Disease Fibroblasts Research Articles

Regulation of pathologic fibroblast functions in digestive diseases: a role for hypoxia?

The recent uncovering of fibroblast heterogeneity has given great insight into the versatility of the stroma. Among other cellular processes, fibroblasts are now thought to contribute to the coordination of immune responses in a range of chronic inflammatory diseases and cancer. While the pathologic roles of myofibroblasts, inflammatory fibroblasts and cancer associated fibroblasts in disease are reasonably well understood, the mechanisms behind their activation remain to be uncovered. In the gastrointestinal (GI) tract, several interleukins and tumour necrosis factor superfamily members have been identified as possible mediators driving the acquisition of inflammatory as well as fibrotic properties in fibroblasts. In addition to cytokines, other microenvironmental factors such as nutrient and oxygen availability are likely contributors to this process. In this respect, the phenomenon of low cellular oxygen levels known as hypoxia is common in a plethora of GI diseases. Indeed, the crosstalk between hypoxia and inflammation is well-documented, with an abundance of studies suggesting that oxygen-sensing enzymes may have regulatory effects on inflammatory signalling pathways such as NF-κB. However, the impact that this has in GI fibroblasts in the context of chronic diseases has not been fully uncovered. Here we discuss the role of fibroblasts in GI diseases, the mediators that have emerged as regulators of their functions and the potential impact of hypoxia in this process, highlighting areas that require further investigation.

P0022 Development of primary human fibroblast cell cultures for fibrosis modelling in Inflammatory Bowel Diseases

Abstract Background Inflammatory Bowel Diseases (IBD) are chronic immune-mediated diseases of the gastrointestinal tract. Fibrosis is frequent in IBD driven by inflammatory cytokines. During fibrosis, extracellular matrix proteins accumulate in the tissue, causing loss of epithelial function and stenosis, leading to malabsorption and intestinal motility problems. This can ultimately lead to serious complications, strictures, fistulas, or ileus. Anti-inflammatory drugs cannot target fibrosis, only surgical intervention can be used. No relevant primary in vitro cell culture model is available for intestinal fibrosis modelling. Therefore, we aimed to establish and optimize human primary fibroblast cell culture from intestinal biopsies of control and IBD patients. We characterized and compared the fibrosis phenotype and gene expression as well. Methods Fibroblast cell cultures were generated from colonic biopsy samples by enzymatic digestion. In a suitable medium, the cells attach to the bottom of the vessel and proliferate, at 90% confluence the cultures were passaged. The phenotype was confirmed by immunofluorescent staining. Target proteins: COL1A1 (Collagen type 1 alpha 1), TGF-ß1 (Transforming growth factor beta), PAI-1 (Plasminogen activator inhibitor type 1), α-SMA (Alpha smooth muscle actin), and PHH3 (Phosphohistone H3). For gene expression analysis RNA was isolated and qRT-PCR technique was used for the following target genes: COL1A1, ACTA2 (Actin alpha 2), TGF-ß1, FN1 (Fibronectin 1), PAI-1, H3C4 (H3 clustered histone 4). We evaluated the collected data and performed statistical analysis. Results We can efficiently create and maintain human colon primary fibroblast cell cultures from colonic biopsies of healthy and CD origins. Cell morphology differences were observed from control and diseased patient samples. At the staining, the fluorescent intensity of fibrotic markers (COL1A1, TGF-ß1, PAI1) and the mitosis marker PHH3 were significantly higher in the CD samples, indicating that the cells maintained fibrotic phenotype, and the diseased fibroblasts had a higher proliferation rate. The myofibroblast marker, α-SMA didn’t show a significant difference. Interestingly, significant differences were not noted between the two groups in the qRT-PCR measurements. Conclusion In conclusion, we can generate primary human intestinal fibroblast cell cultures and maintain them to passage number 3. Fibrotic protein markers and morphology distinguish the diseased samples from the healthy subjects, therefore it could be useable for in vitro fibrosis modelling. However, the gene expression analysis doesn’t discriminate between the two groups at passage number 3. In the future, we would like to observe the fibrotic properties in earlier passage numbers.

