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. 2015 Sep 21;212(10):1497-512.
doi: 10.1084/jem.20150496. Epub 2015 Sep 7.

The Pancreas Anatomy Conditions the Origin and Properties of Resident Macrophages

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Free PMC article

The Pancreas Anatomy Conditions the Origin and Properties of Resident Macrophages

Boris Calderon et al. J Exp Med. .
Free PMC article

Abstract

We examine the features, origin, turnover, and gene expression of pancreatic macrophages under steady state. The data distinguish macrophages within distinct intrapancreatic microenvironments and suggest how macrophage phenotype is imprinted by the local milieu. Macrophages in islets of Langerhans and in the interacinar stroma are distinct in origin and phenotypic properties. In islets, macrophages are the only myeloid cells: they derive from definitive hematopoiesis, exchange to a minimum with blood cells, have a low level of self-replication, and depend on CSF-1. They express Il1b and Tnfa transcripts, indicating classical activation, M1, under steady state. The interacinar stroma contains two macrophage subsets. One is derived from primitive hematopoiesis, with no interchange by blood cells and alternative, M2, activation profile, whereas the second is derived from definitive hematopoiesis and exchanges with circulating myeloid cells but also shows an alternative activation profile. Complete replacement of islet and stromal macrophages by donor stem cells occurred after lethal irradiation with identical profiles as observed under steady state. The extraordinary plasticity of macrophages within the pancreatic organ and the distinct features imprinted by their anatomical localization sets the base for examining these cells in pathological conditions.

