Introduction

Chronic pancreatitis (CP) is a multifactorial, inflammatory disease characterized by progressive destruction of the pancreas, with fibrotic transformation of the tissue as a major morphological feature. The incidence of the disease is steadily increasing, but no specific therapy is currently available, making the identification of new therapeutic targets a priority. Plasminogen activator inhibitor-1 (PAI-1, encoded by SERPINE1) has been implicated in various pathological processes, including fibrosis and inflammation. However, its specific involvement in the pathogenesis of CP is not yet fully understood.

Aims & Methods

We therefore aimed to explore the role of PAI-1 in the development of CP and assess the therapeutic potential of PAI-1 inhibitors as antifibrotic agents to prevent the progression of CP.
​The PAI-1 expression was measured in human pancreatic tissue samples with immunohistochemistry, and human blood serum with ELISA. CP was induced in FVB/N mice with intraperitoneal cerulein injections given every hour, every 3 days for a total of 5 times. PAI-1 activity was inhibited with a specific inhibitor, which was administered per os at a dose of 10 mg/kgbw for 6 consecutive days after the 3rd cerulein treatment. Inflammation and fibrosis were assessed using hematoxylin-eosin and Crossman’s trichrome staining, while biochemical assays measured amylase activity and hydroxyproline concentration. SERPINE1 gene expression was quantified using RT-qPCR, and PAI-1 protein levels and localization were assessed via immunohistochemistry and immunofluorescence co-staining. In vitro, pancreatic fibroblast cells isolated from CP mice were treated with a 10 μM PAI-1 inhibitor.

Results

PAI-1 levels were significantly elevated in both pancreatic tissue and blood samples obtained from CP patients. In CP-induced mice, the body weight and the pancreatic weight/body weight ratio significantly reduced, while histopathology showed significant pancreatic atrophy and fibrosis. This was also confirmed by the increased hydroxyproline concentration and decreased amylase activity of the pancreatic tissue. SERPINE1 gene expression was upregulated in pancreatic tissue and fibroblast cultures in the CP group, while PAI-1 protein levels were elevated in pancreatic tissue. The elevated PAI-1 signal localization and the high overlap with vimentin suggests a possible expression role for activated pancreatic stellate cells (PSC). Administration of a PAI-1 inhibitor reduced body weight and pancreatic weight loss rates, along with significant decreases in pancreatic atrophy, fibrosis, hydroxyproline concentrations, and SERPINE1 gene expression compared to the CP group. In vitro, PAI-1 inhibition reduced cell migration rate and SERPINE1 gene expression in PSC derived from CP mice.

Conclusion

Our results indicate an elevation in PAI-1 expression in CP, with the potential for fibrosis and tissue atrophy reduction by the inhibition of PAI-1. These findings propose PAI-1 as a promising therapeutic target for the treatment of chronic pancreatitis.

Introduction

Acute pancreatitis (AP) is a common clinical emergency, with 20%–30% of cases progressing to severe acute pancreatitis (SAP), which has a mortality rate of up to 40%. Gut microbiota dysbiosis is closely associated with AP severity, yet its underlying mechanisms remain unclear.

Aims & Methods

This study aimed to elucidate how microbiota dysbiosis exacerbates AP.AP was induced in Specific Pathogen-Free (SPF) and Germ-Free (GF) C57BL/6 male mice to investigate the effects of microbiota dysbiosis on neutrophil recruitment, neutrophil extracellular trap (NET) formation, pancreatic injury, and systemic inflammation. Single-cell sequencing of neutrophils from SPF and GF AP mice was conducted to assess microbiota-driven changes in neutrophil distribution, gene expression, and signaling pathways. Serum cfDNA levels were measured in 50 AP patients and 30 healthy controls. Key findings were validated in vivo and in vitro.

