Autoimmune β-cell death leads to type 1 diabetes, and with findings that Ca2+-independent phospholipase A2β (iPLA2β) activation contributes to β-cell death, we assessed the effects of iPLA2β inhibition on diabetes development. Administration of FKGK18, a reversible iPLA2β inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4+ T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) β-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4+ T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA2β-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4+ T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4+ T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA2β-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA2β may be beneficial in ameliorating autoimmune destruction of β-cells and mitigating type 1 diabetes development.

Myofibroblasts are specialized contractile cells that participate in tissue fibrosis and remodeling, including idiopathic pulmonary fibrosis (IPF). Mechanotransduction, a process by which mechanical stimuli are converted into biochemical signals, regulates myofibroblast differentiation. Relaxin is a peptide hormone that mediates antifibrotic effects through regulation of collagen synthesis and turnover. In this study, we demonstrate enhanced myofibroblast contraction in bleomycin-induced lung fibrosis in mice and in fibroblastic foci of human subjects with IPF, using phosphorylation of the regulatory myosin light chain (MLC(20)) as a biomarker of in vivo cellular contractility. Compared with wild-type mice, relaxin knockout mice express higher lung levels of phospho-MLC(20) and develop more severe bleomycin-induced lung fibrosis. Exogenous relaxin inhibits MLC(20) phosphorylation and bleomycin-induced lung fibrosis in both relaxin knockout and wild-type mice. Ex vivo studies of IPF lung myofibroblasts demonstrate decreases in MLC(20) phosphorylation and reduced contractility in response to relaxin. Characterization of the signaling pathway reveals that relaxin regulates MLC(20) dephosphorylation and lung myofibroblast contraction by inactivating RhoA/Rho-associated protein kinase through a nitric oxide/cGMP/protein kinase G-dependent mechanism. These studies identify a novel antifibrotic role of relaxin involving the inhibition of the contractile phenotype of lung myofibroblasts and suggest that targeting myofibroblast contractility with relaxin-like peptides may be of therapeutic benefit in the treatment of fibrotic lung disease.

Insulin release from pancreatic β-cells plays a critical role in blood glucose homeostasis, and β-cell dysfunction leads to the development of diabetes mellitus. In cases of monogenic type 1 diabetes mellitus (T1DM) that involve mutations in the insulin gene, we hypothesized that misfolding of insulin could result in endoplasmic reticulum (ER) stress, oxidant production, and mitochondrial damage. To address this, we used the Akita+/Ins2 T1DM model in which misfolding of the insulin 2 gene leads to ER stress-mediated β-cell death and thapsigargin to induce ER stress in two different β-cell lines and in intact mouse islets. Using transformed pancreatic β-cell lines generated from wild-type Ins2+/+ (WT) and Akita+/Ins2 mice, we evaluated cellular bioenergetics, oxidative stress, mitochondrial protein levels, and autophagic flux to determine whether changes in these processes contribute to β-cell dysfunction. In addition, we induced ER stress pharmacologically using thapsigargin in WT β-cells, INS-1 cells, and intact mouse islets to examine the effects of ER stress on mitochondrial function. Our data reveal that Akita+/Ins2-derived β-cells have increased mitochondrial dysfunction, oxidant production, mtDNA damage, and alterations in mitochondrial protein levels that are not corrected by autophagy. Together, these findings suggest that deterioration in mitochondrial function due to an oxidative environment and ER stress contributes to β-cell dysfunction and could contribute to T1DM in which mutations in insulin occur.

β-cell apoptosis is a significant contributor to β-cell dysfunction in diabetes and ER stress is among the factors that contributes to β-cell death. We previously identified that the Ca2+-independent phospholipase A2β (iPLA2β), which in islets is localized in β-cells, participates in ER stress-induced β-cell apoptosis. Here, direct assessment of iPLA2β role was made using β-cell-specific iPLA2β overexpressing (RIP-iPLA2β-Tg) and globally iPLA2β-deficient (iPLA2β-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2β and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2β, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and β-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, β-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2β deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2β impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in β-cells and (2) both over- or under-expression of iPLA2β is deleterious to the β-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2β in islet β-cells. These findings support the hypothesis that iPLA2β induction under stress, as in diabetes, is a key component to amplifying β-cell death processes.

