HLA-DQ8 Supports Development of Insulitis Mediated by Insulin-Reactive Human TCR-Transgenic T Cells in Nonobese Diabetic Mice.

In an effort to improve HLA-"humanized" mouse models for type 1 diabetes (T1D) therapy development, we previously generated directly in the NOD strain CRISPR/Cas9-mediated deletions of various combinations of murine MHC genes. These new models improved upon previously available platforms by retaining ß2-microglobulin functionality in FcRn and nonclassical MHC class I formation. As proof of concept, we generated H2-Db/H2-Kd double knockout NOD mice expressing human HLA-A*0201 or HLA-B*3906 class I variants that both supported autoreactive diabetogenic CD8+ T cell responses. In this follow-up work, we now describe the creation of 10 new NOD-based mouse models expressing various combinations of HLA genes with and without chimeric transgenic human TCRs reactive to proinsulin/insulin. The new TCR-transgenic models develop differing levels of insulitis mediated by HLA-DQ8-restricted insulin-reactive T cells. Additionally, these transgenic T cells can transfer insulitis to newly developed NSG mice lacking classical murine MHC molecules, but expressing HLA-DQ8. These new models can be used to test potential therapeutics for a possible capacity to reduce islet infiltration or change the phenotype of T cells expressing type 1 diabetes patient-derived ß cell autoantigen-specific TCRs.

Nfkbid Overexpression in Nonobese Diabetic Mice Elicits Complete Type 1 Diabetes Resistance in Part Associated with Enhanced Thymic Deletion of Pathogenic CD8 T Cells and Increased Numbers and Activity of Regulatory T Cells.

Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell-mediated autoimmune destruction of pancreatic ß cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on ß cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.

Lipin 2/3 phosphatidic acid phosphatases maintain phospholipid homeostasis to regulate chylomicron synthesis.

The lipin phosphatidic acid phosphatase (PAP) enzymes are required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate in most mammalian tissues. The 3 lipin proteins (lipin 1, lipin 2, and lipin 3) each have PAP activity, but have distinct tissue distributions, with lipin 1 being the predominant PAP enzyme in many metabolic tissues. One exception is the small intestine, which is unique in expressing exclusively lipin 2 and lipin 3. TAG synthesis in small intestinal enterocytes utilizes 2-monoacylglycerol and does not require the PAP reaction, making the role of lipin proteins in enterocytes unclear. Enterocyte TAGs are stored transiently as cytosolic lipid droplets or incorporated into lipoproteins (chylomicrons) for secretion. We determined that lipin enzymes are critical for chylomicron biogenesis, through regulation of membrane phospholipid composition and association of apolipoprotein B48 with nascent chylomicron particles. Lipin 2/3 deficiency caused phosphatidic acid accumulation and mammalian target of rapamycin complex 1 (mTORC1) activation, which were associated with enhanced protein levels of a key phospholipid biosynthetic enzyme (CTP:phosphocholine cytidylyltransferase α) and altered membrane phospholipid composition. Impaired chylomicron synthesis in lipin 2/3 deficiency could be rescued by normalizing phospholipid synthesis levels. These data implicate lipin 2/3 as a control point for enterocyte phospholipid homeostasis and chylomicron biogenesis.

A Hypermorphic Nfkbid Allele Contributes to Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in NOD Mice.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic ß cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.

Lipin-1 and lipin-3 together determine adiposity in vivo.

The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.

Lipins, lipinopathies, and the modulation of cellular lipid storage and signaling.

Members of the lipin protein family are phosphatidate phosphatase (PAP) enzymes, which catalyze the dephosphorylation of phosphatidic acid to diacylglycerol, the penultimate step in TAG synthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in that they also promote fatty acid oxidation through their activity as co-regulators of gene expression by DNA-bound transcription factors. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins-lipin-1, lipin-2, and lipin-3. In mice and humans, the levels of lipin activity are a determinant of TAG storage in diverse cell types, and humans with deficiency in lipin-1 or lipin-2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of "lipinopathies" in mouse models and in humans has revealed an important role for lipin activity in the regulation of lipid intermediates (phosphatidate and diacylglycerol), which influence fundamental cellular processes including adipocyte and nerve cell differentiation, adipocyte lipolysis, and hepatic insulin signaling. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.

Mouse lipin-1 and lipin-2 cooperate to maintain glycerolipid homeostasis in liver and aging cerebellum.

The three lipin phosphatidate phosphatase (PAP) enzymes catalyze a step in glycerolipid biosynthesis, the conversion of phosphatidate to diacylglycerol. Lipin-1 is critical for lipid synthesis and homeostasis in adipose tissue, liver, muscle, and peripheral nerves. Little is known about the physiological role of lipin-2, the predominant lipin protein present in liver and the deficient gene product in the rare disorder Majeed syndrome. By using lipin-2-deficient mice, we uncovered a functional relationship between lipin-1 and lipin-2 that operates in a tissue-specific and age-dependent manner. In liver, lipin-2 deficiency led to a compensatory increase in hepatic lipin-1 protein and elevated PAP activity, which maintained lipid homeostasis under basal conditions, but led to diet-induced hepatic triglyceride accumulation. As lipin-2-deficient mice aged, they developed ataxia and impaired balance. This was associated with the combination of lipin-2 deficiency and an age-dependent reduction in cerebellar lipin-1 levels, resulting in altered cerebellar phospholipid composition. Similar to patients with Majeed syndrome, lipin-2-deficient mice developed anemia, but did not show evidence of osteomyelitis, suggesting that additional environmental or genetic components contribute to the bone abnormalities observed in patients. Combined lipin-1 and lipin-2 deficiency caused embryonic lethality. Our results reveal functional interactions between members of the lipin family in vivo, and a unique role for lipin-2 in central nervous system biology that may be particularly important with advancing age. Additionally, as has been observed in mice and humans with lipin-1 deficiency, the pathophysiology in lipin-2 deficiency is associated with dysregulation of lipid intermediates.

Lipin proteins and metabolic homeostasis.

The lipin protein family, consisting of three members, was first identified early this century. In the last few years, the lipin proteins have been shown to have important roles in glycerolipid biosynthesis and gene regulation, and mutations in the corresponding genes cause lipodystrophy, myoglobinuria, and inflammatory disorders. Here, we review some of the progress toward elucidating the molecular and physiological functions of the lipin proteins.

Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells.

Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.