scholarly journals TFAM-deficient mouse skin fibroblasts: an ex vivo model of mitochondrial dysfunction

2021 ◽  
Author(s):  
Manuel J. Del Rey ◽  
Carolina Meroño ◽  
Cristina Municio ◽  
Alicia Usategui ◽  
María Mittelbrunn ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Ex Vivo ◽  
Stress Test ◽  
Critical Role ◽  
Substantial Reduction ◽  
Mouse Skin ◽  
Cell Types ◽  
Ex Vivo Model ◽  
Primary Mouse ◽  
Inflammatory Phenotype

Mitochondrial dysfunction in different cell types is associated to several pathological processes and potentially contributes to chronic inflammatory and ageing-related diseases. Mitochondrial Transcription Factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of the Tfamfl/fl UBC-Cre/ERT2+/+ mice, we sought to develop a cellular in vitro system to investigate the role of mitochondrial dysfunction in the stromal cell component. We describe an inducible model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblast (SK-FB) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction of Tfam and mtDNA-encoded mRNA expression in Cre(+) cultured for low (LP) and high passages (HP). Ultimately, Tfam knockout translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction of the mitochondrial respiratory chain (MRC) complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT Cre(+) SK-FB. Functionally, we determined the mitochondrial function and the glycolytic activity by mito-stress and glycolysis-stress test respectively. These analysis showed that mitochondrial dysfunction was developed after long-term 4-OHT treatment in HP Cre(+) SK-FB and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FB showed a senescent and pro-inflammatory phenotype. In conclusion, we have generated and validated the first ex vivo model of fibroblast mitochondrial dysfunction that results in a pro-inflammatory phenotype applicable to explore this process in other cell types in a variety of pathological conditions.

scholarly journals Bile Acids Activate NLRP3 Inflammasome, Promoting Murine Liver Inflammation or Fibrosis in a Cell Type-Specific Manner

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2618
Author(s):  
Theresa Maria Holtmann ◽  
Maria Eugenia Inzaugarat ◽  
Jana Knorr ◽  
Lukas Geisler ◽  
Marten Schulz ◽  
...  
Keyword(s):  
Bile Acids ◽  
Liver Damage ◽  
Nlrp3 Inflammasome ◽  
Lithocholic Acid ◽  
Ex Vivo ◽  
Cell Types ◽  
Inflammasome Activation ◽  
Inflammatory Phenotype ◽  
Murine Liver ◽  
A Cell

Bile acids (BA) as important signaling molecules are considered crucial in development of cholestatic liver injury, but there is limited understanding on the involved cell types and signaling pathways. The aim of this study was to evaluate the inflammatory and fibrotic potential of key BA and the role of distinct liver cell subsets focusing on the NLRP3 inflammasome. C57BL/6 wild-type (WT) and Nlrp3−/− mice were fed with a diet supplemented with cholic (CA), deoxycholic (DCA) or lithocholic acid (LCA) for 7 days. Additionally, primary hepatocytes, Kupffer cells (KC) and hepatic stellate cells (HSC) from WT and Nlrp3−/− mice were stimulated with aforementioned BA ex vivo. LCA feeding led to strong liver damage and activation of NLRP3 inflammasome. Ex vivo KC were the most affected cells by LCA, resulting in a pro-inflammatory phenotype. Liver damage and primary KC activation was both ameliorated in Nlrp3-deficient mice or cells. DCA feeding induced fibrotic alterations. Primary HSC upregulated the NLRP3 inflammasome and early fibrotic markers when stimulated with DCA, but not LCA. Pro-fibrogenic signals in liver and primary HSC were attenuated in Nlrp3−/− mice or cells. The data shows that distinct BA induce NLRP3 inflammasome activation in HSC or KC, promoting fibrosis or inflammation.

scholarly journals Interleukin-6 actions in the hypothalamus protects against obesity and is involved in the regulation of neurogenesis

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Vanessa C. Bobbo ◽  
Daiane F. Engel ◽  
Carlos Poblete Jara ◽  
Natalia F. Mendes ◽  
Roberta Haddad-Tovolli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Interleukin 6 ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Labeling ◽  
Specific Cell ◽  
Related Gene ◽  
Sequencing Data ◽  
Ex Vivo Model ◽  
Diet Induced Obesity

