Extracellular Matrix Deposition Research Articles

Inhibition of RAC1 activator DOCK2 ameliorates cholestatic liver injury via regulating macrophage polarisation and hepatic stellate cell activation

BackgroundThe Rho GTPase Rac family small GTPase 1 (RAC1) is considered a promising fibrotic therapeutic target, but the role of its activator, dedicator of cytokinesis 2 (DOCK2), in liver fibrosis is largely unknown. This study aimed to investigate the expression and role of DOCK2 in cholestasis-induced liver fibrosis and to further explore the potential mechanisms.ResultsCholestasis was induced in male C57BL/6 mice by bile duct ligation (BDL). DOCK2 knockdown was achieved by tail vein injection of adenovirus containing DOCK2-targeting shRNA. The effect of DOCK2 knockdown on cholestatic liver injury was evaluated at different time points after BDL. Hepatic DOCK2 expression gradually increased after BDL. Knockdown of DOCK2 reduced the necrotic area in BDL liver and downregulated serum levels of liver injury indicators. At 3d post-BDL (acute phase), DOCK2 knockdown alleviated M1 macrophage inflammation in the liver, as evidenced by reduced infiltrating iNOS + macrophages and inflammatory cytokines and mitigated NLRP3 inflammasome activation. At 14d post-BDL (chronic phase), DOCK2 knockdown suppressed hepatic stellate cell (HSC) activation and liver fibrosis as indicated by decreased α-SMA + HSCs and extracellular matrix deposition. In vitro experiments further demonstrated that DOCK2 knockdown suppressed M1 macrophage polarisation and HSC to myofibroblast transition, accompanied by inhibition of RAC1 activation.ConclusionsIn summary, this study demonstrates for the first time that the RAC1 activator DOCK2 regulates M1 macrophage polarisation and hepatic stellate cell activation to promote cholestasis-induced liver inflammation and fibrosis, suggesting that DOCK2 may be a potential therapeutic target in cholestatic liver injury.

Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

Sustained β-adrenergic activation induces cardiac fibrosis characterized by excessive deposition of extracellular matrix (ECM). Prostaglandin E2 (PGE2) receptor EP4 is essential for cardiovascular homeostasis. This study aims to investigate the roles of cardiomyocyte (CM) and cardiac fibroblast (CF) EP4 in isoproterenol (ISO)-induced cardiac fibrosis. By crossing the EP4f/f mice with α-MyHC-Cre or S100A4-Cre mice, this work obtains the CM-EP4 knockout (EP4f/f-α-MyHCCre+) or CF-EP4 knockout (EP4f/f-S100A4Cre+) mice. The mice of both genders are subcutaneously injected with ISO (5 mgkg-1day-1) for 7 days. Compared to the control mice, both EP4f/f-α-MyHCCre+ and EP4f/f-S100A4Cre+ mice show a significant improvement in cardiac diastolic function and fibrosis as assessed by echocardiography and histological staining, respectively. In the CMs, inhibition of EP4 suppresses ISO-induced TGF-β1 expression via blocking the cAMP/PKA pathway. In the CFs, inhibition of EP4 reversed TGF-β1-triggers production of ECM via preventing the formation of the TGF-β1/TGF-β receptor complex and blocks CF proliferation via suppressing the ERK1/2 pathway. Furthermore, double knockout of the CM- and CF-EP4 or administration of EP4 antagonist, grapiprant, markedly improves ISO-induced cardiac diastolic dysfunction and fibrosis. Collectively, this study demonstrates that both CM-EP4 and CF-EP4 contribute to β-adrenergic activation-induced cardiac fibrosis. Targeting EP4 may offer a novel therapeutic approach for cardiac fibrosis.

RUNX2 promotes fibrosis via an alveolar-to-pathological fibroblast transition.

A hallmark of pulmonary fibrosis is the aberrant activation of lung fibroblasts into pathological fibroblasts that produce excessive extracellular matrix1-3. Thus, the identification of key regulators that promote the generation of pathological fibroblasts can inform the development of effective countermeasures against disease progression. Here we use two mouse models of pulmonary fibrosis to show that LEPR+ fibroblasts that arise during alveologenesis include SCUBE2+ alveolar fibroblasts as a major constituent. These alveolar fibroblasts in turn contribute substantially to CTHRC1+POSTN+ pathological fibroblasts. Genetic ablation of POSTN+ pathological fibroblasts attenuates fibrosis. Comprehensive analyses of scRNA-seq and scATAC-seq data reveal that RUNX2 is a key regulator of the expression of fibrotic genes. Consistently, conditional deletion of Runx2 with LeprcreERT2 or Scube2creERT2 reduces the generation of pathological fibroblasts, extracellular matrix deposition and pulmonary fibrosis. Therefore, LEPR+ cells that include SCUBE2+ alveolar fibroblasts are a key source of pathological fibroblasts, and targeting Runx2 provides a potential treatment option for pulmonary fibrosis.