Figures

Figure 1.
Figure 1.
Islet-resident leukocyte profile under steady state. Islets from 8-wk-old B6 mice were analyzed by flow cytometry by gating on single CD45+ cells (see Fig. S1). (A and B) Islet-resident myeloid cells were cell surface positive for CD11c, MHC-II, F4/80, CD11b, CD64, lysozyme (LyzM)-GFP, CX3CR1-GFP, and CD68 (A) and negative for CD206, CD301, B220, SiglecH, CD19, and Ly6C (B). Data are representative of six independent experiments. LyzM and CX3CR1 were examined in LyzM-GFP and CX3CR1-GFP reporter mice. (C) Expression of GFP in CD45+ CD11c+ MHC-II+ gated myeloid cells from islets, spleen, and PLNs of Zbtb46-GFP reporter mice. Data are representative of three independent experiments. (D) Shown is the number of myeloid cells (CD11c+) in whole islets isolated from Notch2f/f CD11c-cre reporter mice. Each dot represents the number of CD11c+ cells per individual islet, and bars indicate the median. Data are representative of two independent experiments evaluating 50 islets per group. (E and F) RT-PCR evaluation on sorted islet-resident macrophages (F4/80+) and the nonleukocyte compartment including endocrine and endothelial cells (CD45) for the expression of leukocyte and nonleukocyte genes. Data are pooled from three independent experiments using 10 mice per sort. Bars indicate the mean ± SEM. P-values were calculated using unpaired Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, P ≥ 0.05. Sample size included two to three samples per experimental group.
Figure 2.
Figure 2.
Exocrine pancreas leukocyte profile under steady state. (A) Exocrine pancreas (stroma) gating strategy on live single CD45+ cells and their profile based on F4/80 and CD11b surface expression (F4/80+ CD11b+ profile). (B) The three populations identified by F4/80+ CD11b+ profile (F4/80+ CD11b+, CD11b+ F4/80, and F4/80 CD11b; from A, right) were analyzed further and highlighted in different colors (red, blue, and black, respectively). (C) The populations in B were gated on CD11c+ MHC-II+ for further evaluation. (D) Stromal macrophages (F4/80+ CD11b+, highlighted in red; left) were analyzed for expression of CD206 and CD301 (middle). Control isotype antibodies are shown on the right. Data are representative of six independent experiments. (E) Antigen presentation and T cell activation assay testing sorted macrophages (MΦ, F4/0+ CD11b+) from islets and the two stromal sets (CD206+ and CD206). Macrophages were incubated with no antigen or titrating concentrations of either LLO peptide 190–201 or LLO protein (WW nonhemolytic variant). T cell activation was assessed by IL-2–driven [3H]thymidine uptake and expressed in counts per minute (CPM). Splenic APCs were used for controls. Data presented are representative of two independent experiments performed in duplicate or triplicate. P-values were calculated using the unpaired Student’s t test: **, P < 0.01; ***, P < 0.001; NS, P ≥ 0.05. Sample size included two to three samples per experimental group. (F) Mean fluorescent intensity (MFI) for MHC-II of stromal macrophages (CD206+ and CD206). Results are pooled from three independent experiments. P-values were calculated using the Mann–Whitney U test: *, P < 0.05. Sample size included four independent experiments per group. Bars show the mean ± SEM.
Figure 3.
Figure 3.
Pancreatic macrophage activation profile by RT-PCR under steady state and high carbohydrate and high fat diet. (A) Steady state islet macrophages (F4/80+ CD11b+) and stromal macrophages (F4/80+ CD11b+ CD206+ and F4/80+ CD11b+ CD206) from 8-wk-old B6 mice were sorted and examined by qRT-PCR. CD45 (CD45neg) islet cells were used as controls for comparison. M1 (Il1b and Tnfa), M2 (Il10, Arg1, Mrc1, Mgl1, Mgl2, Ym1, and Fizz1), and control (Ins1, Ptprc, and Emr1) transcripts were assessed. Transcript expression is pooled from three independent experiments. (B) Pancreatic macrophages were maintained on high carbohydrate or high fat diet for 5 wk and assessed as in A for M1 and M2 transcripts. Comparisons of transcript expression for each pancreatic macrophage subset from two experiments are shown. All bars show the mean ± SEM. P-values were calculated using unpaired Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, P ≥ 0.05. Sample size included two to three samples per experimental group.
Figure 4.
Figure 4.
Pancreatic macrophages in B6.CSF-1–deficient mice (op/op). (A) Immunofluorescence microscopy examination of CD11c+ cells in islets of WT littermates and op/op mice at 8 wk of age. Each dot represents the number of CD11c+ cells per individual islet (50 examined islets per group). The experiments shown are representative of four independent evaluations. (B) Islet size was determined for islets shown in A. (C) Islets and pancreatic stroma from 8-wk-old WT littermates (Control) and op/op and IL-34−/− mice analyzed by flow cytometry. Islets and stroma were gated on singlets for CD45+ (top) and plotted for F4/80 and CD11b (middle) and for CD206 and CD301 (bottom). (D and E) Quantitative absolute numbers from pooled independent experiments showing total CD45+ cells (D) and macrophages (gated on F4/80+ CD11b+; E) in islets and stroma of control, op/op, IL-34−/−, and Ccr2−/− mice. (F) Percentages of stromal CD206+ and CD206 macrophages from independent experiments in control, op/op, and Ccr2−/− mouse strains. All scatter plots show bars that represent mean values. P-values were calculated using the Mann–Whitney U test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, P ≥ 0.05.
Figure 5.
Figure 5.