Abstract Background Interleukin-6 (IL6) produced in the context of exercise acts in the hypothalamus reducing obesity-associated inflammation and restoring the control of food intake and energy expenditure. In the hippocampus, some of the beneficial actions of IL6 are attributed to its neurogenesis-inducing properties. However, in the hypothalamus, the putative neurogenic actions of IL6 have never been explored, and its potential to balance energy intake can be an approach to prevent or attenuate obesity. Methods Wild-type (WT) and IL6 knockout (KO) mice were employed to study the capacity of IL6 to induce neurogenesis. We used cell labeling with Bromodeoxyuridine (BrdU), immunofluorescence, and real-time PCR to determine the expression of markers of neurogenesis and neurotransmitters. We prepared hypothalamic neuroprogenitor cells from KO that were treated with IL6 in order to provide an ex vivo model to further characterizing the neurogenic actions of IL6 through differentiation assays. In addition, we analyzed single-cell RNA sequencing data and determined the expression of IL6 and IL6 receptor in specific cell types of the murine hypothalamus. Results IL6 expression in the hypothalamus is low and restricted to microglia and tanycytes, whereas IL6 receptor is expressed in microglia, ependymocytes, endothelial cells, and astrocytes. Exogenous IL6 reduces diet-induced obesity. In outbred mice, obesity-resistance is accompanied by increased expression of IL6 in the hypothalamus. IL6 induces neurogenesis-related gene expression in the hypothalamus and in neuroprogenitor cells, both from WT as well as from KO mice. Conclusion IL6 induces neurogenesis-related gene expression in the hypothalamus of WT mice. In KO mice, the neurogenic actions of IL6 are preserved; however, the appearance of new fully differentiated proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons is either delayed or disturbed.

scholarly journals Runx3 prevents spontaneous colitis by directing differentiation of anti-inflammatory mononuclear phagocytes

2019 ◽  
Author(s):  
Shay Hantisteanu ◽  
Joseph Dicken ◽  
Varda Negreanu ◽  
Dalia Goldenberg ◽  
Ori Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
Early Onset ◽  
Target Genes ◽  
Critical Role ◽  
Substantial Reduction ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Inflammatory Genes ◽  
A Genome ◽  
Anti Inflammatory

ABSTRACTRUNX3 is one of three mammalian Runt-domain transcription factors (TFs) that regulate gene expression in several types of immune cells. Runx3-deficiency in mice is associated with a multitude of defects in the adaptive and innate immunity systems, including the development of early onset colitis. Our study reveals that conditional deletion of Runx3 specifically in mononuclear phagocytes (MNP) (MNPRunx3−/−) but not in T cells, recapitulates the early onset spontaneous colitis seen in Runx3−/− mice.We show that Runx3 is expressed in colonic MNP, including resident macrophages (RM) and the dendritic cell cDC2 subsets and its loss results in impaired differentiation/maturation of both cell types. At the transcriptome level, loss of Runx3 in RM and cDC2 was associated with upregulation of pro-inflammatory genes similar to those in the early onset IBD murine model of RMIl10r−/−. The impaired RM maturation in the absence of Runx3 was associated with a marked decrease in expression of anti-inflammatory and TGFβ-regulated genes. Similarly, the decreased expression of β-catenin signaling associated genes in Runx3-deficient cDC2 indicates their impaired differentiation/maturation. Analysis of ChIP-seq data suggests that in both MNP cell types a significant fraction of these differentially expressed genes are high confidence Runx3 directly regulated genes. Interestingly, several of these putative Runx3 target genes harbor SNPs associated with IBD susceptibility in humans. Remarkably, the impaired maturation and pro-inflammatory phenotype of MNP lacking Runx3 was associated with a substantial reduction in the prevalence of colonic lamina propria Foxp3+ regulatory T cells and an increase in IFNγ-producing CD4+ T cells, underscoring Runx3 critical role in establishing tolerogenic MNP.Together, these data emphasize the dual role of Runx3 in colonic MNP, as a transcriptional repressor of pro-inflammatory genes and an activator of maturation-associated genes including anti-inflammatory genes. Our study highlights the significance of the current MNPRunx3−/− model for understanding of human MNP-associated colitis. It provides new insights into the crucial involvement of Runx3 in intestinal immune tolerance by regulating colonic MNP maturation through TGFβR signaling and anti-inflammatory functions by Il10R signaling, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans including gastrointestinal tract diseases such as celiac and Crohn’s disease.

scholarly journals miRNA Profiling in Human Precision-cut Lung Slices (PCLS))

2021 ◽  
Author(s):  
Monika Niehof ◽  
Stella Reamon-Buettner ◽  
Olga Danow ◽  
Tanja Hansen ◽  
Katherina Sewald
Keyword(s):  
Magnetic Beads ◽  
Ex Vivo ◽  
Rna Extraction ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Published Data ◽  
Ex Vivo Model ◽  
Technical Limitation ◽  
Modified Method ◽  
Precision Cut Lung Slices

Abstract ObjectiveHuman precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA 29 isolation from PCLS to enable miRNA readouts. Results The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human 38 PCLS model.