P0047 Understanding the pathogenic role of inflammatory fibroblasts in Inflammatory Bowel Disease

Abstract Background TWEAK (TNF-like weak inducer of apoptosis), a member of the tumor necrosis factor superfamily, has been found to be upregulated in IBD biopsies and to promote chronic inflammation in in vivo murine models. Previous work by our group has identified that TWEAK represses the expression of homeostatic factors in colonic fibroblasts, and it promotes inflammatory fibroblast-monocyte crosstalk. The aim of this study was to translate in vitro observations into a clinical setting by investigating the presence of TWEAK in the healthy and inflamed gut and its correlation with inflammatory fibroblasts. Methods Fresh colonic biopsies were enzymatically digested and processed to extract single cells. Isolated cells were analysed by flow cytometry to characterise the expression and cellular source of TWEAK, as well as changes in structural cell compartments, including inflammatory polarisation in colonic fibroblasts. Flow cytometry was conducted on 31 colonic samples of patients with known IBD (inflamed n=11, noninflamed n=10, remission, n=10) as well as 4 healthy controls with no history of IBD Results We observed an increased number of TWEAK+ myeloid cells (CD45+/CD11b+/TWEAK+) in inflamed segments compared to non-involved sections, or to biopsies from healthy donors or patients in remission (Figure 1A). Similarly, inflamed biopsies showed an increase in CD90+/PDPN+ stromal cells, which is consistent with an expansion in inflammatory fibroblasts (Figure 1B), and highly correlates with the accumulation of TWEAK+ myeloid cells (Figure 1C). Conclusion The accumulation of TWEAK+ myeloid cells in inflamed tissues is concomitant with the expansion of the inflammatory stroma in ulcerative colitis, positioning TWEAK as a potential driver of inflammatory fibroblast polarisation. Understanding the effect of TWEAK in stromal remodelling is of key importance both to understanding intestinal physiology and to developing potential therapeutic strategies in diseases as diverse as inflammatory bowel disease.

Substrate stiffness dictates unique doxorubicin-induced senescence-associated secretory phenotypes and transcriptomic signatures in human pulmonary fibroblasts.

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-β-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2kPa), fibrotic (50kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21 + cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

Senescent lung fibroblasts in idiopathic pulmonary fibrosis facilitate non-small cell lung cancer progression by secreting exosomal MMP1.

Lung cancer is a fatal complication of idiopathic pulmonary fibrosis (IPF) with a poor prognosis. Current treatments are insufficient in improving the prognosis of lung cancer patients with comorbid idiopathic pulmonary fibrosis (IPF-LC). Senescent fibroblasts, as stromal cells in the tumor microenvironment, influence tumor progression via exosomes. With evidence that fibroblast senescence is an important mechanism of IPF, we investigated the impact of senescent IPF lung fibroblast (diseased human lung fibroblasts, DHLF)-derived exosomes on non-small cell lung cancer (NSCLC). We found DHLF expressed significant senescence markers, and promoted NSCLC proliferation, invasion, and epithelial-mesenchymal transition. Specifically, senescent DHLF showed strong secretion of exosomes, and these exosomes enhanced the proliferation and colony-forming ability of cancer cells. Proteomic analysis showed DHLF-derived exosomes exhibited upregulated senescence-associated secretory phenotype (SASP) factors, notably MMP1, which activates the surface receptor PAR1. Knocking down MMP1 or using PAR1 inhibitors reduced the tumor-promoting effects of DHLF-derived exosomes in vivo and in vitro. Mechanistically, MMP1 acted by activating the PI3K-AKT-mTOR pathway. In conclusion, our results suggest that exosomal MMP1 derived from senescent IPF fibroblasts promotes NSCLC proliferation and colony formation by targeting PAR1 and activating the PI3K-AKT-mTOR pathway. These findings provide a novel therapeutic approach for patients with IPF-LC.

Impact of Photoactivated Curcumin on Proliferation of Experimentally Induced Periodontitis and Diabetic Gingival Fibroblast Cell Line

