Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice

Abstract

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Keywords: Beta cells; Diabetes; Endocrinology; Pharmacology.

Figure 1. The UPR and ISR are active in prediabetic NOD mice and proinflammatory cytokine–treated human islets.

(A) UMAP embeddings of a reanalysis of scRNA-Seq of islets from female NOD mice. (B) GSEA of the endocrine cell population identified in A for UPR. (C) UMAP embeddings of a reanalysis of scRNA-Seq of islets from female NOD mice. (D) GSEA of the β cell population identified in C for UPR. (E) Representative immunoblot of p-eIF2α and total eIF2α; n = 3 biological replicates. (F) Quantification of the immunoblots in E (ANOVA). (G) Representative images of pancreas immunostained as indicated. Scale bars: 50 μm. Dotted lines indicate islets. (H) Quantification of the images in G; each dot represents an islet from n = 3 mice (distinguished by color), with mean values of each mouse shown (ANOVA of means). (I) Representative puromycin immunoblot image (left panel) and corresponding total protein stain (right panel) from islets of 8-week-old female mouse islets. (J) Quantification of the puromycin intensity normalized to total protein stain from I; n = 3 (each replicate represents pooled islets from 3–4 mice) (ANOVA). (K) Representative immunoblot of p-PERK and total PERK from islets of 8-week-old female mice. (L) Quantification of the immunoblot for p-PERK normalized to total PERK from n = 4 (each replicate represents pooled islets from 3–4 mice). (M) Representative puromycin incorporation immunoblot (left panel) and corresponding total protein stain (right panel) from MIN6 cells treated as indicated. (N) Quantification of the puromycin intensity normalized to total protein stain from L; n = 3 independent experiments (RM-ANOVA). PIC, proinflammatory cytokine. (O) Polyribosomal profiling traces of human islets treated as indicated. Data are represented as mean ± SEM.

Figure 2. PERK inhibition delays autoimmune diabetes in NOD mice.

Prediabetic female NOD mice (6 weeks of age) were treated with varying doses of HC-5770 or ISRIB. (A) Experimental design for diabetes incidence study. (B) Diabetes incidence. n = 20 mice per group (Mantel-Cox). (C) Representative H&E stain of pancreata from nondiabetic mice at 25 weeks of age that were treated between 6 and 10 weeks of age. A, acinar; I, islet. Scale bars: 50 μm. (D) Experimental design mechanistic studies. (E) Representative images of pancreata from NOD mice following 2 weeks of HC-5770 administration (6 mg/kg) stained for insulin (brown) and counterstained with hematoxylin (blue). Scale bars: 500 μm. (F) β Cell mass of mice treated with HC-5770 (6 mg/kg) for 2 weeks; n = 4–5 mice per group (ANOVA). (G) Representative images of pancreata from NOD mice following 2 weeks of HC-5770 administration immunostained as indicated. Arrows indicate regions of insulitis. Scale bars: 50 μm. (H) Average insulitis score of mice treated with varying doses of HC-5770 for 2 weeks; n = 4–5 mice per group. NB: these data are replicated in Supplemental Figure 2F for comparative purposes (ANOVA). (I) Experimental design. (J) Representative images of pancreata from NOD mice following 2 weeks of ISRIB administration stained as indicated. Scale bars: 500 μm. (K) β Cell mass of mice treated with ISRIB for 2 weeks; n = 4–5 mice per group (ANOVA). (L) Representative images of pancreata from NOD mice following 2 weeks of ISRIB administration immunostained as indicated; arrows indicate regions of insulitis. Scale bars: 50 μm. (M) Average insulitis score of mice treated with varying doses of ISRIB for 2 weeks; n = 4–5 mice per group (ANOVA). Data are presented as mean ± SEM.

Figure 3. PERK inhibition increases levels of PD-L1 in β cells of NOD mice.

