like receptor 4 (TLR4) in tissue fibroblasts is pro-fibrogenic. After withdrawal of liver injury, we unexpectedly observed a significant delay of fibrosis resolution as TLR4 signaling was pharmacologically inhibited in vivo in two murine models. Single-cell transcriptome analysis of hepatic CD11b+ cells, main producers of matrix metalloproteinases (MMPs), revealed a prominent cluster of restorative Tlr4-expressing Ly6c2-low myeloid cells. Delayed resolution after gut sterilization suggested its microbiome-dependent nature. Enrichment of a metabolic pathway linking to a significant increase of bile salt hydrolase-possessing family Erysipelotrichaceae during resolution. Farnesoid X receptor-stimulating secondary bile acids including 7-oxo-lithocholic acids upregulated MMP12 and TLR4 in myeloid cells in vitro. Fecal material transplant in germ-free mice confirmed phenotypical correlations in vivo. These findings highlight a pro-fibrolytic role of myeloid TLR4 signaling after injury withdrawal and may provide targets for anti-fibrotic therapy.
| S-EPMC9982274 | biostudies-literature
Project description:BackgroundIn vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators.MethodsPrimary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor).ResultsCF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661.ConclusionsCF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.
| S-EPMC7796805 | biostudies-literature
Project description:Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2-/-) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC-/- mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC-/- in Mdr2-/- mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2-/- mice, and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-β1, and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-β1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-β1, and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression.
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Project description:Alzheimer's disease (AD) is characterized by the deposition of amyloid-? (A?) in brain parenchyma and cerebral blood vessels as cerebral amyloid angiopathy (CAA). Clusterin, a chaperon protein associated with A? aggregation, toxicity and transport through blood-brain barrier, may play a key role in the development of AD. Recently, clusterin peptide D-[113-122] was shown to mimic clusterin's function and exerted therapeutic effect in atherosclerosis. In this study, we investigated whether this clusterin peptide also affected (A?) deposition in AD transgenic mouse.Using a micropump, synthetic peptide 113-122 of clusterin protein (20 ?g/200 ?l) was infused into the lateral ventricle of 8-month 5 × FAD transgenic mouse model (Tg6799), for 2 weeks. Water-maze testing showed an improved cognitive function of the Tg6799 mice treated with clusterin. Immunocytochemistry and quantitative analysis revealed that intraventricular (icv) administration of clusterin peptide in Tg6799 mouse reduced A? plaques as well the severity of cerebral amyloid angiopathy. Enzyme-linked immunosorbent assay demonstrated a decreased in the soluble levels of A? (A?40 and A?42) in the brain. Western-blot revealed an increased level of LRP-2 after clusterin peptide treatment.These results suggest that icv infusion of clusterin peptide D-[113-122] offers a promising therapeutic approach to reduce A? deposition as well as CAA. The LRP2-mediated clearance system might be involved in the mechanism of these effects.
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Project description:Parenteral nutrition-associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)-based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD.
| S-EPMC4070735 | biostudies-literature
Project description:Background and aimsBiliary atresia (BA) is a devastating neonatal cholangiopathy that progresses to fibrosis and end-stage liver disease by 2 years of age. Portoenterostomy may reestablish biliary drainage, but, despite drainage, virtually all afflicted patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival.Approach and resultsIn the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling human BA and has been used to study mechanistic aspects of the disease. Unfortunately, nearly all RRV-infected pups succumb by day of life 14. Thus, in this study we generated an RRV-TUCH rotavirus reassortant (designated as TR(VP2,VP4) ) that when injected into newborn mice causes an obstructive jaundice phenotype with lower mortality rates. Of the mice that survived, 63% developed Ishak stage 3-5 fibrosis with histopathological signs of inflammation/fibrosis and bile duct obstruction.ConclusionsThis model of rotavirus-induced neonatal fibrosis will provide an opportunity to study disease pathogenesis and has potential to be used in preclinical studies with an objective to identify therapeutic targets that may alter the course of BA.
| S-EPMC7384231 | biostudies-literature
Project description:BackgroundWe previously reported regenerative therapies for decompensated cirrhosis based on peripheral venous drip infusion using non-cultured whole bone marrow (BM) cells, or the less invasive cultured BM-derived mesenchymal stem cells (BMSCs). Here, we assessed the efficacy and safety of hepatic arterial infusion using cultured autologous BMSCs, comparing it with peripheral infusion, using our established canine liver fibrosis model.MethodsCanine BM cells were harvested and cultured, and the resultant BMSCs were returned to carbon tetrachloride (CCl4)-induced liver cirrhosis model canines via either a peripheral vein (Vein group) or hepatic artery (Artery group). A variety of assays were performed before and 4, 8, and 12 weeks after BMSC infusion, and liver fibrosis and indocyanine green (ICG) half-life (t1/2) were compared to those in a control group that received CCl4 but not BMSCs. The safety of this approach was evaluated by contrast-enhanced computed tomography (CT) and serial blood examinations after infusion.ResultsFour weeks after infusing BMSCs, a significant improvement was observed in the Vein group (n = 8) compared to outcome in the Control group (n = 10), along with a decrease in ICG t1/2. In the Artery group (n = 4), ICG t1/2 was significantly shorter than that in the Vein group at 8 weeks (Δt1/2: -3.8 ± 1.7 min vs. +0.4 ± 2.4 min; p < 0.01) and 12 weeks (Δt1/2: -4.2 ± 1.7 min vs. +0.4 ± 2.7 min; p < 0.01) after BMSC administration. Post-infusion contrast-enhanced CT showed no liver infarction, and blood tests showed no elevations in either serum lactate dehydrogenase concentrations or hypercoagulability.ConclusionsWe confirmed the efficacy and safety of the hepatic arterial infusion of cultured autologous BMSCs using a canine model, thereby providing non-clinical proof-of-concept.
| S-EPMC6343920 | biostudies-literature