Estrogen receptor alpha ablation reverses muscle fibrosis and inguinal hernias.

Fibrosis of the lower abdominal muscle (LAM) contributes to muscle weakening and inguinal hernia formation, an ailment affecting a noteworthy fifty percent of men by age 75, necessitating surgical correction as the singular therapy. Despite its prevalence, the mechanisms driving LAM fibrosis and hernia development remain poorly understood. Utilizing a humanized mouse model that replicates elevated skeletal muscle tissue estrogen concentrations akin to aging men, we identified estrogen receptor alpha (ESR1) as a key driver of LAM fibroblast proliferation, extracellular matrix deposition, and hernia formation. Fibroblast-specific ESR1 ablation effectively prevented muscle fibrosis and herniation, while pharmacological ESR1 inhibition with fulvestrant reversed hernias and restored normal muscle architecture. Multiomic analyses on in vitro LAM fibroblasts unveiled an estrogen/ESR1-mediated activation of a distinct profibrotic cistrome and gene expression signature, concordant with observations in inguinal hernia tissues in human males. Our findings hold significant promise for prospective medical interventions targeting fibrotic conditions and presenting non-surgical avenues for addressing inguinal hernias.

Fraxinellone-mediated targeting of cathepsin B leakage from lysosomes induces ferroptosis in fibroblasts to inhibit hypertrophic scar formation

BackgroundHypertrophic scar (HS) is a common fibrotic skin disorder characterized by the excessive deposition of extracellular matrix (ECM). Fibroblasts are the most important effector cells involved in HS formation. Currently no satisfactory treatment has been developed.MethodsThe impact of fraxinellone (FRA) on the proliferation and migration capacity of human hypertrophic scar-derived fibroblasts (HSFs) was assessed by EdU proliferation, wound healing and transwell assays. Quantitative real-time PCR (qRT‒PCR), Western blot (WB), immunofluorescence staining and collagen gel contraction assays were performed to evaluate the collagen production and activation capacity of HSFs. Oxford Nanopore Technologies long-read RNA sequencing (ONT long-read RNA-seq) revealed the occurrence of ferroptosis in HSF and ferroptosis executioner-cathepsin B (CTSB). The mechanisms underlying FRA-induced HSF ferroptosis were examined through fluorescence staining, qRT‒PCR, WB and molecular docking study. The therapeutic efficacy of FRA was further validated in vivo using a rabbit ear scar model.ResultsFRA treatment significantly suppressed the proliferation, migration, collagen production and activation capacity of HSFs. ONT long-read RNA-seq discovered that FRA modulated the expression of transcripts related to ferroptosis and lysosomes. Mechanistically, FRA treatment reduced the protein expression level of glutathione peroxidase 4 (GPX4) and induced the release of CTSB from lysosomes into the cytoplasm. CTSB further induced ferroptosis via spermidine/spermine-N1-acetyltransferase (SAT1)-mediated lipid peroxidation, mitochondrial damage and mitogen-activated protein kinase (MAPK) signalling pathway activation, eventually affecting the function of HSFs. Moreover, FRA treatment attenuated the formation of HS in rabbit ears via CTSB-mediated ferroptosis. The antifibrotic effects of FRA were abrogated by pretreatment with a CTSB inhibitor (CA-074-me).ConclusionsThis study reveals that FRA ameliorates HS by inducing CTSB leakage from lysosomes, causing SAT1-mediated lipid peroxidation, mitochondrial damage and MAPK signalling pathway activation, thus mediating HSF ferroptosis. Therefore, FRA could be a promising therapeutic agent for treating HS.

Yiqi Juanshen decoction alleviates renal interstitial fibrosis by targeting the LOXL2/PI3K/AKT pathway to suppress EMT and inflammation

