Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes

Abstract

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.

Keywords: CXCL10; alpha-cells; chemokines; pancreas; type 1 diabetes.

Figure 1

CXCL10 is increased in pancreatic islets of new-onset diabetic NOD mice. (a) Immunofluorescence staining of CXCL10 in pancreatic tissue sections of n = 4 normoglycaemic NOD mice and in n = 4 new-onset diabetic NOD mice. Representative images of normoglycaemic (panels A,B) and new-onset diabetic NOD mice (panels C,D) are reported. CXCL10 is reported in red; nuclei in white/gray. Scale bar is 50 μm. Analysis of CXCL10 total voxels absolute volume (b) and normalized per total islet volume (c), in pancreatic islets of n = 4 normoglycaemic and n = 4 NOD new-onset diabetic NOD mice. A total of n = 27 and n = 25 pancreatic islets were individually analyzed in normoglycaemic and new-onset diabetic mice, respectively; individual values for each islet are reported in μm³ (b) or as a volumetric ratio (c). Exact p-values were analyzed using non-parametric Mann–Whitney U test (p < 0.05).

Figure 2

CXCL10 is increased in alpha-cells of new-onset diabetic NOD mice. (a) Representative images of triple immunofluorescence reporting the expression of insulin (INS, green), glucagon (GCG, blue), and CXCL10 (red) in pancreatic islets of normoglycaemic (panels A–E) and of new-onset diabetic NOD mice (panels F–J). In panels E,K, zoom-in of pancreatic islets are shown. Colocalization between insulin and CXCL10 is shown in yellow and indicated by yellow arrows; colocalization between glucagon and CXCL10 is reported in magenta and indicated by red arrows. Scale bar in panels D,I = 50 μm. Scale bar panels E,J = 20 μm. (b) Colocalization rate are reported as the results of Manders's coefficient evaluation between CXCL10 and insulin (CXCL10-INS) (green dots) and CXCL10 and glucagon (CXCL10-GCG) (blue dots) in individual pancreatic islets of n = 4 normoglycaemic NOD mice and n = 4 new-onset diabetic NOD mice. Each dot represents an individual islet. A total of n = 27 pancreatic islets of normoglycaemic and new-onset diabetic NOD mice are reported. Values are reported as the percentage of CXCL10 signal overlapping with total INS or GCG signal. Exact p-values were analyzed using multiple comparison ordinary one-way ANOVA test (p < 0.05). Dotted lines represent mean ± SD. (c) Colocalization plots of CXCL10-Insulin (left) and CXCL10-glucagon (right) of a new-onset diabetic NOD mouse pancreatic islet. Positive pixels for CXCL10 (red), insulin (green), and glucagon (blue), alongside with colocalizing pixels (CXCL10-insulin: yellow; CXCL10-glucagon: magenta), are reported in the plots. Significant colocalizing pixels are within the area delimited by white lines, representing background and threshold levels relative to each channel. Each pixel is reported as a gray-scale RGB intensity value (0–255).

Figure 3

CXCL10 is expressed in ICIs and IDIs of new-onset diabetic individuals and distinguished four pancreatic islet subsets. Triple immunofluorescence analysis of insulin (INS, green), glucagon (GCG, blue), and CXCL10 (red) in pancreatic sections of new-onset T1D DiViD cases. Representative pancreatic islet 40 × confocal microscope images are shown for each channel, alongside with digital zoom-in for each set of panel. (A–E) ICI showing positivity for CXCL10. (F–J) ICI without CXCL10 positivity. (K–O) IDI showing CXCL10 positivity. (P–T) IDI without positivity for CXCL10. Scale bar =100 μm. Scale bar zoom-in = 40 μm.

Figure 4

Distribution of islet subsets in T1D DiViD individuals based on CXCL10 expression and ICI/IDI classification. Histological evaluation of islet subsets distribution in each of the six recent-onset DiViD individuals, based on the analysis of two non-consecutive pancreatic sections/case. Distribution of islets is reported as percentage value of total islets identified per section.

Figure 5

Alpha-cells contribute to CXCL10 expression in islets of new-onset T1D patients. (a) Triple immunofluorescence analysis of insulin (INS, green, panel A), glucagon (GCG, blue, panel B), and CXCL10 (red, panel C) of pancreatic islets in T1D DiViD cases. Panel E: digital zoom-in of overlapping (merge) channels, showing colocalization of CXCL10 and insulin (yellow pixels) indicated by yellow arrow and of CXCL10 and glucagon (magenta pixels) indicated by red arrow. Scale bar = 50 μm. Scale bar zoom-in = 20 μm. (b) Colocalization analysis of CXCL10 and insulin (green dots) and CXCL10 and glucagon (blue dots) in pancreatic islets of T1D DiViD cases. A total of n = 50 ICIs from 6 DiViD cases were analyzed for both CXCL10-insulin and CXCL10-glucagon colocalization rate. Values are reported as the percentage of overlapping CXCL10-insulin or CXCL10-glucagon pixels over total insulin or glucagon positive pixels, according to Mander's Coefficient calculation. Exact p-value was calculated using Wilcoxon matched-pairs signed rank test. (c) Colocalization plots of CXCL10-insulin (left) and CXCL10-glucagon (right) of a recent-onset diabetic DiViD individual ICI (Case-1). Positive pixels for CXCL10 (red), insulin (green), and glucagon (blue), alongside with colocalizing pixels (CXCL10-insulin: yellow; CXCL10-glucagon: magenta), are reported in the plots. Significant colocalizing pixels are within the area delimited by white lines, representing background and threshold levels relative to each channel. Each pixel is reported as a gray-scale RGB intensity value (0–255).