Steven P. Mathis

Role of Leukotriene B4 Receptors in the Development of Atherosclerosis: Potential Mechanisms

Objective—Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1−/− mice. Methods and Results—Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E−/− mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. Conclusions—The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

Deficiency of the Leukotriene B4 Receptor, BLT-1, Protects against Systemic Insulin Resistance in Diet-Induced Obesity

Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b+ monocytes in wild-type mice that express leukotriene B4 receptor, BLT-1, and that this increase is abolished in BLT-1–null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1−/− mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1−/− mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.

Leukotriene B4 receptor-1 mediates intermittent hypoxia-induced atherogenesis.

RATIONALE Obstructive sleep apnea, which is characterized by intermittent hypoxia (IH) during sleep, has emerged as an independent risk factor for cardiovascular disease, including atherosclerosis. Leukotriene B4 (LTB4) production is increased in patients with obstructive sleep apnea and negatively correlates to hypoxic levels during sleep, with continuous positive airway pressure therapy decreasing LTB4 production. OBJECTIVES Determine the potential role of LTB4 in IH-induced atherosclerosis in a monocyte cellular model and a murine model. METHODS THP-1 cells were exposed to IH for 3, 6, 24, and 48 hours. Macrophage transformation and foam cell formation were assessed after IH exposures. Apolipopotein E (ApoE)(-/-) or BLT1(-/-)/ApoE(-/-) mice were fed an atherogenic diet and exposed to IH (alternating 21% and 5.7% O(2) from 7 am to 7 PM each day) for 10 weeks. Atherosclerotic lesion formation in en face aorta was examined by oil red O staining. MEASUREMENTS AND MAIN RESULTS IH increased production of LTB4 and the expression of 5-lipoxygenase and leukotriene A4 hydrolase, the key enzymes for producing LTB4. IH was associated with transformation of monocytes to activated macrophages, as evidenced by increased expression of CD14 and CD68. In addition, IH exposures promoted increased cellular cholesterol accumulation and foam cell formation. The LTB4 receptor 1 (BLT1) antagonist U-75302 markedly attenuated IH-induced changes. Furthermore, IH promoted atherosclerotic lesion formation in ApoE(-/-) mice. IH-induced lesion formation was markedly attenuated in BLT1(-/-)/ApoE(-/-) mice. CONCLUSIONS BLT1-dependent pathways underlie IH-induced atherogenesis, and may become a potential novel therapeutic target for obstructive sleep apnea-associated cardiovascular disease.

Nonredundant Roles for Leukotriene B4 Receptors BLT1 and BLT2 in Inflammatory Arthritis

Lipid mediators derived from arachidonic acid through the cyclooxygenase and lipoxygenase pathways are known to be important mediators of inflammation. Studies in mouse models demonstrated an important role for the high-affinity leukotriene B4 receptor BLT1 in arthritis, atherosclerosis, and asthma. BLT2, a low-affinity leukotriene B4 receptor, was also shown to be a high-affinity receptor for cyclooxygenase-1 derived 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. However, its biochemical activities and physiological roles remain unknown. In this study, we developed mice deficient in BLT2 by targeted disruption. The BLT2−/− mice developed normally, and analysis of immune cells showed that disruption of BLT2 did not alter BLT1 expression or function. Mast cells from the C57BL/6 mice but not from the BLT2−/− mice showed intracellular calcium mobilization in response to 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. In an autoantibody-induced inflammatory arthritis model, the BLT2−/− mice showed reduced incidence and severity of disease, including protection from bone and cartilage loss. Reciprocal bone marrow transplant experiments identified that loss of BLT2 expression on a bone marrow-derived cell lineage offers protection against severe disease. Thus, BLT2, a unique receptor for 5-lipoxygenase– and cyclooxygenase-1–derived lipid mediators, represents a novel target for therapies directed at treating inflammation associated with arthritis.

Role of Leukotriene B4 Receptors in Rheumatoid Arthritis

The purpose of this review is to summarize the role that murine models of arthritis are playing in the understanding of human rheumatoid arthritis and how leukotriene B(4) (LTB(4)) is emerging as an important target in this field. Both the collagen-induced arthritis (CIA) model and the K/BxN serum transfer arthritis model have contributed to outline the potential mechanisms involved in inflammatory arthritis. Indeed, the CIA model has contributed to the development of effective anti-TNFalpha and anti-IL-1beta based treatments for RA that are currently in the clinic. Many recent studies in mouse models have suggested a critical role for LTB(4) and its receptors in the development of inflammatory arthritis. Inhibitors of LTB(4) biosynthesis as well as LTB(4) receptors are protective in mouse models of RA and mice deficient in the LTB(4) biosynthetic enzymes or LTB(4) receptors are resistant to disease development suggesting several promising targets for RA in this pathway.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called “cellular senescence.” It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P1 and S1P2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P2-mediated activation of the lipid phosphatase PTEN.

