Abstract
AimsType 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D.Methods12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15null macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass.Results12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15null mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass.ConclusionsThese results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.
Highlights
Type 1 diabetes (T1D) is a complex autoimmune disease in which immune cells react against insulin-producing beta cells within the pancreatic islets of Langerhans [1,2], leaving affected individuals dependent upon exogenous insulin for life, and at high risk for developing serious cardiovascular and microvascular complications
Characterization of 12/15-LO in Immune Cells and Effects on Gene Expression In order to characterize the mechanism of protection from diabetes progression in NOD- Alox15 null mice, we first investigated several aspects of the timing and cellular location of the Alox15 gene expression in NOD mice, and how alterations in expression might affect inflammation
We investigated how 12/15-LO expression affected macrophage proinflammatory cytokines downstream of 12/15LO, including MCP-1, IL-1b, STAT-4, and IL-12p40
Summary
Type 1 diabetes (T1D) is a complex autoimmune disease in which immune cells react against insulin-producing beta cells within the pancreatic islets of Langerhans [1,2], leaving affected individuals dependent upon exogenous insulin for life, and at high risk for developing serious cardiovascular and microvascular complications. Female NOD mice develop spontaneous type 1like diabetes that mimics human disease [3]. Alox deletion leads to 98 percent protection from diabetes in female NOD mice. Macrophage infiltration and CD4+ T cell infiltrates into the pancreas were significantly reduced in NOD-Alox15null mice [4]. The murine Alox gene encodes the 12/15-lipoxygenase (12/ 15-LO) enzyme, which is involved in the oxygenation of arachidonic and linoleic acids to the inflammatory mediators 12S-hydroperoxyeicosatetraenoic acid and 13-(S)-hydroxy-9Z11Eoctadecadienoic acid. 12-HETE can activate a signaling cascade that can lead to cytokine-induced cell damage [8]. Downstream generation of IL-12 by 12/15-LO products presumably leads to T cell activation and phosphorylation of signal transducers and activators of transcription 4 (STAT4) [9,10].
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