Whole-genome CRISPR screening identifies genetic manipulations to reduce immune rejection of stem cell-derived islets.

Human embryonic stem cells (hESCs) provide opportunities for cell replacement therapy of insulin-dependent diabetes. Therapeutic quantities of human stem cell-derived islets (SC-islets) can be produced by directed differentiation. However, preventing allo-rejection and recurring autoimmunity, without the use of encapsulation or systemic immunosuppressants, remains a challenge. An attractive approach is to transplant SC-islets, genetically modified to reduce the impact of immune rejection. To determine the underlying forces that drive immunogenicity of SC-islets in inflammatory environments, we performed single-cell RNA sequencing (scRNA-seq) and whole-genome CRISPR screen of SC-islets under immune interaction with allogeneic peripheral blood mononuclear cells (PBMCs). Data analysis points to "alarmed" populations of SC-islets that upregulate genes in the interferon (IFN) pathway. The CRISPR screen in vivo confirms that targeting IFNγ-induced mediators has beneficial effects on SC-islet survival under immune attack. Manipulating the IFN response by depleting chemokine ligand 10 (CXCL10) in SC-islet grafts confers improved survival against allo-rejection compared with wild-type grafts in humanized mice. These results offer insights into the nature of immune destruction of SC-islets during allogeneic responses and provide targets for gene editing.

Impact of severe hypoglycemia on the heat shock and related protein response.

Heat shock proteins contribute to diabetes-induced complications and are affected by glycemic control. Our hypothesis was that hypoglycemia-induced heat shock and related protein changes would be amplified in type 2 diabetes (T2D). This prospective, case-control study enrolled 23 T2D patients and 23 control subjects who underwent hyperinsulinemic-induced hypoglycemia (≤ 2.0 mmol/L (36 mg/dl)) with blood sampling at baseline, at hypoglycemia and after a 24-h post-hypoglycemia follow-up period. Proteomic analysis of heat shock-related and pro-inflammatory proteins was performed. At baseline, MAPKAPK5 (p = 0.02) and UBE2G2 (p = 0.003) were elevated and STUB1 decreased (p = 0.007) in T2D. At hypoglycemia: PPP3CA (p < 0.03) was increased and EPHA2 (p = 0.01) reduced in T2D; by contrast, three proteins were reduced in controls [HSPA1A (p = 0.007), HSPB1 (p < 0.02), SMAD3 (p = 0.005)] while only MAPKAPK5 was elevated (p = 0.02). In the post-hypoglycemia follow-up period, most proteins normalized to baseline by 24-h; however, STIP1 (p = 0.003), UBE2N (p = 0.004) and UBE2L3 (p < 0.04) were decreased in controls at 24-h. No protein differed from baseline at 24-h in T2D. Pro-inflammatory interleukin-6 increased at 4-h post-hypoglycemia in controls and T2D (p < 0.05 and p < 0.003, respectively) and correlated with HSPA1A; anti-inflammatory IL-10 decreased 2-h post-hypoglycemia in T2D only. Other pro-inflammatory proteins, IL-1α, IFN-γ and TNF-α, were unchanged. Heat shock and related proteins differed at baseline between T2D and controls, with an exaggerated response of heat shock and related proteins to hypoglycemia that returned to baseline, though with changes at 24-h in controls alone. An increase in pro-inflammatory IL-6, with a decrease in anti-inflammatory IL-10, suggests that the HSP system is overactivated due to underlying inflammation in T2D.Trial registration: ClinicalTrials.gov NCT03102801.

Plasma heat shock protein response to euglycemia in type 2 diabetes.

INTRODUCTION: Glucose variability is associated with mortality and macrovascular diabetes complications. The mechanisms through which glucose variability mediates tissue damage are not well understood, although cellular oxidative stress is likely involved. As heat shock proteins (HSPs) play a role in the pathogenesis of type 2 diabetes (T2D) complications and are rapidly responsive, we hypothesized that HSP-related proteins (HSPRPs) would differ in diabetes and may respond to glucose normalization. RESEARCH DESIGN AND METHODS: A prospective, parallel study in T2D (n=23) and controls (n=23) was undertaken. T2D subjects underwent insulin-induced blood glucose normalization from baseline 7.6±0.4 mmol/L (136.8±7.2 mg/dL) to 4.5±0.07 mmol/L (81±1.2 mg/dL) for 1 hour. Control subjects were maintained at 4.9±0.1 mmol/L (88.2±1.8 mg/dL). Slow Off-rate Modified Aptamer-scan plasma protein measurement determined a panel of HSPRPs. RESULTS: At baseline, E3-ubiquitin-protein ligase (carboxyl-terminus of Hsc70 interacting protein (CHIP) or HSPABP2) was lower (p=0.03) and ubiquitin-conjugating enzyme E2G2 higher (p=0.003) in T2D versus controls. Following glucose normalization, DnaJ homolog subfamily B member 1 (DNAJB1 or HSP40) was reduced (p=0.02) in T2D, with HSP beta-1 (HSPB1) and HSP-70-1A (HSP70-1A) (p=0.07 and p=0.09, respectively) also approaching significance relative to T2D baseline levels. CONCLUSIONS: Key HSPRPs involved in critical protein interactions, CHIP and UBE2G2, were altered in diabetes at baseline. DNAJB1 fell in response to euglycemia, suggesting that HSPs are reacting to basal stress that could be mitigated by tight glucose control with reduction of glucose variability.