ABSTRACT Objective: To evaluate the impact of photoactivated curcumin on proliferation of experimentally induced periodontitis and diabetic gingival fibroblast in laboratory settings. Materials and Methods: Gingival fibroblast (GF) cells were divided into healthy GF (HGF), diabetic GF (DGF), and periodontitis-associated diabetic GF (P-DGF) cells that were treated with a solution of curcumin that was prepared and diluted in autoclave distilled water to obtain a concentration used (1 mg/ml). Gingival fibroblasts with curcumin and without curcumin were seeded in a 96-well plate that was treated with a light-emitting diode curing light with a wavelength of 445 nm using a transparent diffuser tip. All healthy (HGF) as well as diseased (DGF and P-DGF) gingival fibroblasts were thus treated with curcumin (c-only), photoactivation (p-only), and both photoactivated curcumin (pc-both) and were then histologically analyzed for evaluation of cell proliferation and viability of fibroblasts. The number of proliferated cells/proliferative values were calculated by relative fluorescence values displayed by a fluorimeter. The number of viable cells (%viability) correlates with the magnitude of dye reduction and expressed as percentage of Alamar-Blue reduction. Results: HGF group treated with p-alone, c-alone, and pc-both showed a significant increase in the fluorescence value and proliferation in the cells at 24 to 48 and 48 to 72 h, whereas for the DGF group, there was a statistically significant increase in the fluorescence value and proliferation when treated with p-alone from 24 to 72 h, with a significant decrease in the proliferation of the cells when treated with c-alone and pc-both at 24 to 48 h and 48 to 72 h. For the P-DGF group, a significant decrease in the fluorescence value and proliferation was observed when the cells were treated with p-alone and c-alone at 24 to 72 h. Conclusion: Photoactivation (p-alone) was the most efficient in depicting a highly significant increase in the percentage viability for the group P-DGF and DGF at 24 h, whereas a significant increase in the fluorescence value and proliferation was observed for the group P-DGF when treated with photoactivated curcumin (pc-both) from 24 to 72 h.

Inhalation of itraconazole mitigates bleomycin-induced lung fibrosis via regulating SPP1 and C3 signaling pathway pivotal in the interaction between phagocytic macrophages and diseased fibroblasts

BackgroundIdiopathic pulmonary fibrosis (IPF) stands as a significant contributor to global mortality rates. Presently, there exists a dearth of effective anti-fibrotic treatments for this condition. While itraconazole (ITR) has exhibited potential in mitigating pulmonary fibrosis, its oral administration is hampered by unfavorable pharmacokinetics, which elevate the risk of adverse reactions, thus limiting its clinical utility.MethodsAn inhalable formulation of ITR were engineered which aimed at enhancing its pulmonary dispersion. First, pharmacokinetics were conducted to investigate the blood concentration and tissue residue of ITR after inhalation administration. In addition, bleomycin induced mouse pulmonary fibrosis model was used to compare the therapeutic effects of ITR administered by inhalation and intragastric administration. Finally, single-cell RNA sequencing (scRNAseq) was used to explore the mechanism of ITR inhalation administration.ResultsWe found that a large amount of drugs accumulated in the lung tissue for a long time after inhalation administration, thus maximizing the therapeutic effect of drugs. Inhalation of ITR daily at for 21 days significantly attenuated bleomycin-induced lung fibrosis and inflammation in murine models. Additionally, our findings revealed that ITR inhalation diminished the proportion of diseased fibroblasts while promoting reparative fibroblast populations in the murine model. Furthermore, it effectively reversed the proportion of activated phagocytic macrophages. Mechanistically, ITR inhalation exerted its effects by regulating SPP1 and C3 signaling pathway pivotal in the interaction between phagocytic macrophages and diseased fibroblasts.ConclusionsThese insights into the molecular mechanisms underlying ITR’s therapeutic effects on IPF underscore the favorable pharmacokinetic profile conferred by inhalation, thus presenting a promising formulation poised for clinical translation.

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts.

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-β-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

Mechanics-activated fibroblasts promote pulmonary group 2 innate lymphoid cell plasticity propelling silicosis progression

Crystalline silica (CS) particle exposure leads to silicosis which is characterized as progressive fibrosis. Fibroblasts are vital effector cells in fibrogenesis. Emerging studies have identified immune sentinel roles for fibroblasts in chronic disease, while their immune-modulatory roles in silicosis remain unclear. Herein, we show that group 2 innate lymphoid cell (ILC2) conversion to ILC1s is closely involved in silicosis progression, which is mediated by activated fibroblasts via interleukin (IL)−18. Mechanistically, Notch3 signaling in mechanics-activated fibroblasts modulates IL-18 production via caspase 1 activity. The mouse-specific Notch3 knockout in fibroblasts retards pulmonary fibrosis progression that is linked to attenuated ILC conversion. Our results indicate that activated fibroblasts in silicotic lungs are regulators of ILC2–ILC1 conversion, associated with silicosis progression via the Notch3–IL-18 signaling axis. This finding broadens our understanding of immune-modulatory mechanisms in silicosis, and indicates potential therapeutic targets for lung fibrotic diseases.