Development and characterization of a novel meniscal extracellular matrix-derived scaffold

2017 ◽  
Author(s):  
◽  
Farrah Ann Monibi
Keyword(s):  
Extracellular Matrix ◽  
Tissue Repair ◽  
Cellular Proliferation ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Critical Role ◽  
Meniscal Repair ◽  
Biomechanical Properties ◽  
Ex Vivo Model

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Musculoskeletal injuries are a common and significant problem in orthopaedic practice. Despite advances in orthopaedic surgery, effective treatments for injuries to the knee meniscus remain a common and significant clinical challenge. Tissue engineering is a developing field that aims to regenerate injured tissues with a combination of cells, scaffolds, and signals. Many natural and synthetic scaffold materials have been developed and tested for the repair and restoration of a number of musculoskeletal tissues. Among these, biological scaffolds derived from extracellular matrix (ECM) have been developed and tested given the critical role of the ECM for maintaining the biological and biomechanical properties, structure, and function of native tissues. Decellularized scaffolds composed of ECM hold promise for repair and regeneration of the meniscus given the potential for ECM-based biomaterials to aid in cell recruitment, infiltration, and differentiation. The objectives of this research were to decellularize canine menisci in order to fabricate a micronized, ECM-derived scaffold, and to determine the cytocompatibility and repair potential of the scaffold ex vivo by developing an in vitro model for meniscal repair. In the first series of experiments, menisci were decellularized with a combination of physical agitation and chemical treatments. For scaffold fabrication, decellularized menisci were cryoground into a powder and the size and morphology of the ECM particles were evaluated using scanning electron microscopy. Histologic and biochemical analyses of the scaffold confirmed effective decellularization with loss of proteoglycan from the tissue but no significant reduction in collagen content. When washed effectively, the decellularized scaffold was cytocompatible to meniscal fibrochondrocytes, synoviocytes, and whole meniscal tissue based on the resazurin reduction assay, fluorescent live/dead staining, and histologic evaluation. Further, the scaffold supported cellular attachment and proliferation when combined with platelet rich plasma, and promoted an upregulation of genes associated with meniscal ECM synthesis and tissue repair. In an ex vivo model for meniscal repair, radial tears repaired and augmented with the scaffold demonstrated increased cellular proliferation and tissue repair compared to non-augmented repairs. Therefore, a micronized scaffold derived from decellularized meniscus may be a viable biomaterial for promoting avascular meniscal healing. However, further studies are necessary to determine an optimal carrier for delivery of the scaffold, and to examine the potential for the scaffold to induce cellular differentiation and functional meniscal fibrochondrogenesis.

scholarly journals Expression of α2-macroglobulin by the interaction between hepatocytes and endothelial cells in coculture

1998 ◽  
Vol 275 (1) ◽  
pp. R203-R211
Author(s):  
Mark A. Talamini ◽  
Michael P. McCluskey ◽  
Timothy G. Buchman ◽  
Antonio De Maio
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Apparent Molecular Weight ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Hepatic Function ◽  
Cellular Interactions ◽  
Ex Vivo Model ◽  
Extracellular Medium ◽  
Isolated Rat Hepatocytes

The interaction between distinct cell types within the liver seems to be important in regulating hepatic function. However, these interactions have not been well characterized because of difficulty in reproducing the hepatic environment in an ex vivo model. In the present study a coculture system of hepatocytes and endothelial cells was established to investigate the communication between parenchymal and nonparenchymal cells. Freshly isolated rat hepatocytes were placed onto a monolayer of primary aortic rat endothelial cells. Analysis of the proteins secreted into the extracellular medium after pulse labeling with radioactive amino acids revealed the presence of a 180,000-apparent molecular weight glycoprotein, BBB-180, which was not detected in the extracellular medium of hepatocytes or endothelial cells when they were cultured separately. This glycoprotein was identified as α2-macroglobulin after sequencing of the proteolytic peptides derived from the purified protein. This finding was confirmed by Northern and Western blotting, immunoprecipitation, and RT-PCR. The expression of α2-macroglobulin required direct contact between hepatocytes and viable endothelial cells. These findings suggest that endothelial cells modulate hepatocyte gene expression by direct cellular interactions.

scholarly journals Preclinical and clinical characterization of the RORγt inhibitor JNJ-61803534

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaohua Xue ◽  
Aimee De Leon-Tabaldo ◽  
Rosa Luna-Roman ◽  
Glenda Castro ◽  
Michael Albers ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Phase 1 ◽  
Critical Role ◽  
Mouse Skin ◽  
Orphan Receptor ◽  
Double Blind Study ◽  
Healthy Human ◽  
Adaptive Immune Cells ◽  
Dose Dependent

