The kinetics of plasmacytoid dendritic cell accumulation in the pancreas of the NOD mouse during the early phases of insulitis.

In non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, plasmacytoid dendritic cells (pDCs) have a diabetes-promoting role through IFN-α production on one hand, while a diabetes-inhibiting role through indoleamine 2,3-dioxygenase (IDO) production on the other. Little is known about the kinetics and phenotype of pDCs in the NOD pancreas during the development of autoimmune diabetes. While para/peri-insular accumulation of conventional dendritic cells (cDCs) could be observed from 4 weeks of age onwards in NOD mice, pDCs only started to accumulate around the islets of Langerhans from 10 weeks onwards, which is concomitant with the influx of lymphocytes. NOD pancreatic pDCs showed a tolerogenic phenotype as assessed by their high expression of IDO and non-detectable levels of IFN-α and MxA. Furthermore, expression of the pDC-attracting chemokines CXCL10 and CXCL12 was significantly increased in the NOD pancreas at 10 weeks and the circulating pDC numbers were increased at 4 and 10 weeks. Our data suggest that a simultaneous accumulation of IDO(+) pDCs and lymphocytes in the pancreas in 10 weeks old NOD mice, which may reflect both an immunogenic influx of T cells as well as a tolerogenic attempt to control these immunogenic T cells

Accumulation of mDCs and pDCs in the NOD pancreas.

<p>The localization of mDCs and pDCs in the C57BL/6, NOR and NOD pancreas at the age of 4, 10 and 20 weeks was determined by immunohistochemical detection. Pictures show CD11c and Siglec-H expression in the pancreas of mice from 4, 10 and 20 weeks of age, magnification 200x (A). Bar graphs represent the mean insulitis score of CD11c⁺ (B) and Siglec-H⁺ cells (C) in the pancreas. Data are presented as average+SEM, n = 5 mice, * p<0.04, ** p<0.01, *** p<0.001 as determined by the unpaired Mann-Whitney U test.</p

Expression of pDC receptors and chemokines in the pancreas.

<p>Histograms represent the CXCR3 and CXCR4 expression on pDCs (CD11b⁻PDCA-1⁺ cells) in the pancreas of 10 weeks of age (A). Bar graph represents the geometric MFI of CXCR4 on pDCs in the pancreas of 10 weeks of age (B). Bar graphs represent the protein level of CXCL9 (C57BL/6 and NOD), CXCL10 and CXCL12 (C57BL/6, NOR and NOD) in pancreas lysates of 4 and 10 weeks (C-E). Data are presented as average+SEM, n = 5–10 mice, * p<0.04, ** p<0.01 as determined by the unpaired Mann-Whitney U test.</p

Increased pDC numbers in the NOD pancreas at 10 weeks of age.

<p>The presence of pDCs in the C57BL/6, NOR and NOD pancreas at the age of 4 and 10 weeks was determined by flow cytometry. Dot plots show the CD11b and PDCA-1 expression on sorted CD45⁺ cells from the pancreas (A). Bar graphs represent the percentage and the absolute number of CD11b⁻PDCA-1⁺ cells in the pancreas at 4 (B–C) and 10 weeks (D–E). Histograms represent the B220, CD80 and CD86 expression on pDCs (CD11b⁻PDCA-1⁺ cells) in the pancreas of 10 weeks (F). Bar graphs represent the geometric MFI of CD80 (G) and CD86 (H) on pDCs. Data are presented as average+SEM, n = 5–6 mice, *p<0.04, ** p<0.01 as determined by the unpaired Mann-Whitney U test.</p

Enhanced IDO and a decreased PD-L1 expression in NOD pancreas pDCs.