Reduced WNT4 expression in normal skin fibroblasts leads to ‘Dupuytren-like’ changes in the transcriptome

BackgroundDupuytren's disease (DD) is a fibro-proliferative disorder of unknown aetiology. Previous studies have implicated multiple WNT signalling genes/proteins in Dupuytren pathology, including WNT4. However, it is not yet clear whether WNT signalling dysregulation plays an important role in the initiation of the disease or progression. The aim of this study was to determine if loss of WNT4 expression triggered ‘Dupuytren-like’ changes in the transcriptome of healthy skin fibroblasts. MethodsFibroblasts were isolated from the wrists of healthy adult males and from the wrists and disease cord tissue from males in a family positive for Dupuytren's disease. Normal skin fibroblasts from healthy controls were treated with WNT4 siRNA and scrambled controls. RNASeq was used to analyse the transcriptomes of disease and non-disease fibroblasts from patients with Dupuytren's as well as in siRNA treated and non-treated control fibroblasts. ResultsAnalysis of the transcriptomes from DD patient and normal skin fibroblasts showed significant differences, including in WNT4 expression. Downregulation of WNT4 in normal skin fibroblasts using siRNA led to ‘DD-like’ changes in the transcriptome. ConclusionIn people susceptible to DD WNT4 is downregulated even in non-fibrotic fibroblasts. Knockdown of WNT4 in normal fibroblasts led to changes that made cells ‘DD-like’. This study shows that WNT4 is down regulated in ‘non-disease’ cells, and that downregulating WNT4 in normal skin fibroblasts leads to widespread ‘DD like’ changes in the transcriptome, suggesting WNT4 downregulation is a key driver of DD.

MSU crystallization promotes fibroblast proliferation and renal fibrosis in diabetic nephropathy via the ROS/SHP2/TGFβ pathway

Monosodium urate (MSU) crystallisation deposited in local tissues and organs induce inflammatory reactions, resulting in diseases such as gout. MSU has been recognized as a common and prevalent pathology in various clinical conditions. In this study, we investigated the role of MSU in the pathogenesis of diabetic kidney disease (DKD). We induced renal injury in diabetic kidney disease mice using streptozotocin (STZ) and assessed renal histopathological damage using Masson's trichrome staining and Collagen III immunofluorescence staining. We measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and uric acid (UA) using ELISA. Protein expression levels of NLRP3, p-NF-κB, SHP2, p-STAT3, and p-ERK1/2 were analyzed by Western blot. To further investigate the role of MSU in diabetic kidney disease, we conducted in vitro experiments. In our in vivo experiments, we found that compared to the Model group, there was a significant increase in interstitial fibrosis in the kidneys of mice after treatment with MSU, accompanied by elevated levels of MDA, SOD, and UA. Furthermore, the protein expression of NLRP3, p-NF-NB, SHP2, p-STAT3, and p-ERK1/2 was upregulated. In our subsequent studies on mouse fibroblasts (L929 cells), we discovered that high glucose, MSU, and TGF-β could promote the expression of P22, GP91, NLRP3, NF-κB, p-NF-κB, p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins. Additionally, we found that SHP2 could counteract the upregulation trend induced by MSU on the expression of p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins, and inhibitors YQ128, NAC, and Cetuximab exhibited similar effects. Furthermore, immunofluorescence results indicated that SHP2 could inhibit the expression of the fibrosis marker α-SMA in L929 cells. These findings suggest that MSU can promote renal fibroblast SHP2 expression, induce oxidative stress, activate the NLRP3/NF-κB pathway, and enhance diabetic kidney disease fibroblast proliferation through the TGFβ/STAT3/ERK1/2 signaling pathway, leading to renal fibrosis.

P099 Neutrophil extracellular traps enhance profibrotic activity of intestinal fibroblasts in Crohn's disease through TLR2/NF-kB pathway

Abstract Background Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD). Methods NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4fl/flMRP8Cre+) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development. Results We found spatial tissue distribution of clusters of NETs adjacent to FAP+ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p<0.0001), slower wound healing capability (p<0.0001) and higher collagen release in the medium (p<0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p<0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4fl/flMRP8Cre+ mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation. Conclusion NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

BackgroundLesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells.MethodsWe prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts.ResultsLND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND.ConclusionsA complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