Chronic kidney disease (CKD) is a major health concern, with renal interstitial fibrosis (RIF) as a key feature. Effective management of RIF is crucial for treating CKD. Yiqi Juanshen decoction (YQJSD), as traditional Chinese medicine, has shown promising results in CKD treatment. This study evaluates YQJSD’s effectiveness in ameliorating RIF and explores the underlying molecular mechanisms using the unilateral ureteral obstruction (UUO) model. YQJSD has been shown to effectively reduce serum creatinine and blood urea nitrogen levels, decrease extracellular matrix deposition, and down-regulate the expression of α-SMA, COL4α1, Fibronectin (FN). Mechanistically, YQJSD exerts its effects by modulating multiple pathways: it inhibits the NF-κB signaling pathway, inhibiting the expression of pro-inflammatory cytokines like NF-κB1, IL-1β, TNF-α, and CCR1. Simultaneously, YQJSD suppresses the epithelial-mesenchymal transition (EMT) by downregulating the expression of Snail1, Vimentin, Twist1, and FSP1, while increasing E-cadherin expression. Moreover, YQJSD can regulate the PI3K/AKT signaling pathway by decreasing the expression of LOXL2 and PIK3R1, along with p-AKT1/2/3. This modulation of the LOXL2/PI3K/AKT pathway contributes to the inhibition of both EMT and inflammation, highlighting a critical role in the therapeutic intervention against RIF. These findings suggest that YQJSD may serve as a promising therapeutic management of RIF in CKD patients.

Inhibition of peptidyl arginine deiminase-4 ameliorated pulmonary fibrosis via modulating M1/M2 polarisation of macrophages.

Pulmonary fibrosis (PF) arises from dysregulated wound healing, leading to excessive extracellular matrix (ECM) deposition and impaired lung function. Macrophages exhibit high plasticity, polarizing to pro-inflammatory M1 during early inflammation and anti-inflammatory, fibrosis-inducing M2 during later stages of PF. Additionally, neutrophils and neutrophil extracellular traps (NETs) release mediated by peptidyl arginine deiminase (PAD-4), also play a key role in PF progression. PAD-4 inhibitor chloro-amidine (CLA) has shown anti-fibrotic effects in bleomycin (BLM) induced PF mouse model in our earlier study. Here, we have demonstrated that CLA also exhibited inhibition of macrophage polarisation in in-vitro in THP-1 monocytes and in-vivo in BLM induced PF. THP-1 monocytes were exposed to NETs isolated from phorbol 12-myristate-13-acetate (PMA) stimulated and PMA plus CLA treated differentiated HL-60 (dHL-60) cells. Monocytes exposed to stimulated NETs resulted in increased oxidative stress, disrupted mitochondrial membrane potential and increased M1 and M2 macrophage markers. These alterations were abrogated in THP-1 cells upon exposure to CLA treated NETs. Further, CLA treatment in BLM induced mice improved abnormal BALF, biochemical, and histological parameters in line with our previous findings. Additionally, CLA also reduced M1 and M2 markers time-dependently, as shown by immunofluorescence (IF), western blot, and RT-PCR analysis. CLA treatment led to decreased expression of PAD-4, M1-related pro-inflammatory cytokines and M2-related pro-fibrotic cytokines and mediators, as confirmed by western blot and ELISA analysis. Thus, it is established that inhibition of PAD-4 lead to mitigation of macrophage polarisation and a combined anti-fibrotic effect is achieved which can be explored further.

Oral submucous fibrosis: pathogenesis and therapeutic approaches.

Oral submucous fibrosis (OSF), characterized by excessive deposition of extracellular matrix (ECM) that causes oral mucosal tissue sclerosis, and even cancer transformation, is a chronic, progressive fibrosis disease. However, despite some advancements in recent years, no targeted antifibrotic strategies for OSF have been approved; likely because the complicated mechanisms that initiate and drive fibrosis remain to be determined. In this review, we briefly introduce the epidemiology and etiology of OSF. Then, we highlight how cell-intrinsic changes in significant structural cells can drive fibrotic response by regulating biological behaviors, secretion function, and activation of ECM-producing myofibroblasts. In addition, we also discuss the role of innate and adaptive immune cells and how they contribute to the pathogenesis of OSF. Finally, we summarize strategies to interrupt key mechanisms that cause OSF, including modulation of the ECM, inhibition of inflammation, improvement of vascular disturbance. This review will provide potential routes for developing novel anti-OSF therapeutics.

Interplay between Skeletal Muscle Catabolism and Remodeling of Arteriovenous Fistula via YAP1 Signaling.

