Fig 2: Chronic pancreatitis leads to pancreatic tissue remodeling.a Flow cytometry analysis of isolated leukocytes from pancreatic tissue showed a significant increase of CCR2+ cells (p = 0.0032, con n = 7/CP n = 15). b CD11b+ labeling identified that also cells of the innate immune system infiltrate into the damaged pancreas (p < 0.0001, con n = 7/CP n = 15). c Most of these cells were positive for CD206 (p < 0.0001, con n = 7/CP n = 15) or CD163 (p < 0.0001, con n = 7/CP n = 15), which characterized them as alternatively activated macrophages. d Labeling of CP tissue for CD206 illustrates their increase in pancreatic tissue of mice. A quantative analysis showed that ~9% of all cells were positive for CD206 in CP tissue (p < 0.0001, con n = 5/CP n = 9), scale bars represent 20 µm. e IL-33 expression of CD206+ macrophages was demonstrated by immunofluorescent labeling, scale bars represent 20 µm. f This cytokine is a strong inducer of ILC2s (lin-CD45+CD127+CD90+GATA3+) which were also significantly elevated in the pancreas of CP mice (p = 0.0002, con n = 7/CP n = 15). g The replacement of exocrine pancreas by fibrotic tissue in CP was illustrated by azan blue staining of the pancreas, scale bars represent 50 µm. h Flow cytometry analysis of isolated pancreatic cells for PSC marker proteins CD271 (p < 0.0001, con n = 7/CP n = 15) and GFAP (p < 0.0001, con n = 7/CP n = 15) identified a significant increase. i Serum collagen levels were significantly elevated in CP (p = 0.0433, con n = 7/CP n = 13) and collagen deposition and tissue fibrosis were observed in histology. j Masson Goldner trichrome staining was utilized to evaluate the tissue remodeling during CP, scale bars represent 50 µm. k Area quantification (in%) by pattern quant software illustrates the loss of exocrine and endocrine tissue (red) and the increase of fibrotic area (green) and of free space, which reflects fatty tissue and edema (yellow). l Area quantification of exocrine and endocrine active tissue (p < 0.0001, con n = 6/CP n = 10) versus fibrosis (p < 0.0001, con n = 6/CP n = 10) demonstrated the significant changes in organ architecture. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk (a–f, h, i, l). Source data are provided as a Source Data file.

Fig 3: Immunofluorescence detection of Runx3 co-localization with CCR2+ immune cells in IAV-infected mouse lungs. (A–C) Immunofluorescence (IF) detection of Runx3 in IAV-infected mouse lungs. Lung sections from saline-treated (Mock, A) or H1N1-infected mice on day 7 pi (B,C) were subjected to IF staining with a Runx3 monoclonal antibody followed by Alexa Fluor 568-labelled goat anti-mouse secondary IgG (n = 6/group). Runx3 immunoreactivities are stained red (B, green arrows), cell nuclei stained blue by DAPI, and merged pink (C). (D–F) Co-localization of Runx3 with CCR2+ immune cells in IAV-infected mouse lungs. Lung sections from H1N1-infected mice were subjected to IF staining with a FITC-labeled CCR2 polyclonal antibody and a Runx3 monoclonal antibody followed by Alexa Fluor 568-labelled goat anti-mouse secondary IgG (n = 4). CCR2 is stained green (D, white arrows), Runx3 red (E, green arrows), and merged yellow (F, white arrows).

Fig 4: Distribution and localization of identified macrophage subgroups by flow cytometry and immunofluorescence.(A) Bar diagram showing the percentage of F4/80+ cells within the CD45+ population throughout the epididymal regions, assessed by flow cytometry (n=8, mean ± SD). (B) Stacked bar diagram displaying the percentages of identified macrophage subtypes within the F4/80+ population throughout epididymal regions assessed by flow cytometry. Markers were selected based on single-cell RNA sequencing (scRNASeq) results (n=4). (C) Confocal microscopy images of F4/80 staining (purple) on Cx3cr1GFPCcr2RFP adult reporter mice. The majority of interstitial and intraepithelial CX3CR1+ cells were F4/80+. Arrowheads indicate a small fraction of intraepithelial CX3CR1+ F4/80 cells found within the initial segment (IS). Arrows indicate interstitial F4/80+ cells that were CX3CR1- and CCR2+ within caput, corpus, and cauda epididymides. Asterisks (*) label a small fraction of F4/80 single positive cells (CX3CR1-CCR2-) found in the corpus and cauda. Scale bar 50 µm (L=lumen). (D) Confocal microscopy images of MHC-II staining (purple) on Cx3cr1GFPCcr2RFP adult reporter mice. Asterisks (*) indicate intraepithelial CX3CR1+MHC-II- cells within the IS and caput epididymides. Arrowheads indicate CX3CR1+MHC-II+ cells, lining the epididymal duct within the IS and situated within the epithelium within caput, corpus, and cauda epididymides. Arrows indicate interstitial CX3CR1+MHC-II+CCR2+ cells additionally found within corpus and cauda epididymides. Scale bar 50 µm (L=lumen). (E) Confocal microscopy images of CD163 staining (purple) on Cx3cr1GFPCcr2RFP adult reporter mice in corpus and cauda epididymides. Arrows indicate CD163 single positive cells that were found in close proximity to vessels within the corpus and cauda. Arrowheads indicate CD163+CCR2+ cells found solitarily distributed within the interstitium in the corpus and cauda. Scale bar 50 µm.

Fig 5: Quantification and localization of identified immune cell populations among epididymal regions (scale bar 20 µm).(A) Distribution (assessed by flow cytometry n=4-8, bar diagram showing mean ± SD) of total leukocytes (CD45+ cells) and localization within the initial segment and corpus, as shown by immunostaining of CD45. (B–H) Quantification and localization of the following immune cell populations were assessed by flow cytometry and immunostaining using selected markers (n=4–8, mean ± SD). The following markers were used: CD45, F4/80, CD11B, Ly6C, MHC-II, CLEC9A, CD209A, CD163, CCR2, CX3CR1 for identifying myeloid cell populations, and CD45, B220/CD45R, CD3, TCRß, TCR?d, NK1.1 for lymphoid cell populations (further panel information and gating strategies are displayed in the Methods section and supplemental material, respectively). Representative immunofluorescence images are displayed from the corpus (CS) regions: (B) total macrophages (F4/80+, red), located in the interstitial, intraepithelial, and peritubular compartments, (C) monocytes (Ly-6C+), located in the peritubular compartment, (D and E) conventional dendritic cells cDC 1 (Clec9a+) and 2 (DC-Sign/CD209a+), (F) NK cells (NK1.1+ for flow cytometry and NCR1 for immunostaining), located in the intraepithelial compartment, (G) B cells (B220/CD45R+ for flow cytometry and CD19+ for immunostaining), (H) T cells that were further segregated into aß T cells (TCRß+, red) and ?d T cells (TCR?d+, green), scale bar 20 µm.