Increased Expression of Viral Sensor MDA5 in Pancreatic Islets and in Hormone-Negative Endocrine Cells in Recent Onset Type 1 Diabetic Donors

Abstract

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic β-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as melanoma differentiation associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of nondiabetic and T1D donors is still missing. In the present study, we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 nondiabetic autoantibody-negative, 2 nondiabetic autoantibody-positive, 8 recent-onset, and 2 long-standing T1D). In nondiabetic control donors, MDA5 was expressed both in α- and β-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in α-cells. In T1D donors, we observed an increased colocalization rate of MDA5-glucagon with respect to MDA5-insulin in comparison to nondiabetic controls; such increase was more pronounced in recent-onset with respect to long-standing T1D donors. Of note, an increased colocalization rate of MDA5-glucagon was found in insulin-deficient-islets (IDIs) with respect to insulin-containing-islets (ICIs). Strikingly, we detected the presence of MDA5-positive/hormone-negative endocrine islet-like clusters in T1D donors, presumably due to dedifferentiation or neogenesis phenomena. These clusters were identified exclusively in donors with recent disease onset and not in autoantibody-positive nondiabetic donors or donors with long-standing T1D. In conclusion, we showed that MDA5 is preferentially expressed in α-cells, and its expression is increased in recent-onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

Keywords: MDA5; dedifferentiation; enteroviruses; innate immunity; pancreas; pancreatic islet; type 1 diabetes; viral sensor.

Figure 1

Triple immunofluorescence analysis of insulin, glucagon, and MDA5 in human pancreata of nondiabetic, Aab+, and T1D multiorgan donors. (A) Representative images showing fluorescence confocal microscopy imaging analysis of FFPE pancreatic tissue sections derived from nondiabetic donors (panels A–E) (n=10) (ND), autoantibody-positive donors (panels F–J) (n=2: 6027, 6167) (Aab+), type 1 diabetic recent-onset donors (panels K–O) (T1D-RO) (n=8: DiViD cases -1, -2, -3, -4, -5, -6, nPOD 6113, 6087), and type 1 diabetic long-standing donors (panels P–T) (T1D-LS) (n=2: EUnPOD 060217, 120718), stained for insulin (red, panels A, F, K, P), glucagon (blue, panels B, G, L, Q), and MDA5 (green, panels C, H, M, R). Channel merge shows the colocalization of insulin and MDA5 in yellow/orange and of glucagon and MDA5 in turquoise color. A zoom-in inset reports details of pancreatic islets of channel merge images. Scale bar in panels D, I, N, S is 75 μm. (B) Bar plot graphs showing the colocalization rate analysis of MDA5-insulin (orange), MDA5-glucagon (light gray) in nondiabetic controls (ND) (n=10), autoantibody-positive (Aab+) (n=2), T1D-RO (n=8), and T1D-LS (n=2). Each dot represents an individual pancreatic islet reported as a colocalization rate value between MDA5-glucagon and MDA5-insulin. Median with interquartile range values are reported as a histogram plot. Statistics performed using ANOVA followed by Kruskal–Wallis multiple comparison test (*p<0.05). (C) Bar plot graph showing the colocalization rate analysis of MDA5-insulin (orange), MDA5-glucagon in insulin-containing islets (ICIs) (light gray), MDA5-glucagon in insulin-deficient islets (IDIs) (dark gray) in nondiabetic controls (ND) (n=10) and in T1D donors (n=10, RO+LS). Each dot represents an individual pancreatic islet reported as a colocalization rate value of MDA5-glucagon or MDA5-insulin. Median with interquartile range values are reported. Statistics performed using ANOVA followed by Kruskal–Wallis multiple comparison test (*p<0.05).

Figure 2

Immunofluorescence detection of MDA5-positive/hormone-negative islet-like structures in the pancreas of T1D donors. (A) Representative images reporting triple immunofluorescence analysis of insulin (red, panels A, F, K), glucagon (blue, panels B, G, L), MDA5 (green, panels C, H, M), DAPI (white) in T1D-RO donors (nPOD 6113: panels A–E; nPOD 6087: panels F–J; DiViD case 3: panels K–O). Zoom-in insets (panels E, J, O) report details of channel merge images. Scale bar in panels D, I, N is 50 µm. (B) Representative images of quadruple immunofluorescence analysis of insulin (red, panels A, G, M), glucagon (blue, panels B, H, N), somatostatin (white, panels C, I, O), and MDA5 (green, panels D, J, P) in T1D-RO donors. Zoom-in insets (F, L, R) report details of channel merge images. Scale bar in panels E, K, Q is 50 µm. (C) Representative images of quadruple immunofluorescence analysis of chromogranin-A (blue, panels A, G, M), insulin (red, panels B, H, N), glucagon (white, panels C, I, O), and MDA5 (green, panels D, J, P) in T1D-RO donors. Zoom-in insets (F, L, R) report details of channel merge images. Scale bar in panels E, K, Q = 50 µm.

Figure 3

Whole-slide imaging islet distribution analysis based on MDA5 positivity in ND and T1D donors. (A) Representative whole-slide image of T1D case-3 (DiViD) of triple immunofluorescence staining for insulin (INS, red), glucagon (GCG, blue), MDA5 (green), and DAPI (white) analyzed using a Nanozoomer S60 slide scanner. The image (panel A) reports the localization of 5 different histological structures identified in T1D pancreatic sections. Zoom-in insets for each type of islet or islet-like structure are reported on the left. Blue square (panel B): insulin-containing islet (ICI)-MDA5 positive; red square (panel D): insulin-containing islet-MDA5 negative; green square (panel C): insulin-deficient islet (IDI)-MDA5 positive; magenta square (panel E): insulin-deficient islet-MDA5 negative; orange square: MDA5-only positive islet-like structures. (B) Table showing islet distribution based on MDA5 positivity in pancreatic tissue sections of ND, T1D-Recent Onset (RO), and T1D-Long Standing (LS). Results are reported as percentage values (alongside with absolute values) over the total number of islets detected. Donor types as well as case IDs are reported. (C) Graphical representation of the distribution of pancreatic islets based on MDA5 positivity in pancreatic tissue sections of ND, T1D-RO (DiViD cases), and T1D-LS.