Arteriovenous (AV) fistulas are the preferred access for dialysis but have a high incidence of failure. This study aims to understand the crosstalk between skeletal muscle catabolism and AV fistula maturation failure. Skeletal muscle metabolism and AV fistula maturation were evaluated in mice with chronic kidney disease (CKD). The roles of myostatin and YAP1 in regulating the transdifferentiation of adventitial mesenchymal stem cells (mesenchymal stem cells) and intima hyperplasia in AV fistula were investigated. Nanoparticles carrying a YAP1 inhibitor, verteporfin, with light irradiation-controlled release were synthesized and applied to AV fistula. Increased trichrome signals and stenosis were observed in AV fistulas from mice treated with myostatin and from mice with CKD. In contrast, blocking myostatin function with an anti-myostatin peptibody not only improved body weight and muscle size in CKD mice but also decreased neointima formation in AV fistulas. In cultured mesenchymal stem cells, myostatin induced YAP1 expression, promoting the differentiation of mesenchymal stem cells into myofibroblasts and inducing extracellular matrix deposition. Red light irradiation-controlled release of verteporfin from nanoparticles blocked YAP1 activation and alleviated myostatin-induced mesenchymal stem cell activation. Periadventitial application and red-light irradiation of nanoparticles carrying verteporfin significantly suppressed stiffening and neointima formation in AV fistula. CKD induced muscle wasting leading to increased production of myostatin, which stimulated mesenchymal stem cell activation and vascular fibrosis linked to AV fistula stenosis. YAP1 signaling was activated in these processes. Red-light irradiation-controlled release of verteporfin offered a feasible approach for local vascular drug intervention to improve AV fistula maturation.

A Novel Gene DUSP8 Missense Mutation Causes Nonsyndromic Hereditary Gingival Fibromatosis by Dysregulating Lysine Lactylation.

The goal of this study was to explore new candidate genes and pathogenesis mechanisms of nonsyndromic hereditary gingival fibromatosis (nsHGF) and to provide an experimental basis for the diagnosis of nsHGF. Whole-exome sequencing (WES) was performed on peripheral blood DNA from three nsHGF family members to screen for new candidate genes, and Sanger sequencing and related databases were used to verify the pathogenicity of this gene deficiency. Moreover, the effects of gene deficiency on the biological characteristics of human gingival fibroblasts (HGFs) were evaluated via cell proliferation assays, extracellular matrix (ECM) deposition detection, cell apoptosis and cell cycle assessment, cell migration and gene expression analyses. A novel missense mutation in dual-specificity phosphatase 8 (DUSP8, c.1348C>T, p.R450C), which is in the nsHGF-related GINGF4 locus, was identified via WES analysis. A functional study revealed that knocking down DUSP8 expression increased cell proliferation, cell migration and the expression of profibrotic factors (particularly COL1A1), inhibited cell apoptosis, and ultimately resulted in nsHGF. Similarly, this DUSP8 mutation inhibited the expression of the encoded protein and promoted cell proliferation and the expression of profibrotic factors. In addition, both DUSP8 knockdown and DUSP8 mutation induced nsHGF by accelerating glycolysis and panlysine lactylation (Kla) to promote cell proliferation and the expression of ECM-related factors. DUSP8 deficiency might be a novel pathogenic factor that contributes to nsHGF.

Pathophysiology of Benign Prostatic Hyperplasia: Cellular and Molecular Mechanisms

Benign prostatic hyperplasia (BPH) is a prevalent condition among aging males, characterized by non-malignant enlargement of the prostate gland, which can lead to lower urinary tract symptoms (LUTS) and a significant reduction in quality of life. This review explores the cellular and molecular mechanisms underpinning the pathophysiology of BPH, highlighting the interplay of hormonal, inflammatory, and growth factor signaling pathways. Testosterone and dihydrotestosterone (DHT) serve as pivotal regulators of prostate growth, driving hyperplasia through androgen receptor-mediated transcriptional activity. Additionally, chronic inflammation, mediated by immune cell infiltration and cytokine release, contributes to tissue remodeling and stromal proliferation. Dysregulated growth factors such as fibroblast growth factors (FGFs) and transforming growth factor-beta (TGF-β) further amplify cellular proliferation and extracellular matrix deposition. Emerging evidence underscores the role of oxidative stress and mitochondrial dysfunction in exacerbating prostatic cellular senescence and genomic instability. This review also addresses the influence of stromal-epithelial interactions and the contribution of stem/progenitor cell niches in maintaining aberrant growth. Understanding these intricate mechanisms provides a foundation for identifying novel therapeutic targets aimed at mitigating BPH progression and associated symptoms. Keywords: Benign prostatic hyperplasia, cellular mechanisms, hormonal regulation, inflammation, growth factors, molecular pathways, therapeutic targets.

Human subcutaneous and visceral adipocyte atlases uncover classical and nonclassical adipocytes and depot-specific patterns.

Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA sequencing (snRNA-seq) analyses largely uncovered overlapping or similar cell-type landscapes. We hypothesized that adipocyte subtypes, differentiation trajectories and/or intercellular communication patterns could illuminate this depot similarity-difference gap. For this, we performed snRNA-seq of human subcutaneous or visceral adipose tissues (five or ten samples, respectively). Of 27,665 adipocyte nuclei in both depots, most were 'classical', namely enriched in lipid metabolism pathways. However, we also observed 'nonclassical' adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization or angiogenesis or ribosomal and mitochondrial processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via nonclassical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Last, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation and intercellular communication patterns to human fat depot differences.

