Brian L. Harry

Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis. Methods and Results—Bone marrow from 12/15-LO−/−/apoE−/− mice was transplanted into apoE−/− mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE−/− mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2&agr;-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO−/−/apoE−/− mice regained the severity of atherosclerotic lesion typical of apoE−/− mice after replacement of their bone marrow cells with bone marrow from apoE−/− mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO−/− mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL. Conclusions—We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.

CXCR6 Promotes Atherosclerosis by Supporting T-Cell Homing, Interferon-γ Production, and Macrophage Accumulation in the Aortic Wall

Background— T lymphocytes are thought to be important in atherosclerosis, but very little is known about the mechanisms of lymphocyte recruitment into atherosclerosis-prone aortas. In this study we tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of CD4+ T helper 1 cells and natural killer T cells, is involved in lymphocyte homing into the aortic wall and modulates the development and progression of atherosclerosis. Methods and Results— To investigate the role of CXCR6 in the development and progression of atherosclerosis, we bred CXCR6-deficient (CXCR6GFP/GFP) mice with apolipoprotein E–deficient (ApoE−/−) mice. We found that CXCR6GFP/GFP/ApoE−/− mice fed a Western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE−/− controls. Flow cytometry analysis of the aortas from CXCR6GFP/GFP/ApoE−/− mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas resulted in significantly diminished production of interferon-&ggr; and reduction of CD11b+/CD68+ macrophages in the aorta. Conclusions— These data provide evidence for a proatherosclerotic role of CXCR6. Absence of CXCR6 alters the recruitment of CXCR6+ leukocytes and modulates the local immune response within the aortic wall.

Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.

Endothelial Cell PECAM-1 Promotes Atherosclerotic Lesions in Areas of Disturbed Flow in ApoE-Deficient Mice

Objective—Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. Methods and Results—We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-&kgr;B were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-&kgr;B activity and VCAM-1 expression under conditions of atheroprone flow. Conclusion—These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-&kgr;B–mediated gene expression.

Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization

Eliminating paternal mitochondria During fertilization, the oocyte and sperm each bring their mitochondria to the union. Shortly afterward, the paternal mitochondria are degraded, and only the maternal mitochondria are conveyed to the progeny. Zhou et al. observed that the integrity of the inner membrane of paternal mitochondria is compromised, which apparently marks them for degradation (see the Perspective by van der Bliek). Autophagy commences by mitochondrial endonuclease G relocating from the intermembrane space into the matrix and subsequently degrading the paternal mitochondrial DNA. Any delay in this process increases embryonic lethality. Science, this issue p. 394; see also p. 351 A mitochondrial enzyme promotes the destruction and removal of sperm-derived mitochondria in nematode worm embryos. Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.

DARC on RBC limits lung injury by balancing compartmental distribution of CXC chemokines

The Duffy antigen receptor for chemokines (DARC) has a high affinity for CC and CXC chemokines. However, it lacks the ability to induce cell responses that are typical for classical chemokine receptors. The role of DARC in inflammatory conditions remains to be elucidated. We studied the role of DARC in a murine model of acute lung injury. We found that in Darc‐gene‐deficient (Darc−/−) mice, LPS‐induced PMN migration into the alveolar space was elevated more than twofold. In contrast, PMN adhesion to endothelial cells and within the interstitial space was reduced in Darc−/− mice. Darc−/− mice also exhibited increased microvascular permeability. Elevated PMN migration in Darc−/− mice was associated with increased concentrations of two essential CXCR2 ligands, CXCL1 and CXCL2/3 in the alveolar space. In the blood, CXCL1 was mostly associated with RBC in WT mice and with plasma in Darc−/− mice. We found that DARC on RBC prevented excessive PMN migration into the alveolar space. In contrast, DARC on non‐hematopoietic cells appeared to have only minor effects on leukocyte trafficking in this model. These findings show how DARC regulates lung inflammation by controlling the distribution and presentation of chemokines that bind CXCR2.

Improved Survival and Reduced Vascular Permeability by Eliminating or Blocking 12/15-Lipoxygenase in Mouse Models of Acute Lung Injury (ALI)

Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15−/− mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15−/− mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans

HBx is a multifunctional hepatitis B virus (HBV) protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. However, the host targets and mechanisms of action of HBx are poorly characterized. We show here that expression of HBx in Caenorhabditis elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV. Genetic and biochemical analyses indicate that HBx interacts directly with the B-cell lymphoma 2 (Bcl-2) homolog CED-9 (cell death abnormal) through a Bcl-2 homology 3 (BH3)-like motif to trigger both cytosolic Ca2+ increase and cell death. Importantly, Bcl-2 can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2 are conserved cellular targets of HBx. A genetic suppressor screen of HBx-induced cell death has produced many mutations, including mutations in key regulators from both apoptosis and necrosis pathways, indicating that this screen can identify new apoptosis and necrosis genes. Our results suggest that C. elegans could serve as an animal model for identifying crucial host factors and signaling pathways of HBx and aid in development of strategies to treat HBV-induced liver disorders.

JAK2 inhibition for the treatment of hematologic and solid malignancies

Introduction: Mutations in Janus kinase 2 (JAK2), and in particular JAK2 V617F, are common in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In the past several years, JAK2 inhibitors have been rapidly developed as targeted therapies for MPNs. Areas covered: JAK2 mutations, including JAK2 V617F and unique fusion proteins, are critical for oncogenesis of some hematologic malignancies. Although JAK2 mutations are extremely rare in solid cancers, pathophysiological JAK2/STAT signaling can still promote tumor cell growth, proliferation, migration, invasion and angiogenesis. JAK2 inhibition can curtail malignant cellular behaviors and thus may be a promising therapeutic strategy. Expert opinion: The involvement of oncogenic JAK2 mutations in hematologic malignancies indicates that JAK2 inhibition has the potential to be a highly successful treatment option. The exact role of JAK2 signaling in solid cancers is unclear, but JAK2 inhibition may prevent disease progression by restricting malignant cell phenotypes. JAK2 inhibitors in development for the treatment of MPNs have demonstrated clinical activity with minimal toxicity. This class of agents should be investigated more rigorously for the treatment of other malignancies with aberrant JAK2 signaling with or without JAK2 mutations.