Fig 2: Blockage of the IL1R1 signaling pathway by Anakinra reduces fibrosis in a mouse model of CP. (A) Chronic pancreatitis was induced by repetitive caerulein injections over 4 weeks in wild type mice. The treatment group received a single application of 10 mg/kg bodyweight Anakinra, always before the first caerulein injection, whereas the control group received PBS (n = 10 each group). (B) H&E histology and azan blue staining of CP tissue sections illustrate the tissue architecture and fibrosis. (C) Quantification of azan blue fibrotic area in CP tissue. (D) Immunofluorescent labeling of fibroblasts in CP tissue sections of mice by FGFR (green), FAP (red), and αSMA (white). (E) Quantification of immunofluorescent labeling of FGFR+ cells and FAP+ cells in CP tissue of Anakinra and PBS‐treated mice. (F) Immunofluorescent labeling of CD206+ macrophage and CCR2+ immune cells in CP tissue sections of mice. (G) Quantification of immunofluorescence‐labeled CD206+ and CCR2+ cells in CP tissue. Differences were tested for statistical significance by unpaired student's t‐test for independent samples, and significance levels of p<0.05 are marked by an asterisk. (H) Labeling and quantification of α‐amylase+ cells in CP tissue. (I) Measurement of fecal elastase and chymotrypsin activity in RFU/µg faeces (CP groups n = 10, untreated control group n = 5). (J) Differences in body weight during induction of CP in mice (CP groups n = 10, untreated control group n = 5). (K–L) Quantitative RT‐PCR of cells isolated from wild type CP mice, Il1a‐/‐ CP mice and Anakinra treated CP mice (n = 5) for Acta2, a marker for activated stellate cells, and the genes Col3a1, Col6a5 and Col1a1 encoding extracellular matrix proteins and genes encoding pro‐fibrotic receptors such as Il13ra1, Pdgfrb and Tgfbr3. Differences were tested for statistical significance by one way ANOVA followed by Dunnett's multiple comparisons test. Significance levels of p < 0.05 are marked by an asterisk. (M) Graphical abstract summarizes the impact of IL‐1α and its blockage on disease severity of AP as well as fibrinogenesis during CP.

Fig 3: IL‐1α activates crosstalk between pancreatic fibroblast and immune cells. (A) Flow cytometry analysis of blood leukocytes, isolated from wild‐type and Il1a‐/‐ mice at eight and 24 h after onset of AP. The population of CD11b+/Ly6g− reflects monocytes; CD11b+/Ly6g+ cells were identified as neutrophils, whereas CD4+ cells marked the population of T helper cells (n = 4‐5). (B, C) Histogram and dot plot illustrate the expression of CCR2 and CD18 on the surface of CD11b+ cells in blood, 8 h after onset of pancreatitis (n = 5). (D) Measurement of the chemokines CCL2 and CXCL5 in serum of mice 8 h after onset of disease (n = 10). (E) Measurement of the cytokines IL‐6 and TNF‐α in serum of mice 8 h after induction of pancreatitis (n = 10). (F) Measurement of chemokines and cytokines in the culture media of isolated pancreatic fibroblasts which were stimulated with 10 ng/mL IL‐1α for 24 h (n = 5). (G) The supernatant of isolated pancreatic fibroblasts ± IL‐1α stimulation for 24 h was added to BMDMs for an additional 24 h. RNA was isolated from the BMDMs culture, and expression of Ccr2, Sell, Cd80, Cd86, Cd44, and Ccr5 was analysed by RT‐qPCR (n = 4). Differences were tested for statistical significance by unpaired student's t‐test for independent samples, and significance levels of p < 0.05 are marked by an asterisk.

Fig 4: Deficiency of IL‐1α ameliorates the disease severity of acute pancreatitis in mice. (A) Acute pancreatitis was induced by hourly i.p. injections of caerulein in wild‐type and Il1a‐/‐ mice as shown schematically (n = 10 for 8 and 24 h each strain). (B) H&E staining of pancreatic tissue sections. (C) Evaluation of H&E histology by histoscore. (D) Measurement of amylase activity in serum of mice. (E) Immunofluorescent labeling of CD68 and CCR2 in pancreatic tissue sections. (F) Quantification of CD68+ and CCR2+ cells in pancreatic tissue. (G) Measurement of myeloperoxidase (MPO) activity in lung tissue homogenates. (H) H&E staining of lung tissue sections. Differences were tested for statistical significance by unpaired Student's t‐test for independent samples, and significance levels of p < 0.05 are marked by an asterisk.

Fig 5: Damaged acinar cells release Interleukin 1 alpha but not Interleukin 1 beta. (A) Pancreatic acinar cells were isolated from wild type mice and stimulated with 0.001 mm CCK for 60 min. Activity of trypsin, amylase, calpain, and caspase 1 was measured in cell homogenate (n = 5). (B) Determination of acinar cell death by measurement of cell count and propidium iodide (PI) uptake in acinar cells 60 min after stimulation with 0.001 mm CCK (n = 4). Caspase 3 activity was determined 60 min after CCK stimulation in cell homogenate (n = 4). (C) Reverse Transcription‐quantitative PCR (RT‐qPCR)‐analysis of Il1a and Il1b was performed from isolated pancreatic acinar cells following stimulation with 0.001 mm CCK for 1 h and 2 h (n = 5). (D) The release of IL‐1α and IL‐1β into medium was measured in acinar cells isolated from wild type and Il1a‐/‐ mice, after stimulation with 0.001 mm CCK for 1 h and 2 h (n = 5). Differences were tested for statistical significance by unpaired student's t‐test for independent samples, and significance levels of p < 0.05 are marked by an asterisk. (E) Immunohistochemical labeling of IL‐1α on healthy pancreas tissue sections of wild‐type and IL‐1α deficient mice (Il1a‐/‐). (F) IL‐1β release was measured in the supernatant of bone marrow‐derived macrophages (BMDMs) after stimulation with LPS and nigericin. Furthermore, IL‐1β release was measured in the supernatant of CCK‐stimulated acinar cells and BMDMs co‐incubated with these damaged acinar cells (n = 4). Differences were tested for statistical significance by one‐way ANOVA corrected for multiple testing by Tukey's Post‐hoc‐test, significance levels of p < 0.05 are marked by an asterisk.