Fibrosis and Src Signalling in Glaucoma: From Molecular Pathways to Therapeutic Prospects

Glaucoma, a leading cause of irreversible blindness, is characterised by progressive optic nerve damage, with elevated intraocular pressure (IOP) and extracellular matrix (ECM) remodelling in the lamina cribrosa (LC) contributing to its pathophysiology. While current treatments focus on IOP reduction, they fail to address the underlying fibrotic changes that perpetuate neurodegeneration. The Src proto-oncogene, a non-receptor tyrosine kinase, has emerged as a key regulator of cellular processes, including fibroblast activation, ECM deposition, and metabolism, making it a promising target for glaucoma therapy. Beyond its well-established roles in cancer and fibrosis, Src influences pathways critical to trabecular meshwork function, aqueous humour outflow, and neurodegeneration. However, the complexity of Src signalling networks remains a challenge, necessitating further investigation into the role of Src in glaucoma pathogenesis. This paper explores the therapeutic potential of Src inhibition to mitigate fibrotic remodelling and elevated IOP in glaucoma, offering a novel approach to halting disease progression.

P0029 Abnormal glycerophospholipid metabolism in CCL2+DPP4+ mesenchymal stem cells may contributes to creeping fat fibrosis in Crohn's Disease

Abstract Background Creeping fat (CF), an aberrant mesenteric adipose tissue (MAT), is characterized by abnormal lipid metabolism, inflammatory cell infiltration, and extracellular matrix (ECM) deposition in the intestines affected by Crohn's disease (CD). Our previous single-cell RNA-seq analysis identified a CCL2+DPP4+ subset of mesenchymal stem cells (MSCs) that accelerates the abnormal formation of CF. Building on these findings, this study investigates the role of CCL2+DPP4+ MSCs in CF fibrosis formation. Methods We employed H&E and Masson staining to assess histopathological changes and collagen deposition. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was conducted on CCL2+DPP4+ MSCs isolated from CF biopsies of CD patients (n=5) and MAT from controls without CD (n=5). Results We observed ECM deposition in CF and an upregulation of fibrosis-related genes (COL1A1, COL1A2, and FN1)（Figure1）. Analysis of our previous single-cell data revealed that CCL2+DPP4+ MSCs play a significant role in the abnormal accumulation of the ECM (Figure2). Lipidomic profiling revealed differential lipid levels between the CF and healthy MAT（H-MAT） groups, with downregulation of specific lipids in CCL2+DPP4+ MSCs derived from CF, including PE (20:5_16:1), PC (14:0_22:6), and PE (16:1_14:0). KEGG pathway enrichment analysis highlighted significant enrichment in Glycerophospholipid metabolism. Phospholipids (PLs), crucial for membrane structure and function, were found to be regulated by key enzymes PISD(Phosphatidylserine decarboxylase) and LPCAT3(Lysophosphatidylcholine acyltransferase 3), which convert PS（phosphatidylserine） to PE（phosphatidylethanolamine） and integrate polyunsaturated fatty acids into lysophospholipids, respectively. Quantitative real-time PCR confirmed reduced expression of these enzymes and increased fibrosis gene expression in CF-derived CCL2+DPP4+ MSCs(Figure3). Conclusion Our findings suggest that the CCL2+DPP4+ MSC subset in CF exhibits abnormal glycerophospholipid metabolism, which is associated with decreased PISD and LPCAT3 expression. This may contribute to enhanced ECM formation and promotion of CF fibrosis. Understanding these mechanisms could lead to novel therapeutic approaches for CD.

The fibronectin-targeting PEG-FUD imaging probe shows enhanced uptake during fibrogenesis in experimental lung fibrosis

Progressive forms of interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF), are deadly disorders lacking non-invasive biomarkers for assessment of early disease activity, which presents a major obstacle in disease management. Excessive extracellular matrix (ECM) deposition is a hallmark of these disorders, with fibronectin being an abundant ECM glycoprotein that is highly upregulated in early fibrosis and serves as a scaffold for the deposition of other matrix proteins. Due to its role in active fibrosis, we are targeting fibronectin as a biomarker of early lung fibrosis disease activity via the PEGylated fibronectin-binding polypeptide (PEG-FUD). In this work, we demonstrate the binding of PEG-FUD to the fibrotic lung throughout the course of bleomycin-induced murine model of pulmonary fibrosis. We first analyzed the binding of radiolabeled PEG-FUD following direct incubation to precision cut lung slices from mice at different stages of experimental lung fibrosis. Then, we administered fluorescently labeled PEG-FUD subcutaneously to mice over the course of bleomycin-induced pulmonary fibrosis and assessed peptide uptake 24 h later through ex vivo tissue imaging. Using both methods, we found that peptide targeting to the fibrotic lung is increased during the fibrogenic phase of the single dose bleomycin lung fibrosis model (days 7 and 14 post-bleomycin). At these timepoints we found a correlative relationship between peptide uptake and fibrotic burden. These data suggest that PEG-FUD targets fibronectin associated with active fibrogenesis in this model, making it a promising candidate for a clinically translatable molecular imaging probe to non-invasively determine pulmonary fibrosis disease activity, enabling accelerated therapeutic decision-making.

