Fig 2: Depletion of Treg cells strengthen the pancreatic remodeling during CP.a–d Quantification of infiltrating immune cells by evaluation of immunofluorescence labeling of CD4 (p = 0.0172, PBS n = 5/DT n = 5), CD8a (p = 0.0035, PBS n = 6/DT n = 4) and CD206 (p = 0.0451, PBS n = 7/DT n = 8) shows that their numbers were significantly elevated in DT-treated animals, scale bars represent 20 µm. e Flow cytometry analysis of leukocytes isolated from pancreatic tissue provides evidence for increased alternatively activated CD206+ M2 macrophages in DT-treated mice (p = 0.0003, PBS n = 8/DT n = 11). f Gene expression analysis by RT-qPCR confirmed elevated expression of Mrc1 (p = 0.0064, PBS n = 9/DT n = 7) and Ym1 (p = 0.0003, PBS n = 11/DT n = 6) encoding anti-inflammatory macrophage markers, increased transcript levels for the anti-inflammatory cytokine IL-10 (p = 0.0020, PBS n = 8/DT n = 6) whereas mRNA amounts of the pro-inflammatory IL-1ß cytokine (PBS n = 6/DT n = 5) underlines this observation. Transcript levels as determined by RT-qPCR were normalized using Rn5s as internal calibrator gene and were related to the corresponding mRNA amounts in control mice. g M2-like macrophages in DT-treated mice trigger pancreatic stellate cells (PSC), as shown by staining of aSMA and collagen 1, scale bars represent 20 µm. h, i Flow cytometry analysis of dissociated pancreatic tissue shows a significant higher number of GFAP+ (p = 0.0059, PBS n = 7/DT n = 11) and CD271+ cells (p = 0.0140, PBS n = 8/DT n = 11), j and gene expression analysis of aSMA confirms activation of PSC after depletion of Treg cells during CP (p = 0.0373, PBS n = 12/DT n = 8). k Finally, the higher PSC number causes increased fibrosis and pronounced loss of acinar cells, scale bars represent 100 µm. l, m Quantitative analyses performed by Masson Goldner demonstrate a significant increase in fibrosis (p = 0.0042, PBS n = 6/DT n = 7) accompanied by decreased acinar cell numbers (p = 0.0008, PBS n = 6/DT n = 7) in the absence of Treg cells. 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 (b–f, h–j, l, m). Source data are provided as a Source Data file.

Fig 3: Infant episodic learning increases neuronal glutathione reductase (GR) activity.(a) Quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) of reduced (GSH) and oxidized (GSSG) forms of glutathione and the GSH/GSSG ratio in whole hippocampus from PN17, PN24, and PN80, in the untrained condition and 1 hr after inhibitory avoidance (IA) training (n = 7 rats per group). Data are expressed as mean percentage ± s.e.m. of the value in naive group at each age. ***p < 0.001 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). (b) Representative examples and densitometric Western blot analyses of GR, glutathione peroxidase isozyme 1 (GPx), glutamate-cysteine ligase (catalytic subunit GCLC, and regulatory subunit GCLM), and glutathione synthetase (GSy), carried out with whole-protein extracts of dHC from postnatal day 17 naive rats (PN17-N) and rats trained in IA at PN17 and euthanized 1 hr (Tr-1h) or 24 hr (Tr-24h) after training (n = 5–6 rats per group; two independent experiments). Actin was used as the loading control. Data are expressed as mean percentage ± s.e.m. of the value in PN17 naive rats. p > 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test). (c) Mean activity of GR, GPx, GCL, and GSy (expressed as the quantity, in nmol, of substrate transformed per minute and normalized to the total quantity of proteins in each sample, in mg) assayed in dHC protein extracts obtained from rats trained in IA at PN17 and euthanized 15 min, 1 hr, 24 hr, or 7 days after training (Tr-15min, Tr-1h, Tr-24h, and Tr-7d, respectively) (n = 6 rats per group; two independent experiments). To account for developmental differences, two groups of naive (N) rats were used: PN17 and PN24 (n = 6 rats per group). Data are expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, ***p < 0.001: significance vs. PN17 naive rats (one-way ANOVA followed by Dunnett’s multiple comparisons test); p > 0.05 for the comparison between PN24-N and Tr-7d groups (two-tailed unpaired Student’s t-test). (d) GR activity carried out with dHC protein extracts obtained from PN17 naive rats (PN17-N), from rats exposed to an immediate footshock without IA-context exposure (shock-only) and euthanized 15 min later (SO-15min) or from rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). (e) GR activity assayed in dHC protein extracts obtained from PN24 naive rats (PN24-N) and rats trained in IA at PN24 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN24 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). (f) GR activity assayed in dHC protein extracts obtained from PN80 naive rats (PN80-N) and rats trained in IA at PN80 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN80 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). (g) Representative examples and densitometric Western blot analyses of NeuN and GFAP carried out with whole-protein extracts of fluorescence-activated cell sorting (FACS)-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled counter-selected cells from the dHC of PN17 naive rats (n = 4 x 3 rats per group; two independent experiments). Actin was used as the loading control. NeuN intensity values are expressed as mean percentage ± s.e.m. of the value in neurons (NeuN+) group; GFAP intensity values are expressed as mean percentage ± s.e.m. of the value in astrocytes (GFAP+) group. ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). GR activity carried out with whole-protein extracts of FACS-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled cells from the dHC of PN17 naive rats (PN17-N) and of rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 5 x 3 rats per group; two independent experiments). GR activity is expressed as mean nmol/min/mg protein± s.e.m. *p < 0.05 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). See Figure 7—source data 1, Figure 7—source data 2 for numerical values and detailed statistical information. Figure 7—source data 1.Numerical values. Figure 7—source data 2.Detailed statistical information.

Fig 4: 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.