12-Lipoxygenase Activates the Integrated Stress Response in Pancreatic Islets, Suppresses PD-L1 Production, and Promotes the Development of T1D

Presenter
Abhishek Kulkarni (University of Chicago)

Authors
Abhishek Kulkarni, Sarah A. Tersey, Fei Huang, Annie R. Pineros, Farooq Syed, Hongyu Gao, Kara Orr, Yunlong Liu, Maureen Gannon, Marcia McDuffie, Jerry L. Nadler, Margaret A. Morris, Raghavendra G. Mirmira

Purpose
In recent years, type 1 diabetes (T1D) has become viewed as a disease initiated and propagated by islet β cells. 12-lipoxygenase (12-LOX), an enzyme in arachidonic acid metabolism, is expressed in β cells and macrophages, and prior studies have shown that its deletion globally can protect against T1D in the NOD mouse model. In this study, we hypothesized that 12-LOX within the β cell may be an initiator of autoimmunity in T1D through activation of the integrated stress response (ISR).

Methods
We interrogated NOD mice with islet-specific deletion of the gene encoding 12-LOX for activation of the ISR and diabetes outcome, and leveraged tissues from the nPOD repository for interrogation of the ISR in human T1D. In addition, we utilized an inhibitor of the human 12-LOX enzyme (ML355) in studies of human islets and in studies using NOD mice containing targeted replacement of the mouse enzyme with the human enzyme (“humanized mice”).

Summary of Results
We backcrossed both mice with floxed alleles of the gene encoding 12-LOX (Alox15) and mice with the PdxPB-CreERT transgene onto the NOD background at greater than 99.7% of genomic loci. Tamoxifen-induced deletion of islet Alox15 at 6 weeks of age led to preservation of β-cell mass, suppression of insulitis, and near-complete protection against autoimmune diabetes. Single cell RNA-sequencing and mass cytometry analyses revealed that the loss of islet Alox15 led to an increase in a population of β cells expressing Cd274, encoding the immune checkpoint protein PD-L1 [5], and promoted the reprogramming of the immune response. The reprogramming included the expansion of anti-inflammatory macrophages and regulatory T cells and suppression of cytotoxic T cells. The increase in PD-L1 protein was coincident with the suppression of the integrated stress response (ISR) in these mouse islets, as evidenced by reductions in stress granules and levels of phospho-eIF2α. Administration of a PD-L1 blocking antibody led to recovery of the diabetes phenotype in islet Alox15 knockout mice. In humans, increases in islet 12-LOX and the ISR (evidenced by formation of stress granules) was observed in pancreas tissue sections from AAb+ donors. In human islets, inhibition of either 12-LOX using ML355 or the ISR using ISRIB resulted in the upregulation of PD-L1 levels on both β cells and in their liberated exosomes. To assess if inhibition of 12-LOX using the human-specific 12-LOX inhibitor modifies T1D progression, we generated mice in which the gene encoding mouse 12-LOX (Alox15) was replaced by the gene encoding human 12-LOX (ALOX12). These “humanized” mice were subsequently backcrossed onto the NOD background, then treated in the prediabetic phase with the 12-LOX inhibitor ML355. Male and female humanized mice developed T1D at the expected frequency, but those receiving ML-355 showed significant delay of T1D.

Conclusions
Our results support several key, previously unappreciated findings: (a) that cell-autonomous inflammatory signaling via 12-LOX in the β-cell dictates a dialog that propagates innate and adaptive immunity, likely via the suppression of the immune checkpoint PD-L1, (b) 12-LOX activity is linked to the ISR in T1D, and that the ISR may be increasing the susceptibility of β cells to autoimmune attack, and (c) targeting of the 12-LOX pathway and the ISR provides an opportunity for the potential modification of T1D disease progression.