AbstractThe nuclear receptor retinoid-related orphan receptor gamma t (RORγt) plays a critical role in driving Th17 cell differentiation and expansion, as well as IL-17 production in innate and adaptive immune cells. The IL-23/IL-17 axis is implicated in several autoimmune and inflammatory diseases, and biologics targeting IL-23 and IL-17 have shown significant clinical efficacy in treating psoriasis and psoriatic arthritis. JNJ-61803534 is a potent RORγt inverse agonist, selectively inhibiting RORγt-driven transcription versus closely-related family members, RORα and RORβ. JNJ-61803534 inhibited IL-17A production in human CD4+ T cells under Th17 differentiation conditions, but did not inhibit IFNγ production under Th1 differentiation conditions, and had no impact on in vitro differentiation of regulatory T cells (Treg), nor on the suppressive activity of natural Tregs. In the mouse collagen-induced arthritis model, JNJ-61803534 dose-dependently attenuated inflammation, achieving ~ 90% maximum inhibition of clinical score. JNJ-61803534 significantly inhibited disease score in the imiquimod-induced mouse skin inflammation model, and dose-dependently inhibited the expression of RORγt-regulated genes, including IL-17A, IL-17F, IL-22 and IL-23R. Preclinical 1-month toxicity studies in rats and dogs identified doses that were well tolerated supporting progression into first-in-human studies. An oral formulation of JNJ-61803534 was studied in a phase 1 randomized double-blind study in healthy human volunteers to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated in single ascending doses (SAD) up to 200 mg, and exhibited dose-dependent increases in exposure upon oral dosing, with a plasma half-life of 164 to 170 h. In addition, dose-dependent inhibition of ex vivo stimulated IL-17A production in whole blood was observed, demonstrating in vivo target engagement. In conclusion, JNJ-61803534 is a potent and selective RORγt inhibitor that exhibited acceptable preclinical safety and efficacy, as well as an acceptable safety profile in a healthy volunteer SAD study, with clear evidence of a pharmacodynamic effect in humans.

Thrombin and protease-activated receptors (PARs) in atherothrombosis

2008 ◽  
Vol 99 (02) ◽  
pp. 305-315 ◽  
Author(s):  
Lluis Martorell ◽  
Cristina Rodríguez ◽  
Maurizio Gentile ◽  
Olivier Calvayrac ◽  
Lina Badimon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Platelets ◽  
Critical Role ◽  
Secretory Activity ◽  
Bleeding Risk ◽  
Cell Types ◽  
Protease Activated Receptors ◽  
Cellular Effects ◽  
Mediated Effects ◽  
Inflammatory Phenotype

SummaryThrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes,T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extra-cellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation inVSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overallTRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.

scholarly journals Localization of IL-1α Responsive Dermal Populations in an Ex Vivo Human Model of Wound Healing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrej Sikoski ◽  
Krish Jayapranu ◽  
Hong-Ming Zhou ◽  
Yunglong Liu ◽  
Xiaoling Xuei ◽  
...  
Keyword(s):  
Wound Healing ◽  
Human Skin ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Critical Role ◽  
Mouse Skin ◽  
Human Model ◽  
Cell Populations ◽  
Skin Culture ◽  
Inflammatory Phase

While playing a critical role in skin wound healing, the inflammatory phase of this process is poorly understood. To gain a better understanding of the inflammatory phase of wound healing, we developed an ex vivo skin culture model of skin injury-induced inflammation. Previous work in our laboratory showed ex vivo culture of human skin induces an interleukin 1 alpha (IL-1α)-dependent response characterized by increased transcript and protein levels for the inflammatory cytokines/chemokines, IL-6, CXCL1, and CSF3. However, the cellular sources of these factors in ex vivo cultured human skin have not been determined. Prior work with ex vivo cultured mouse skin and single cell RNA sequencing suggested fibroblasts and endothelial cells were potential cellular sources for these inflammatory mediators. The current studies used spatial transcriptomics analysis of ex vivo cultured human skin to localize the IL-1α target cell populations/skin tissue regions that produce IL-6, CXCL1 and CSF3. The Visium Gene Expression Solution platform (10x Genomics Inc.) was used to generate spatial transcriptomics data from skin specimens preserved immediately after biopsy or after skin culture for 24 hours. Loupe Browser version 5.1.0 (10x Genomics Inc) was used for data analysis to identify and characterize cell populations/regions expressing IL6, CXCL1, and CSF3 and associated differentially expressed genes (including cell type-specific transcripts). Notably, these IL-1α-induced transcripts were localized to the parent dermis region cluster. Analysis of subclusters in the dermal region showed differential expression of these inflammatory transcripts in regions enriched with either or both fibroblast and endothelial cell specific-type markers. Potential novel markers of this inflammatory response, like SOD2, were identified and warrant future investigation. Subsequent studies in identifying the targets of IL-1α in skin inflammation is called for, as they may lead to better understanding of this processes in wound healing and better clinical outcomes.

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