<p>Pictures show the immunohistochemical detection of IDO in the pancreas of NOD mice at 4, 10 and 20 weeks of age, magnification 200x (A). The pancreas of NOD mice was stained for Siglec-H (green), IDO (red) and DAPI (blue) by immunofluorescence, magnification 400x (B). Histogram represents the PD-L1 expression on pDCs (CD11b⁻PDCA-1⁺ cells) in the pancreas of C57BL/6, NOR and NOD mice of 10 weeks of age (C). Bar graph represents the geometric MFI of PD-L1 on pDCs (D). Data are presented as average+SEM, n = 5 mice, * p<0.04, ** p<0.01 as determined by the unpaired Mann-Whitney U test.</p

Increased percentage of pDCs in blood of NOR and NOD mice.

<p>The presence of pDCs in the blood of C57BL/6, NOD and NOR of 4 and 10 weeks was determined by flow cytometry. Dot plots show the CD11b and PDCA-1 expression (A). Bar graphs represent the percentage of pDCs (CD11b⁻PDCA-1⁺ cells) at 4 weeks (B) and 10 weeks (C). Histograms represent the B220, CD80, CD86, CXCR3, CXCR4 and PD-L1 expression on pDCs in the pancreas of 10 weeks (D). Bar graphs represent the MFI of CD80 (E) and CD86 expression (F) on pDCs at 10 weeks. Data are presented as average+SEM, n = 4–10 mice, * p<0.04, ** p<0.01, *** p<0.001 as determined by the unpaired Mann-Whitney U test.</p

Cytokine production by <i>in vitro</i> LPS-stimulated pancreas DCs.

<p>DCs were isolated from C57BL/6 and NOD pancreas at an age of 5 weeks. DCs were cultured for 18 h in the presence of LPS or under control conditions (PBS). IL-6, IL-10, IL-12p70 and TNF-α were measured in the supernatant. Bars are represented as median with IQR with N = 4 of 10 pooled mice per sample, P-values were determined by Mann-Whitney U test; *p<0.05.</p

Differentially expressed genes among the <i>in vitro</i> LPS-stimulated pancreatic C57BL/6 and NOD CD8α⁻ DCs.

<p>Differentially expressed genes (DEGs) among the <i>in vitro</i> LPS-stimulated pancreatic C57BL/6 and NOD CD8α⁻ DCs were identified by using a multivariate permutation test. The table shows a list of highly significant DEGs summarized per functional category. Annotation and functional category were provided by Ingenuity Pathway Analysis. Genes in <b>bold</b> were also significant in the q-PCR validation.</p><p>Differentially expressed genes among the <i>in vitro</i> LPS-stimulated pancreatic C57BL/6 and NOD CD8α⁻ DCs.</p

LPS inducible genes in NOD and C57BL/6 pancreatic DCs.

<p>DCs were <i>in vitro</i> stimulated for 18 h with LPS (or PBS). LPS-inducible genes for each strain were identified were identified by using a multivariate permutation test. The venn diagram shows the total number of LPS-inducible genes for each strain, the common LPS-inducible genes and the unique LPS-inducible genes per strain (A). DCs were isolated from C57BL/6 and NOD pancreas at an age of 5 weeks. DCs were cultured for 18 h in the presence of LPS or under control conditions (PBS). Relative expression of Ki67, Reg1, CCR2 and CCR5 were measured by Q-PCR (B). The box represents the interquartile distance, with a line on the median. The wiskers represent the 5 to 95 percentiles; N = 6 of 10 pooled mice per sample, P-values were determined by Mann-Whitney U test; *p<0.05.</p

Graphical representation of the samples.

<p>Dendrogram of the samples after hierarchical clustering (Euclidean distance with average linkage) of the genome-wide gene expression profiles of the pancreatic CD8α⁻ DCs under steady-state conditions and after <i>in vitro</i> LPS stimulation (A). PCA analysis was used to compare all groups. Separation of the groups by principle components 1–3 that show 65.3% of the variance (B). Heatmap with hierarchical clustering (Euclidean distance with average linkage) of the DEG genes among the NOD (Cyan) and C57BL/6 (Orange) pancreatic CD8α⁻ DCs (C). Normalized 2 log-transformed probeset expression values are visualized as a gradient from low (blue) to high (red) expression.</p