P0282 Fibroblast activation protein (FAP)-cleaved type III Collagen [C3F] is a potential marker for intestinal fibrosis in patients with Crohn’s Disease

Abstract Background Crohn ‘s disease (CD) is characterized by chronic inflammation in the gut, where severe complications such as fibrotic strictures require surgical resection. Stricture development involves excessive extracellular matrix (ECM) deposition due to continuous activation of intestinal myofibroblasts, that further contribute to intestinal fibrogenesis. Emerging data suggests that stricture-related intestinal myofibroblasts overexpress a serine protease called fibroblast activation protein (FAP). Furthermore, type III collagen deposition is increased during fibrosis in all layers of the intestinal tract, and studies have shown elevated collagen degradation in serum from patients with CD. The present study aimed at evaluating the potential of FAP-cleaved type III collagen [C3F] as a serum marker for intestinal fibrosis in CD. Methods The cohort consisted of 49 patients with luminal CD and 62 patients with stenotic CD. Clinical assessment was conducted for all patients at baseline and 12 months post study initiation. For the luminal phenotype, endoscopy was performed at both time points. For the stenotic group, radiographical assessment was done prior to surgery and endoscopy was performed at 12 months. C3F was measured in serum at all timepoints for baseline, post-operative day (POD), 1, 3, 6 and 12 months. To compare C3F levels at baseline for luminal vs stenotic phenotype, Mann-Whitney U-test was conducted. For the stenotic group, mixed-effect analysis, with a Dunnett’s multiple comparison, was conducted to compare C3F levels for baseline vs POD and Spearman’s rank correlation was applied. Results C3F baseline levels were significantly higher in patients with stenotic CD compared to luminal CD (Figure 1). In the stenotic group, C3F was significantly increased in patients at POD compared to baseline (Figure 1). Furthermore, C3F showed a positive correlation with C-reactive protein (CRP) (ρ=0.28, p=0.006) and a significant negative correlation with disease duration (ρ= -0.257, p=0.048) for patients with the stenotic CD phenotype (Table 1). Conclusion C3F was elevated in patients with stenotic CD, with even higher levels after the surgical resection. The data suggests that C3F reflects fibroblast activity during fibrostenosis and tissue remodeling following surgery. The positive correlation with CRP supports the association with inflammation leading to collagen deposition and fibrosis. Interestingly, C3F shows negative correlation with disease duration suggesting that C3F could reflect fibrosis at early stages of stenotic CD. These findings highlight the potential use of C3F as a novel biomarker for intestinal fibrosis in CD.

P0014 Mesenteric inflammation-associated fibroblasts are critical in the pathogenesis of Crohn’s disease

Abstract Background The Mesentery, initially considered as a structure support of abdominal organs, has emerged as an active participant in the inflammatory processes of Crohn's disease (CD). It serves as a central hub for immune cell activity, including macrophages, T cells, and B cells, driving the propagation of inflammation. Additionally, mesenteric fibroblasts play a crucial role in CD-associated fibrosis. These fibroblasts are activated by pro-fibrotic cytokines such as TGF-β and IL-13, leading to excessive extracellular matrix (ECM) deposition, which contributes to intestinal wall thickening and the formation of strictures. In this study, we aim to explore the specific role of mesenteric fibroblasts in the pathogenesis of CD. Methods Magnetic-activated cell sorting was utilized to isolate mesenteric inflammation-associated fibroblasts (IAFs) and normal fibroblasts (NFs) from surgical samples of patients with Crohn's disease (CD) and noncancerous colonic polyps (control group). Various cell-based assays, including cell viability, invasion, and collagen contraction assays, were conducted to investigate phenotypic differences between NFs and IAFs. To assess the impact of NFs and IAFs on colonic epithelium, Co-culture experiments were performed with the isolated fibroblasts and Caco-2 and HT-29 colorectal cancer cell lines using transwell and 3D Matrigel culture systems. Results IAFs exhibited a significantly faster growth rate than NFs (Figure 1B; fold change [FC] 1.60 ± 0.04, P < 0.001). Furthermore, IAFs showed markedly higher wound healing (Figure 1C; FC 1.44 ± 0.15, P = 0.004), invasion (Figure 1D; FC 1.45 ± 4.7, P = 0.035), and collagen contraction activities (Figure 1E; FC 1.26 ± 4.73, P = 0.007) compared to NFs. In a 3D Matrigel co-culture system with Caco-2 spheres, abnormal lumen formation with invasive structures of Caco-2 spheres was significantly higher in IAFs compared to NFs (Figure 2A; FC 1.3 ± 0.08, P < 0.001). Similarly, a fluorescein transport assay using a transwell co-culture system revealed that IAFs caused significantly greater epithelial disruption compared to NFs (Figure 2B; FC 1.25 ± 0.04, P = 0.04). Conditioned media from IAFs upregulated the expression of mesenchymal markers, such as ZEB1 (FC 2.67 ± 0.2, P < 0.01) and CDH2 (FC 2.11 ± 0.54, P < 0.05), in Caco-2 cells when compared with NFs (Figure 2C). Conclusion These findings suggest that mesenteric IAFs play an active role in the inflammatory and fibrotic processes in the pathogenesis of CD. Targeting mesenteric IAFs and their associated pathways may provide a promising therapeutic approach for managing CD.