Prediabetic female NOD mice (6 weeks of age) were treated with HC-5770 for 2 weeks, isolated islets were subjected to scRNA-Seq, and pancreas tissue was subjected to NanoString spatial proteomics. (A) UMAP embeddings of merged scRNA-Seq profiles from islets. n = 3 mice per group. (B) GO analysis of all cell clusters (pseudo-bulk analysis). (C) GSEA of β cell clusters showing UPR. (D) GSEA of β cell clusters showing PERK-meditated UPR. (E) Percentages of T, B, and myeloid cells identified within the immune cell clusters. (F) Percentages of α, β, δ, and PP cells identified within the islet cell clusters. (G) Example of the insulin-positive area and the insulitic area used to collect spatial tissue-based proteomics. (H) Heatmap of identified proteins in the insulitic area (left panel) and insulin-positive area (right panel); n = 10–11 ROI from 2 mice per group. *P < 0.05, t test. (I) Representative images of pancreata from mice following 2 weeks of treatment with vehicle or HC-5770 immunostained as indicated; dotted lines indicate islets. Scale bars: 50 μm. (J) Quantification of PD-L1 intensity in the β cells of I; each dot represents an islet from n = 4 mice (distinguished by color), with mean values of each mouse shown (t test for the means). (K) Six-week-old female NOD mice were treated as indicated with HC-5770 for 2 weeks, then administered either anti-PD-L1 or IgG control, followed by another 2 weeks of HC-5770 treatment; glucose was measured on alternate days after injection; n = 5 mice per group. (L) AUC analysis of the data in K (ANOVA). Data are represented as mean ± SEM.

Figure 4. GOLM1 stabilizes PD-L1.

(A) PD-L1 and GOLM1 protein levels were identified using proteomics of EndoC-βH1 human β cells treated with or without proinflammatory cytokines; n = 3 biological replicates. t test. (B) Representative immunoblot analysis of PD-L1 and GOLM1 from EndoC-βH1 cells treated with or without PIC, HC-5770, and ISRIB (left panel) with quantification of PD-L1 levels (middle panel) and GOLM1 levels (right panel); n = 3 biological replicates (ANOVA). (C and D) Relative CD274 and GOLM1 mRNA levels by quantitative reverse-transcriptase PCR (RT-PCR) normalized to ACTB in EndoC-βH1 cells treated as indicated; n = 4–7 biological replicates (ANOVA). (E) Relative GOLM1 RNA levels normalized to ACTB in EndoC-βH1 cells treated as indicated; n = 3 biological replicates (ANOVA). (F) Representative immunoblot analysis of PD-L1 and GOLM1 in EndoC-βH1 cells treated as indicated (left panel) with quantification of PD-L1 levels (right panel); n = 3 biological replicates (ANOVA). (G) Relative CD274 mRNA levels by quantitative RT-PCR normalized to ACTB in EndoC-βH1 cells treated as indicated; n = 3 biological replicates (ANOVA). (H) Representative immunoblot analysis of HLA-I from EndoC-βH1 cells treated as indicated (left panel) with quantification of HLA-I levels (right panel); n = 3 biological replicates (ANOVA). (I) Relative HLA-I mRNA levels by quantitative RT-PCR normalized to ACTB in EndoC-βH1 cells treated as indicated; n = 3 biological replicates (ANOVA). (J) Representative immunoblot analysis of PD-L1 in EndoC-βH1 cells treated as indicated. (K) Immunoblot analysis of ubiquitin following immunoprecipitation for PD-L1 from HEK-293 cells treated as indicated. (L) Immunoblot analysis for GOLM1 following immunoprecipitation for PD-L1 from HEK-293 cells. Data are presented as mean ± SEM.

Figure 5. PERK inhibition increases GOLM1 levels in β cells.

(A) Representative images of pancreata from 8-week-old female CD1, NSG, and NOD mice immunostained as indicated. Scale bars: 50 μm. Dotted lines indicate islets. (B) Quantification of the GOLM1 fluorescence intensity from data in A; each dot represents an islet. n = 3 mice (distinguished by color), with mean values for each mouse shown (ANOVA for means). (C) Representative images from 6-, 8-, 10-, and 12-week-old female NOD mice immunostained for GOLM1 (magenta), insulin (cyan), and nuclei (blue). Scale bars: 50 μm. Dotted lines indicate islets. (D) Quantification of GOLM1 fluorescence intensity from data in C; each dot represents an islet from n = 3–4 mice (distinguished by color), with mean values of each mouse shown (ANOVA for means). (E) Representative images of pancreata from 8-week-old female NOD mice that have been treated with vehicle or HC-5770 for 2 weeks, immunostained as indicated. Scale bars: 50 μm. Dotted lines indicate islets. (F) Quantification of the GOLM1 fluorescence intensity from data in E; each dot represents an islet from n = 4 mice (distinguished by color), with mean values of each mouse shown (t test for means). (G) Dot plot analysis of scRNA-Seq data in the HPAP of residual β cells for GOLM1 and CD274. The size of the dots indicates the percentage of cells that express the studied gene. The color scale shows the change of normalized and centered average gene expression within the different groups. No diabetes (ND): n = 15; single autoantibody positive (AAb1⁺): n = 8; double-autoantibody positive (AAb2⁺): n = 2; T1D: n = 9. Data are represented as mean ± SEM.