Disease Fibroblasts Research Articles

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.

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.

Enhanced Efficiency of the Basal and Induced Apoptosis Process in Mucopolysaccharidosis IVA and IVB Human Fibroblasts.

Morquio disease, also called mucopolysaccharidosis IV (MPS IV), belongs to the group of lysosomal storage diseases (LSD). Due to deficiencies in the activities of galactose-6-sulfate sulfatase (in type A) or β-galactosidase (in type B), arising from mutations in GALNS or GLB1, respectively, keratan sulfate (one of glycosaminoglycans, GAGs) cannot be degraded efficiently and accumulates in lysosomes. This primary defect leads to many cellular dysfunctions which then cause specific disease symptoms. Recent works have indicated that different secondary effects of GAG accumulation might significantly contribute to the pathomechanisms of MPS. Apoptosis is among the cellular processes that were discovered to be affected in MPS cells on the basis of transcriptomic studies and some cell biology experiments. However, Morquio disease is the MPS type which is the least studied in light of apoptosis dysregulation, while RNA-seq analyses suggested considerable changes in the expression of genes involved in apoptosis in MPS IVA and IVB fibroblasts. Here we demonstrate that cytochrome c release from mitochondria is more efficient in MPS IVA and IVB fibroblasts relative to control cells, both under the standard cultivation conditions and after treatment with staurosporine, an apoptosis inducer. This indication of apoptosis stimulation was corroborated by measurements of the levels of caspases 9, 3, 6, and 7, as well as PARP, cleaved at specific sites, in Morquio disease and control fibroblasts. The more detailed analyses of the transcriptomic data revealed which genes related to apoptosis are down- and up-regulated in MPS IVA and IVB fibroblasts. We conclude that apoptosis is stimulated in Morquio disease under both standard cell culture conditions and after induction with staurosporine which may contribute to the pathomechanism of this disorder. Dysregulation of apoptosis in other MPS types is discussed.

Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3β phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.

Secreted phospholipase A2 XIIA triggers a mitochondrial damage-induced senescence in chronic obstructive pulmonary disease fibroblasts

RationaleLung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. ObjectivesThe aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. MethodsFibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. Main resultsIn comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. ConclusionsPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.

E076 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M. Turner: None.

Fibroblasts: New players in the central nervous system?

Fibroblasts are typically described as cells that produce extracellular matrix, contribute to the formation of connective tissue, and maintain the structural framework of tissues. Fibroblasts are the first cell type to be transdifferentiated into inducible pluripotent stem cells (iPSCs), demonstrating their versatility and reprogrammability. Currently, there is relatively extensive characterization of the anatomical, molecular, and functional diversity of fibroblasts in different peripheral organs and tissues. With recent advances in single cell RNA sequencing, heterogeneity and diversity of fibroblasts in the central nervous system (CNS) have also begun to emerge. Based on their distinct anatomical locations in the meninges, perivascular space, and choroid plexus, as well as their molecular diversity, important roles for fibroblasts in the CNS have been proposed. Here, we draw inspirations from what is known about fibroblasts in peripheral tissues, in combination with their currently identified CNS locations and molecular characterizations, to propose potential functions of CNS fibroblasts in health and disease. Future studies, using a combination of technologies, will be needed to determine the bona fide in vivo functions of fibroblasts in the CNS.

CDC20 inhibition alleviates fibrotic response of renal tubular epithelial cells and fibroblasts by regulating nuclear translocation of β-catenin

Fibrosis is a common pathological phenomenon in progressive kidney disease leading to eventual loss of kidney function. Previous studies demonstrated that CDC20 plays a role in cancers by regulating epithelial-mesenchymal transition (EMT) and the infiltration of fibroblasts, suggesting the potential of CDC20 in regulating fibrotic response. However, the role of CDC20 in renal fibrosis is yet unclear. Herein, we reported that renal CDC20 was remarkably upregulated in renal tubular epithelial cells and fibroblasts in chronic kidney disease (CKD) patients, which was in line with a positive correlation with the severity of kidney fibrosis. In mice with unilateral urinary obstruction, CDC20 was also strikingly enhanced, and treatment with Apcin, an inhibitor of CDC20, ameliorated kidney fibrosis. Consistently, the pharmacological inhibition of CDC20 in mouse proximal tubular epithelial cells and rat fibroblasts attenuated TGF-β1-induced fibrotic responses, while overexpression of CDC20 aggravated such responses. Additional studies revealed that CDC20 induces nuclear translocation of β-catenin, which in turn initiates and promotes the pathological process of fibrosis in CKD. Thus, enhanced CDC20 in renal tubular cells and fibroblasts promotes renal fibrosis by activating β-catenin, and CDC20 inhibition may serve as a promising strategy for the prevention and treatment of renal fibrosis.

Retargeting phenylbutyrate, ursodeoxycholic acid, pyrimethamine and betaine for beta-glucocerebrosidase recovery in Gaucher disease fibroblasts resulting from homozygous p.L483P mutation

Retargeting phenylbutyrate, ursodeoxycholic acid, pyrimethamine and betaine for beta-glucocerebrosidase recovery in Gaucher disease fibroblasts resulting from homozygous p.L483P mutation

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new ‘gland-associated immune niche’ has implications for respiratory health.