Islet expression of type I interferon response sensors is associated with immune infiltration and viral infection in type 1 diabetes

Abstract

Previous results indicate the presence of an interferon (IFN) signature in type 1 diabetes (T1D), capable of inducing chronic inflammation and compromising b cell function. Here, we determined the expression of the IFN response markers MxA, PKR, and HLA-I in the islets of autoantibody-positive and T1D donors. We found that these markers can be coexpressed in the same islet, are more abundant in insulin-containing islets, are highly expressed in islets with insulitis, and their expression levels are correlated with the presence of the enteroviral protein VP1. The expression of these markers was associated with down-regulation of multiple genes in the insulin secretion pathway. The coexistence of an IFN response and a microbial stress response is likely to prime islets for immune destruction. This study highlights the importance of therapeutic interventions aimed at eliminating potentially persistent infections and diminishing inflammation in individuals with T1D.

Fig. 1. HLA-I hyperexpression is present in the islets of double AAb⁺ and T1D donors.

(A and C) Representative immunofluorescence images from islets (A) and exocrine tissue (C) showing normal (top), elevated (middle), and hyperexpression (bottom) of HLA-I. The right panels display heatmaps based on the relative fluorescence intensity unit of each cell. The maximum and minimum values are displayed in red and blue, respectively. Scale bars, 100 μm. (B) Box plots showing median and first and third quartile for the percentage of islets showing normal, elevated, or hyperexpression of HLA-I. The upper and lower whiskers extend from the hinges to the largest or lowest value no further than 1.5 times the interquartile range from the hinge. Data beyond the end of the whiskers are called “outlying” points. Each dot represents a donor. (D) Density plots showing the average HLA-I intensity in the exocrine tissue for a representative number of donors in each donor group. The level of expression is shown in green (normal), light orange (elevated), and dark orange (hyperexpression). *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Fig. 2. MxA and PKR are expressed in a significantly higher percentage of islets in T1D donors.

(A) Representative immunofluorescence images of islets positive (+) and negative (−) for PKR (top), MxA (middle), and dsRNA (bottom). Scale bars, 25 μm. (B) Box plots showing median and first and third quartile for the percentage of islets positive for MxA, PKR, and dsRNA. The upper and lower whiskers extend from the hinges to the largest or lowest value no further than 1.5 times the interquartile range from the hinge. Data beyond the end of the whiskers are called outlying points. Each dot represents a donor. (C) Percentage of islets positive for MxA, PKR, and dsRNA for each individual donor. *P < 0.05; ***P < 0.001; ns, not significant.

Fig. 3. IFN response markers are coexpressed in a significant number of islets in AAb⁺ and T1D donors.

(A) Representative immunofluorescence images of entire tissue sections from a nondiabetic (top, donor no. 6278) and a T1D donor (bottom, donor no. 6362) stained for HLA-I, insulin, glucagon, and Hoechst. Each positive islet was assigned a colored circle based on the number of expressed markers as follows: red for the expression of one marker, yellow for two markers, purple for three markers, and blue for four markers. Scale bars, 290 μm. (B) Box plots showing median and first and third quartile show the percentage of islets positive for one, two, three, or four markers for each donor group. The upper and lower whiskers extend from the hinges to the largest or lowest value no further than 1.5 times the interquartile range from the hinge. Data beyond the end of the whiskers are called outlying points. Each dot represents a donor. (C) Percentage of islets positive for one, two, three, or four markers in individual donors. *P < 0.05; ***P < 0.001; ns, not significant.

Fig. 4. IFN response markers are preferentially expressed in ICIs in T1D donors.

(A) Bar graphs (mean) and individual paired values are shown for the percentage of islets that express HLA-I, MxA, PKR, and dsRNA in ICIs and IDIs in T1D donors. (B) Bar graphs (mean) and individual paired values for the percentage of islets expressing none or one, two, three, or four markers in ICIs and IDIs in T1D donors. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 5. In T1D donors, islets that express HLA-I, MxA, or PKR, either alone or in combination are highly infiltrated.

(A) Representative immunofluorescence images of a highly infiltrated islet stained for CD3, glucagon, and Hoechst. Scale bars, 50 μm. (B) Bar graph (mean) for CD3⁺ cells/mm² in each donor. The dots represent individual islets. (C) Box plots showing median and first and third quartile for the distribution of CD3⁺ cells/mm² for each donor group. The upper and lower whiskers extend from the hinges to the largest or lowest value no further than 1.5 times the interquartile range from the hinge. Data beyond the end of the whiskers are called outlying points. Each dot represents a donor. (D) Box plots (median, first and third quartile, and range) for the proportion of islets with at least six CD3⁺ cells. Each dot represents a donor. (E) Dot plots for the proportion of islets infiltrated by at least six CD3⁺ cells expressing none (green) or one (red), two (yellow), three (purple), or four (blue) markers for each donor group. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, not significant.

Fig. 6. Viral footprints can be found in the islets of T1D donors and are correlated with the expression of IFN response markers.

(A) Representative immunohistochemistry images of negative, weak, and strong VP1⁺ islets. Scale bars, 25 μm. (B and C) Box plots (median, first and third quartile, and range) for the percentage of islets with weak (B) and strong (C) VP1⁺ cells in each donor group. (D) Heatmap and hierarchical clustering for the percentage of islets expressing none or one, two, or three markers and weak and strong VP1. **P < 0.01.