OP21 MIF-CD74 signaling promotes intestinal LTi-like ILC3s activation and gut fibrosis via upregulating pSTAT3

Abstract Background Intestinal fibrosis is a common complication of inflammatory bowel disease, and its core pathogenesis is fibroblast activation and massive extracellular matrix (ECM) deposition. In recent years, innate lymphoid cells (ILCs) have been shown to be an important source of fibroblast activation signals in fibrotic diseases. This study aims to explore the heterogeneity of ILC3 (the main ILC subgroup in the human intestine) in the intestinal fibrosis and the mechanism of its interaction with stromal cells. Methods The intestinal fibrosis model was constructed with ILC3 deficiency mice (Rorc knock out) treated with chronic DSS/TNBS. Flow cytometry was performed to detect the proportion of ILC3 subsets and cytokine expression level. ILC3 were sorted out for transcriptome sequencing and co-culture experiment with primary intestinal fibroblasts. Results In chronic DSS/TNBS models, the colon CCR6+ILC3 significantly expanded (p<0.05), and the expression of cytokine IL-22 was upregulated (p<0.05). ILC3 deficiency mice showed lower degree of intestinal fibrosis, including decrease in collagen involvement area (p<0.05) and decrease in ECM-related genes expression(p<0.05). After adoptively transferred of CCR6+ILC3, the severity of intestinal fibrosis in mice increases again. Flow cytometry revealed that CCR6+ILC3 was the main source of IL-22 in the mouse colon. CCR6+ILC3 cells were then sorted by flow cytometry, and co-cultured with mouse intestinal fibroblasts in vitro. Results shown that CCR6+ILC3 can activate fibroblasts. The expression level of α-SMA protein was significantly upregulated (p<0.05). Cell migration ability was also improved in the scratch experiment (p<0.05). Blocking IL-22 could inhibit the effects above. Transcriptome sequencing analysis of sorted ILC3 revealed a significant upregulation in signaling pathways related to cell activation, proliferation and adhesion, with CD74 existing in all of them. Flow cytometry confirmed the expression of CD74 on ILC3 for the first time, which specifically present on CCR6+ILC3 within RORγt+ cells. Cell stimulation experiments in vitro showed that macrophage migration inhibitory factor (MIF)-CD74 signal upregulated IL-22 level of ILC3 cells and was associated with increased phosphorylation of STAT3 signaling pathway. And MIF, the CD74 ligand, significantly increased in both mouse intestinal fibrosis models and stenosis tissue samples from CD patients (p<0.05). Conclusion CCR6+ILC3 is enriched in the intestinal fibrotic microenvironment and can continuously promote disease progression by activating fibroblasts through IL-22. CCR6+ILC3 can sense the pathogenic inflammatory factor MIF in chronic colitis through CD74, and increase IL-22 expression by promote STAT3 phosphorylation.