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Type 1 diabetes (T1D) is a consequence of autoimmune-mediated destruction of pancreatic β-cells, and the leading causes for this process are incompletely understood. Our previous work revealed that Ca2+-independent phospholipase A2β (iPLA2β), which hydrolyzes membrane phospholipids at the sn-2 position and releases bioactive lipids, modulates polarization of macrophages (MΦ). Several of the iPLA2β derived lipid signals (iDLs) are proinflammatory, which can initiate immune cell infiltration and β-cell damage. Our recent work suggests that MΦ-derived from spontaneous-T1D prone nonobese diabetic mice (NOD) produce a proinflammatory lipids including (PGE2, 5-HETEs, 20-HETEs, DHETs, LTB4) at the early stage of the disease (4 weeks), interestingly, the proinflammatory lipid signature is similar to a high-risk T1D individuals. Here, we examined the effects of MΦ-iPLA2β iDLs by generating a select conditional decrease in iPLA2β in NOD MΦ (NOD.cMiPLA2𝛽;;;;+/-). We found that (1) that the selective decrease of iPLA2β in MΦ significantly reduces T1D incidence and immune cell infiltration to the islets in the NOD mice. (2) NOD.cMiPLA2𝛽;;;;+/- bone marrow-derived (BMD) MΦ are skewed towards an anti-inflammatory phenotype in comparison to NOD BMD MΦ, favoring an anti-inflammatory phenotype. (4) Selective inhibition of (PGE2 and DHETs) shifted NOD MΦ towards an anti-inflammatory phenotype. These findings suggest that MΦ-iDLs contribute to T1D development, and inhibition of select iDLs production can be targeted to counter T1D development. Disclosure A. Almutairi: None. Y. Gai: None. X. Y. Lei: None. D. Stephenson: None. C. Chalfant: None. S. Ramanadham: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK110292); National Institute of Allergy and Infectious Diseases (R21AI146743)

Type 1 diabetes (T1D) is a consequence of autoimmune-mediated destruction of pancreatic β-cells and the leading causes for this process are incompletely understood. Our previous work suggests the involvement of lipid signaling from immune cells on T1D development. Relevant lipids were shown to be generated by the Ca2+-independent phospholipase A2β (iPLA2β), which is ubiquitously expressed and hydrolyzes membrane phospholipids at the sn-2 position to release bioactive lysophospholipids and free fatty acids such as arachidonic acid. Arachidonic acid can be metabolized to generate eicosanoids, many of which are pro-inflammatory. We found that iPLA2β activation promotes pro-inflammatory M1 macrophage phenotype and that selective inhibition of iPLA2β preserves β-cell mass and reduces T1D incidence and insulitis in the NOD mice. Herein, we report that NOD macrophages generate significantly higher pro-inflammatory lipids and reduced anti-inflammatory lipids than C57 macrophages during the prediabetic phase. Such changes in the lipidome are mitigated with reduced expression of iPLA2β. Specifically, lipidomics analyses revealed that NOD.iPLA2β+/- macrophage production of multiple pro-inflammatory lipids (PGE2, leukotrienes, 12-HETE, DHETs) is decreased, and of anti-inflammatory lipids increased, relative to NOD-derived macrophages. The mitigated inflammatory lipid signature during the prediabetic phase the NOD.iPLA2β+/- contributed to a reduced T1D incidence. These findings suggest a role for macrophage iPLA2β-derived lipids (iDLs) in T1D development. In support, adoptive transfer of NOD.iPLA2β+/- macrophages reduced T1D incidence and improved glucose tolerance; and conditional knockout of iPLA2β in macrophages reduced T1D incidence in the NOD mice. We hypothesize that iDLs produced by macrophages contribute to T1D development and that these could be targeted to prevent onset/progression of T1D. Disclosure A. Almutairi: None. Y. Gai: None. X.Y. Lei: None. M.A. Park: None. C. Chalfant: None. S. Ramanadham: None. D. Stephenson: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK110292); National Institute of Allergy and Infectious Diseases (R21AI146743)