Corticosteroids enhance CD8+ T cell–mediated airway hyperresponsiveness and allergic inflammation by upregulating leukotriene B4 receptor 1

BACKGROUND Leukotriene B4 (LTB4) is a potent inflammatory lipid mediator that binds to LTB4 receptor 1 (BLT1). Ligation of BLT1 by LTB4 plays an important role in the recruitment of effector memory CD8+ T cells into the airways of sensitized and challenged mice. OBJECTIVES The effects of the corticosteroid dexamethasone (DEX) on BLT1-expressing effector memory CD8+ T cells and effector memory CD8+ T cell-mediated airway hyperresponsiveness (AHR) and allergic inflammation were determined. METHODS Effector memory CD8+ T cells were generated from ovalbumin(257-264)-primed mononuclear cells from OT-1 mice in the presence of IL-2. In some cultures DEX was added. The effects of DEX on BLT1 expression, LTB4-induced Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, chemotaxis, and effector memory CD8+ T cell-mediated AHR were examined. RESULTS DEX-treated effector memory CD8+ T cells showed significant increases in surface expression of BLT1, LTB4-induced intracellular Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, and chemotaxis. Upregulation of BLT1 by DEX was accompanied by increased IL-2 receptor expression. Adoptive transfer of DEX-treated effector memory CD8+ T cells into ovalbumin-sensitized and ovalbumin-challenged CD8-/- mice resulted in significant increases in AHR, allergic inflammation, goblet cell metaplasia, and numbers of both CD8+ and CD4+ T cells in the bronchoalveolar lavage fluid and lungs. CONCLUSIONS Corticosteroids upregulate BLT1 on effector memory CD8+ T cells and related signaling pathways and potentiate allergic airway inflammation and AHR induced by these cells.

Critical role for polar residues in coupling Leukotriene B4 binding to signal transduction in BLT1

Leukotriene B4 (LTB4) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB4 binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB4 binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.

Leukotriene B4-receptor-1 mediated host response shapes gut microbiota and controls colon tumor progression

ABSTRACT Inflammation and infection are key promoters of colon cancer but the molecular interplay between these events is largely unknown. Mice deficient in leukotriene B4 receptor1 (BLT1) are protected in inflammatory disease models of arthritis, asthma and atherosclerosis. In this study, we show that BLT1−/− mice when bred onto a spontaneous tumor (ApcMin/+) model displayed an increase in the rate of intestinal tumor development and mortality. A paradoxical increase in inflammation in the tumors from the BLT1−/−ApcMin/+ mice is coincidental with defective host response to infection. Germ-free BLT1−/−ApcMin/+ mice are free from colon tumors that reappeared upon fecal transplantation. Analysis of microbiota showed defective host response in BLT1−/− ApcMin/+ mice reshapes the gut microbiota to promote colon tumor development. The BLT1−/−MyD88−/− double deficient mice are susceptible to lethal neonatal infections. Broad-spectrum antibiotic treatment eliminated neonatal lethality in BLT1−/−MyD88−/− mice and the BLT1−/−MyD88−/−ApcMin+ mice are protected from colon tumor development. These results identify a novel interplay between the Toll-like receptor mediated microbial sensing mechanisms and BLT1-mediated host response in the control of colon tumor development.

Mast Cell–Dependent CD8+ T-cell Recruitment Mediates Immune Surveillance of Intestinal Tumors in ApcMin/+ Mice

Mast cells, depending on the microenvironment, can promote or suppress tumor progression. Chemokine-mediated recruitment of mast cells was an essential prerequisite for the initiation of LTB4/BLT1-regulated CD8+ T-cell homing and generation of antitumor immunity against intestinal tumors. The presence of mast cells in some human colorectal cancers is a positive prognostic factor, but the basis for this association is incompletely understood. Here, we found that mice with a heterozygous mutation in the adenomatous polyposis coli gene (ApcMin/+) displayed reduced intestinal tumor burdens and increased survival in a chemokine decoy receptor, ACKR2-null background, which led to discovery of a critical role for mast cells in tumor defense. ACKR2–/–ApcMin/+ tumors showed increased infiltration of mast cells, their survival advantage was lost in mast cell–deficient ACKR2–/–SA–/–ApcMin/+ mice as the tumors grew rapidly, and adoptive transfer of mast cells restored control of tumor growth. Mast cells from ACKR2–/– mice showed elevated CCR2 and CCR5 expression and were also efficient in antigen presentation and activation of CD8+ T cells. Mast cell–derived leukotriene B4 (LTB4) was found to be required for CD8+ T lymphocyte recruitment, as mice lacking the LTB4 receptor (ACKR2–/–BLT1–/–ApcMin/+) were highly susceptible to intestinal tumor-induced mortality. Taken together, these data demonstrate that chemokine-mediated recruitment of mast cells is essential for initiating LTB4/BLT1-regulated CD8+ T-cell homing and generation of effective antitumor immunity against intestinal tumors. We speculate that the pathway reported here underlies the positive prognostic significance of mast cells in selected human tumors. Cancer Immunol Res; 6(3); 332–47. ©2018 AACR.