P0135 Effect of human recombinant IL-33 on fibrosis markers in colonic fibroblasts

Abstract Background IBD is frequently complicated by intestinal fibrosis and strictures formation, occurring in more than 50% of Crohn’s disease (CD) and 5% of ulcerative colitis. Intestinal fibrosis leads to surgery in CD and strictures frequently recur leading to repeated surgeries1. There is currently no specific therapy to prevent or inhibit intestinal fibrosis and this therefore constitutes a major treatment challenge. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM) synthesized by intestinal myofibroblasts1. Interleukin-33 (IL-33) is a pleiotropic nuclear cytokine involved in immunoregulation and tissue repair2. Following cellular injury, IL-33 is released by epithelial, endothelial or fibroblast cells. Binding to the ST2 (suppressor of tumorigenicity 2) receptor, it induces translocation of the transcription factor NF-□□□□B, which regulates the expression of pro-inflammatory genes2. IBD patients have increased colonic expression of IL-33 and ST2 in epithelial cells compared to control patients3. Neutralization of IL-33 enables the repair of lung epithelial cells by limiting the release of pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α in a mouse model of pulmonary epithelial lesions4. We hypothesized that IL-33 may be involved in IBD-associated intestinal fibrosis. We therefore aimed to understand the role of IL-33 in a human colonic fibroblast cell line (CCD-18Co). Methods First, we measured IL-33 and ST2 mRNA expression in CCD-18Co in response to TGF-ß1 (10 ng/mL) and/or TNF-ɑ (100 ng/mL) for 24h. Then, CCD-18Co were induced by increasing doses of IL-33 treatment (10, 50, 100 ng/mL) for 24h and inflammatory and fibrosis markers were measured. Results TGF-ß1 significantly induced mRNA levels encoding ECM-associated proteins such as α-SMA (***p<0.001) or COL1A1 (*p<0.05, Figure 1).Treatment with TGF-ß1 and/or TNF-α had no effect on the expression of mRNAs encoding IL-33 and ST2 in CCD-18Co in our experimental conditions. IL-33 treatment significantly increased mRNA levels for IL-1ß and ECM-associated proteins (COL3A1, CTGF and VIM) (*p<0.05, Figure 1) in CCD-18Co but these effects are abolished in presence of TGF-ß1 and/or TNF-α treatment. Conclusion Treatment with TGF-ß1 and/or TNF-α had no significative impact on IL-33 mRNA levels in the CCD-18Co in our experimental conditions. IL-33 treatment increased ECM-associated proteins in CCD-18Co line under basal conditions but not in TGF-ß1 and/or TNF-α conditions.

DOP073 Gli1+ Mesenchymal Cells Drive Intestinal Fibrostenosis by Secreting SMOC2: A Biomarker and Therapeutic Target

Abstract Background Intestinal fibrostenosis in Crohn’s disease (CD) represents a significant clinical challenge, driven by the absence of reliable early biomarkers, limited therapeutic options, and high postoperative recurrence rates, all of which contribute to poor patient outcomes. This study aims to elucidate the role of Gli1+ mesenchymal cells in the pathogenesis of intestinal fibrostenosis and to explore their potential as a novel therapeutic target. Methods CD patients who underwent surgery for intestinal obstruction due to fibrosis were included in this study, and intestinal tissues were collected from both fibrotic and normal sites. Dextrose sodium sulfate (DSS)-induced and 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced chronic colitis mouse models and their normal controls were also included. Single-cell RNA sequencing was performed on both human and murine tissues. Gli1-CreERT2; R26-tdTomato mice were generated for lineage tracing. Cell sorting combined with Smart-seq was used to isolate Gli1+ mesenchymal cells, revealing their pathogenic determinants. Results Single-cell RNA sequencing data and immunofluorescence staining from surgical patients revealed an enrichment of Gli1+ mesenchymal cells at fibrotic sites. Lineage tracing of Gli1+ mesenchymal cells demonstrated their proliferation and acquisition of a myofibroblast phenotype following chronic colitis. In vivo ablation of Gli1+ mesenchymal cells attenuated the severity of intestinal fibrosis. Compared to Gli1- cells, Gli1+ mesenchymal cells expressed a distinct gene expression profile, including markers of extracellular matrix (ECM)-related genes and Smoc2 (SPARC Related Modular Calcium Binding 2, encodes an extracellular matrix protein). Elevated Smoc2 expression was confirmed in both human and murine tissues, and Smoc2 knockdown further supported its role in promoting intestinal fibrosis. Mechanistically, Gli1+ mesenchymal cells establish a pro-fibrotic niche through the endocrine and exocrine functions of SMOC2, promoting ECM deposition and the activation of other fibroblasts. Additionally, a predictive and diagnostic model was established by assessing SMOC2 levels in tissue and plasma samples from CD patients with intestinal fibrosis, highlighting its potential as a biomarker for postoperative recurrence and as a therapeutic target. Conclusion Our results demonstrate that Gli1+ mesenchymal cells are key drivers of intestinal fibrostenosis, forming a pro-fibrotic niche through their unique secretion of the SMOC2 protein at fibrotic sites. SMOC2 could therefore be used as a predictive biomarker for postoperative recurrence and as a potential therapeutic target.