Pancreatic macrophages in early postnatal life. Islets and stromal macrophages from 1-, 2-, and 4-wk-old mice (gated on live singlets for CD45+) were evaluated for expression of F4/80, CD11b, CD11c, MHC-II, Ly6C, CD206, and CD301. Islet macrophages were gated from all CD45+ islet cells, whereas pancreatic stroma macrophages were gated on F4/80+ CD11b+ profile (indicated by arrows). Data are representative of two independent experiments at 1 wk and three independent experiments at 2 and 4 wk, each having four to eight mice per time point.
Figure 6.
Figure 6.
Ontogeny of pancreatic macrophages. (A) Flt3-cre × RosamTmG reporter mice (13–14 wk of age) were analyzed by flow cytometry and gated on singlets for CD45+ cells and macrophages (F4/80+ CD11b+) from islets, stroma, and PLNs to evaluate GFP and tdTomato (TdTom+) expression. Pooled results (right) from seven independent evaluations are shown. (B) Csf1r-Mer-iCre-Mer × RosamTmG reporter mice were gavaged with tamoxifen at E8.5 to label the progeny of yolk sac macrophages (TdTomato GFP+). Islets, stroma, and PLN macrophages were evaluated at 10 wk of age to determine whether yolk sac progeny persisted into adulthood. Pooled results (right) from two experiments show the percentage of labeled (GFP+) macrophages (islets, stroma, and PLNs), brain microglia, and blood monocytes. Bars indicate the mean ± SEM. (C) Pancreatic stroma of Flt3-cre × RosamTmG reporter mice (13–14 wk of age) were analyzed by flow cytometry and gated on macrophages (F4/80+ CD11b+) for their TdTom+ or GFP+ expression (R1 and R2 gates, respectively) to evaluate CD206 and CD301 profiles. Data are representative of two independent experiments. (D) Immunofluorescence examination of pancreatic sections from 10-wk-old mice showing stromal macrophage distribution (stained for CD68 in red), their positive or negative expression for CD206 (green), and nuclear staining by DAPI (blue). Higher magnifications of the periductal area (boxes) are shown below. Images are representative of 8–10 sections from two examined mice. Bars, 100 µm. (right) Quantitative analysis showing distribution of CD206+ and CD206 macrophages in stroma and ductal-associated areas. Bars indicate the mean. P-values were calculated using the Mann–Whitney U test: ***, P < 0.001; NS, P ≥ 0.05.
Figure 7.
Figure 7.
Pancreatic macrophages after parabiosis and in situ maintenance. (A) Parabiotically joined CD45.2+ (left) and congenic CD45.1+ mice (right) were examined at 6 wk of parabiosis. Islets and stroma macrophages (gated on F4/80+ CD11b+, and CD206+ CD301+ or CD206 CD301 expression [R1 and R2 gates, respectively]: middle and bottom) along with their CD45.1+ or CD45.2+ expression are shown. The top scatter dot plot panel shows pooled quantitative pancreatic macrophage chimerism percentages at 6 wk of parabiosis for islet chimerism (total of 12 mice from three independent experiments, pooling 2 mice per parabiont side) and stromal CD206+ and CD206 macrophages (from two independent experiments using single parabionts). The bottom scatter dot plot panel shows pooled quantitative tissue macrophage chimerism percentages (PLNs and spleen) from the same experiments and compared with blood monocyte chimerism for each parabiont. Bars represent mean values. (B) Islet, pancreatic stroma (CD206+ and CD206), PLN, and BM macrophage (MΦ) proliferation was assessed after 7 d of ad libitum BrdU treatment. Histograms of macrophages (gated on CD45+; F4/80+ and CD11b+) with or without BrdU treatment (blue and red histogram lines, respectively) are shown. Bars indicate percentages of BrdU incorporation. (C) Pooled results for BrdU incorporation as shown in B from three independent experiments. Bars indicate the mean ± SEM. P-values were calculated using the Mann–Whitney U test: *, P < 0.05; NS, P ≥ 0.05.
Figure 8.
Figure 8.
Replacement of pancreatic macrophages after genotoxic insult. Lethally irradiated CD45.2 B6 mice received CD45.1 congenic B6 BM transplant. (A and B) CD45+ gated islet cells (A) and pancreatic stroma cells (B) were examined at different chimerism time points. (A, top) CD45+ cells showing F4/80 and CD11b profile. (middle) CD45+ cells evaluated for the presence of CD45.1+ (donor) and CD45.1 (host). (bottom) Histograms of CD45.1+ gated cells (donor) for Ly6C expression. (B, top) CD45+ cells showing F4/80 and CD11b profile. (middle) F4/80+ CD11b+ gated cells evaluated for CD45.1+ (donor) and CD45.1 (hosts) expression. (bottom) Histograms of CD45.1+ F4/80+ CD11b+ gated cells (donor macrophages) for Ly6C expression. Data are a representative of three independent experiments.
Figure 9.
Figure 9.
Pancreatic macrophage profiles after genotoxic insult. (A and B) Stroma from WT CD45.2 (A) and CD45.2 mice receiving CD45.1 BMT (CD45.1 BMT; B) after genotoxic insult and evaluated at 9 wk after transplant. (top) Stroma cells (gated on singlets CD45+) showing F4/80 and CD11b profile. (middle) Histograms of F4/80+ CD11b+ gated cells (from top panels) evaluated for the presence of CD45.1+ (donor) and CD45.1 (host) macrophages. (bottom) Histograms of F4/80+ CD11b+ gated cells from CD45.2 WT and CD45.1 BMT showing CD206 and CD301 expression. Data are a representative of two independent experiments. (C) Sorted islet macrophages (CD45+ F4/80+ CD11b+) and stromal macrophages (CD45+ F4/80+ CD11b+ CD206+ and CD45+ F4/80+ CD11b+ CD206) from CD45.2 WT and CD45.1 BMT mice at 9 wk after transplant. CD45-negative (CD45neg) islet cells were used as controls for comparison. M1-like (Il1b and Tnfa), M2-like (Il10, Arg1, Mrc1, Mgl1, Mgl2, Ym1, and Fizz1), and control (Ins1, Ptprc, and Emr1) transcripts were assessed. Transcript expression is pooled from two independent experiments performed in duplicate. Bars represent the mean ± SEM. P-values were calculated using the unpaired Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, P ≥ 0.05. Sample size included two samples per experimental group.

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