Results

Serum cfDNA (NETs) levels were significantly elevated in AP patients compared to healthy controls and decreased during recovery. GF-treated mice exhibited reduced pancreatic edema, inflammatory infiltration, and acinar cell necrosis, with lower pathology scores. Immunohistochemistry confirmed decreased MPO and F4/80-positive cells in pancreatic tissues, while ELISA showed reduced levels of amylase, lipase, IL-6, and TNF-α in GF-treated mice. Single-cell sequencing revealed a lower proportion of pancreatic neutrophils and an increase in acinar cells in GF-treated mice, whereas the NET formation pathway was activated in SPF-treated mice. The TLR2/MYD88/NLRP3/Caspase-1 signaling cascade, which promotes NET formation, was also activated in SPF mice. Consistent with pathway analysis, CXCL1, CCL9, and CXCR2 expression was upregulated, while Reg3β expression was downregulated in SPF_AP mice. Dual immunofluorescence demonstrated reduced MPO/CitH3 (NETs) formation in GF-treated mice. Neutrophils from GF-treated mice released significantly less cfDNA upon PMA stimulation. Serum cfDNA levels were elevated in both AP-induced mice and AP patients.

Conclusion

Gut microbiota dysbiosis exacerbates AP severity by promoting neutrophil recruitment, activating the TLR2/NLRP3/MYD88/Caspase-1 signaling cascade, and enhancing NET formation, leading to aggravated pancreatic injury and systemic inflammation. These findings highlight microbiota modulation as a potential therapeutic strategy to prevent AP progression.

Disclosure

The authors declare that they have no conflict of interests.

Introduction

Growing evidence suggests an increased morbidity and mortality following an episode of acute pancreatitis (AP)¹. To date, there are no robust biomarkers predicting recurrent acute pancreatitis (RAP), occurrence of post discharge mortality, and the development of diabetes mellitus and pancreatic exocrine insufficiency (PEI). In a European-wide multicenter study the orointestinal microbiome could be used to predict severity of AP². Here, we now aim to assess the early-phase microbiome to predict post-discharge mortality and morbidity.

Aims & Methods

Between 2019 and 2022, buccal and rectal swabs were prospectively collected from 424 patients with acute pancreatitis within 72h after hospital admission in 15 European centers (P-MAPS-study)². Follow-up data regarding the outcome after discharge of the index AP were collected using a RedCAP database until December 2024 via telephone interviews and/or from patient medical records. Endpoints included the association of the buccal and rectal microbiome with post discharge mortality, development of RAP, PEI, diabetes, development of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDA). Existing 16S rRNA (buccal) and metagenomic (rectal) Nanopore sequencing data were reclassified with an updated library. For α- and β-diversity statistics, multiple regression models or distance-based redundancy analysis (db-RDA) were used controlling for potential confounders, such as age, body mass index, medication and concomitant diseases.

Results

Post discharge data were available for 294 participants with a median time of follow-up of 2.7 years. During this period 66 patients (22.4%) developed RAP, 16 (5.4%) CP, 23 (7.8%) PEI, 25 (8.5%) diabetes, 4 (1.4%) PDA, and 20 (6.8%) died after hospital discharge. Sequencing data from 267 buccal and 265 rectal samples were deemed suitable for subsequent analysis. Notably, preliminary analysis revealed significant associations between the buccal and rectal microbiome and post discharge development of diabetes (db-RDA p-value 0.021* and 0.016*) buccal samples and post discharge mortality (db-RDA p-value 0.049*) and rectal samples and RAP (db-RDA p-value 0.011*). No significant differences were found regarding CP, PEI and PDA.

Conclusion

Microbial sequencing collected at the initial phase of acute pancreatitis in the emergency room show significant association with long-term complications after AP and post-discharge mortality emphasizing the role of the orointestinal microbiota in acute pancreatitis.

References

1. Czapári D., Váradi A. & Farkas N. Detailed Characteristics of Post-discharge Mortality in Acute Pancreatitis. Gastroenterology 2023 Sep;165(3):682-695
2. Ammer-Herrmenau C., Antweiler K. & Asendorf T. Gut microbiota predicts severity and reveals novel metabolic signatures in acute pancreatitis. Gut. 2024 Feb 23;73(3):485-495