THE EPIGENETIC LANDSCAPE IN SOFT-TISSUE FIBROSIS

Dupuytren's disease (DD) is a fibroproliferative soft tissue disease affecting the palmar fascia of the hand causing permanent and irreversible flexion contracture. Aberrant fibrosis is likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in soft tissue fibrosis in diseases such as DD is not well established. Therefore, we conducted a comprehensive multi-omic study investigating the epigenetic profiles that influence gene expression in DD pathology. Using control (patients undergoing carpal tunnel release) and diseased fibroblasts (patients undergoing Dupuytren's fasciectomy), we conducted ATAC-seq to assess differential chromatin accessibility between control and diseased fibroblasts. Additionally, ChIP-seq mapped common histone modifications (histone H4; H3K4me3, H3K9me3, H3K27me3, H4K16Ac, H4K20Me3) associated with fibrosis. Furthermore, we extracted RNA from control and DD tissue and performed bulk RNA-seq.ATAC-seq analysis identified 2470 accessible genomic loci significantly more accessible in diseased fibroblasts compared to control. Comparison between diseased and control cells identified numerous significantly different peaks in histone modifications (H4K20me3, H3K27me3, H3K9me3) associated with gene repression in control cells but not in diseased cells. Pathway analysis demonstrated a substantial overlap in genes being de-repressed across these histone modifications (Figure 1). Both, ATAC-seq and ChIP-seq analysis indicated pathways such as cell adhesion, differentiation, and extracellular matrix organisation were dysregulated as a result of epigenetic changes. Moreover, de novo motif enrichment analysis identified transcription factors that possibly contributed to the differential gene expression between control and diseased tissue, including HIC1, NFATC1 and TEAD2. RNA-seq analysis found that these transcription factors were upregulated in DD tissue compared to control tissue.The current epigenetic study provides insights into the aberrant fibrotic processes associated with soft tissue diseases such as DD and indicates that epigenetic-targeted therapies may be an interesting viable treatment option in future.For any figures or tables, please contact the authors directly.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3–4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air–liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

ERCC6L2 Disease and Shwachman-Diamond Syndrome Exhibit Alterations in Metabolomes in Patient Fibroblasts

Introduction Metabolomics is an emerging tool to study metabolic alterations in cancers. It has also the potential to identify clinically relevant biomarkers for diagnosis, risk appraisal, and targeted drug development. In hematology, metabolic reprogramming is essential for normal regulation of hematopoiesis. Association of discrete metabolic alterations with explicit somatic mutations have also been reported in hematological malignancies. We have previously observed glutaminase (GLS) overexpression and improved oxygen consumption rate with excess glutamine in low glucose conditions in patient-derived fibroblasts in ERCC6L2 disease (unpublished). This prompted us to study metabolomic alterations in ERCC6L2 disease and Shwachman-Diamond syndrome (SDS) induced strictly by the germline ERCC6L2 and SBDS mutations. Both inherited bone marrow failure (BMF) syndromes carry considerable tendency of somatic TP53 mutagenesis predisposing patients to a high risk myeloid malignancy. Our aim here was to identify potential drug targets by targeted metabolomics. Methods We examined patient-derived skin fibroblasts (ERCC6L2 disease, n=4; SDS, n=4) and healthy controls (n=4) and performed targeted metabolomics with U-13C-D-glucose and U-13C-L-glutamine labeling. Fibroblasts were seeded on six-well plates and on the following day exposed to 13C-glucose (25mM) or 13C-glutamine (2mM) label containing medium for 30 or 120 minutes. Next, metabolites were extracted and quantified with liquid chromatography mass spectrometer. The results were normalized to total metabolite intensity per sample. Results In 13C-glucose labeled samples, we detected statistically significantly lower intensity of pyruvate M+3 in both ERCC6L2 and SDS fibroblasts compared to control at 30 minutes, which persisted at 120 minutes in SDS cells. In the TCA cycle, malate M+3 labeling indicates pyruvate carboxylase (PC) activity and M+2 labeling pyruvate dehydrogenase activity (PDH) (Figure 1). We found a significantly higher portion of malate M+3 than M+2 in SDS cells at all time points, suggesting lower PDH and higher PC activity in SDS cells. Additionally, we detected higher levels of alanine M+3 in SDS samples. As pyruvate is a source for alanine synthesis, we suspect an alteration in pyruvate metabolism; potentially to overcome challenges in pyruvate oxidation due to either decrease in the respiratory chain function or PDH activity. In 13C-glutamine labeled samples, we detected statistically significantly higher intensity of glutamine M+5 and M+4 in ERCC6L2 samples at 30 minutes compared to control and SDS cells. Glutamine is first converted to glutamate by glutaminase (GLS), after which it enters the TCA cycle as ketoglutarate (KG) (Figure 1). In addition to glutamine, we detected an increase, although not statistically significant, in glutamate M+5 and M+4, as well as KG M+5, in ERCC6L2 cells at 30 minutes. A similar trend was observed in the subsequent TCA cycle intermediates succinic acid M+4, fumarate M+4, and malate M+4. At 120 minutes, there was no difference in metabolite intensities between the groups, suggesting a potentially faster glutamine uptake by ERCC6L2 cells. Conclusions Our analysis using non-hematopoietic tissue provides understanding of metabolic weak links not confounded by somatic events occurring early in the disease course in ERCC6L2 and SDS patients. Our findings imply increased glutamine utilization in ERCC6L2 cells and altered pyruvate metabolism in SDS cells. Therefore, these metabolic pathways are attractive drug targets (summarized in Figure 1). Further studies are needed to determine their potential in supporting the compromised hematopoiesis to reduce the stress for somatic mutagenesis.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential

BackgroundHuman mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype.MethodshiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer.ResultsReprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential.ConclusionOverall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

GLUT-1 changes in paediatric Huntington disease brain cortex and fibroblasts: an observational case-control study

Paediatric Huntington disease with highly expanded mutations (HE-PHD; >80 CAG repeats) presents atypically, compared to adult-onset Huntington disease (AOHD), with neurodevelopmental delay, epilepsy, abnormal brain glucose metabolism, early striatal damage, and reduced lifespan. Since genetic GLUT-1 deficiency syndrome shows a symptom spectrum similar to HE-PHD, we investigated the potential role of the two main glucose transporters, GLUT-1 and GLUT-3, in HE-PHD. We compared GLUT-1 and GLUT-3 protein expression in HE-PHD, juvenile-onset (JOHD), and AOHD brains (n=2; n=3; n=6) and periphery (n=3; n=2; n=2) versus healthy adult controls (n=6; n=6). We also investigated mitochondrial complexes and hexokinase-II protein expression. GLUT-1 and GLUT-3 expression were significantly lower in HE-PHD frontal cortex (p=0.009, 95% [CI 13.4, 14.7]; p=0.017, 95% [CI 14.2, 14.5]) versus controls. In fibroblasts, GLUT-1 and GLUT-3 expression were lower compared to controls (p<0.0001, 95% [CI 0.91, 1.09]; p=0.046, 95% [CI 0.93, 1.07]). In the frontal cortex, this occurred without evidence of extensive neuronal degeneration. Patients with HE-PHD had deregulated mitochondrial complex expression, particularly complexes II-III, levels of which were lower in frontal cortex versus controls (p=0.027, 95% [CI 17.1, 17.6]; p=0.002, 95% CI [16.6, 16.9]) and patients with AOHD (p=0.052, 95% [CI 17.0, 17.6]; p=0.002, 95% [CI 16.6, 16.7]). Hexokinase-II expression was also lower in HE-PHD frontal cortex and striatum versus controls (p=0.010, 95% [CI 17.8, 18.2]; p=0.045, 95% [CI 18.6, 18.7]) and in frontal cortex versus patients with AOHD (p=0.013, 95% [CI 17.7, 18.1]). Expression JOHD levels were consistently different to those of HE-PHD but similar to those of AOHD. Our data suggest a dysfunctional hypometabolic state occurring specifically in paediatric Huntington disease brains. '5×1000' Personal Income Tax donation to LIRH Foundation; Italian Ministry of HealthRC2301MH04 and RF-2016-02364123 to CSS.

The diverse nature of intestinal fibroblasts in development, homeostasis, and disease.

Only in recent years have we begun to appreciate the involvement of fibroblasts in intestinal development, tissue homeostasis, and disease. These insights followed the advent of single-cell transcriptomics that allowed researchers to explore the heterogeneity of intestinal fibroblasts in unprecedented detail. Since researchers often defined cell types and their associated function based on the biological process they studied, there are a plethora of partially overlapping markers for different intestinal fibroblast populations. This ambiguity complicates putting different research findings into context. Here, we provide a census on the function and identity of intestinal fibroblasts in mouse and human. We propose a simplified framework consisting of three colonic and four small intestinal fibroblast populations to aid navigating the diversity of intestinal fibroblasts.

Parallel neurodegenerative phenotypes in sporadic Parkinson's disease fibroblasts and midbrain dopamine neurons.

Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they differed in their nature and scale. Furthermore, statistical analysis revealed potential novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.