Bhama Ramkhelawon

CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation

Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1β (IL-1β) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimers disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-β and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1β production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1β and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.

TGF-β activity protects against inflammatory aortic aneurysm progression and complications in angiotensin II–infused mice

Complicated abdominal aortic aneurysm (AAA) is a major cause of mortality in elderly men. Ang II-dependent TGF-beta activity promotes aortic aneurysm progression in experimental Marfan syndrome. However, the role of TGF-beta in experimental models of AAA has not been comprehensively assessed. Here, we show that systemic neutralization of TGF-beta activity breaks the resistance of normocholesterolemic C57BL/6 mice to Ang II-induced AAA formation and markedly increases their susceptibility to the disease. These aneurysms displayed a large spectrum of complications on echography, including fissuration, double channel formation, and rupture, leading to death from aneurysm complications. The disease was refractory to inhibition of IFN-gamma, IL-4, IL-6, or TNF-alpha signaling. Genetic deletion of T and B cells or inhibition of the CX3CR1 pathway resulted in partial protection. Interestingly, neutralization of TGF-beta activity enhanced monocyte invasiveness, and monocyte depletion markedly inhibited aneurysm progression and complications. Finally, TGF-beta neutralization increased MMP-12 activity, and MMP-12 deficiency prevented aneurysm rupture. These results clearly identify a critical role for TGF-beta in the taming of the innate immune response and the preservation of vessel integrity in C57BL/6 mice, which contrasts with its reported pathogenic role in Marfan syndrome.

The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Microparticles From Human Atherosclerotic Plaques Promote Endothelial ICAM-1–Dependent Monocyte Adhesion and Transendothelial Migration

Rationale and Objective: Membrane-shed submicron microparticles (MPs) released following cell activation or apoptosis accumulate in atherosclerotic plaques, where they stimulate endothelial proliferation and neovessel formation. The aim of the study was to assess whether or not MPs isolated from human atherosclerotic plaques contribute to increased endothelial adhesion molecules expression and monocyte recruitment. Method and Results: Human umbilical vein and coronary artery endothelial cells were exposed to MPs isolated from endarterectomy specimens (n=62) and characterized by externalized phosphatidylserine. Endothelial exposure to plaque, but not circulating, MPs increased ICAM-1 levels in a concentration-dependant manner (3.4-fold increase) without affecting ICAM-1 mRNA levels. Plaque MPs harbored ICAM-1 and transferred this adhesion molecule to endothelial cell membrane in a phosphatidylserine-dependent manner. MP-borne ICAM-1 was functionally integrated into cell membrane as demonstrated by the increased ERK1/2 phosphorylation following ICAM-1 ligation. Plaque MPs stimulated endothelial monocyte adhesion both in culture and in isolated perfused mouse carotid. This effect was also observed under flow condition and was prevented by anti–LFA-1 and anti–ICAM-1 neutralizing antibodies. MPs isolated from symptomatic plaques were more potent in stimulating monocyte adhesion than MPs from asymptomatic patients. Plaque MPs did not affect the release of interleukin-6, interleukin-8, or MCP-1, nor the expression of VCAM-1 and E-selectin. Conclusion: These results demonstrate that MPs isolated from human atherosclerotic plaques transfer ICAM-1 to endothelial cells to recruit inflammatory cells and suggest that plaque MPs promote atherosclerotic plaque progression.

MicroRNA-33–dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4(+) T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3(+) Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Netrin-1 promotes adipose tissue macrophage retention and insulin resistance in obesity

During obesity, macrophage accumulation in adipose tissue propagates the chronic inflammation and insulin resistance associated with type 2 diabetes. The factors, however, that regulate the accrual of macrophages in adipose tissue are not well understood. Here we show that the neuroimmune guidance cue netrin-1 is highly expressed in obese but not lean adipose tissue of humans and mice, where it directs the retention of macrophages. Netrin-1, whose expression is induced in macrophages by the saturated fatty acid palmitate, acts via its receptor Unc5b to block their migration. In a mouse model of diet-induced obesity, we show that adipose tissue macrophages exhibit reduced migratory capacity, which can be restored by blocking netrin-1. Furthermore, hematopoietic deletion of Ntn1 facilitates adipose tissue macrophage emigration, reduces inflammation and improves insulin sensitivity. Collectively, these findings identify netrin-1 as a macrophage retention signal in adipose tissue during obesity that promotes chronic inflammation and insulin resistance.

Effects of Native and Myeloperoxidase-Modified Apolipoprotein A-I on Reverse Cholesterol Transport and Atherosclerosis in Mice

Objective—Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. Approach and Results—ApoA-I−/− or apolipoprotein E–deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I−/− mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E–deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. Conclusions—Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

Neuroimmune Guidance Cue Semaphorin 3E Is Expressed in Atherosclerotic Plaques and Regulates Macrophage Retention

Objective—The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. Approach and Results—Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe–/– mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. Conclusions—Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

Hypoxia Induces Netrin-1 and Unc5b in Atherosclerotic Plaques Mechanism for Macrophage Retention and Survival

Objective— Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 ( Ntn1 ) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. Approach and Results— We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro , Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1 - and Unc5b -promoter activities are induced by oxidized low-density lipoprotein and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α–dependent manner. Conclusions— These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1α–induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages. # Significance {#article-title-39}Objective—Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells. Approach and Results—We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1&agr;, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1&agr;. Correspondingly, J774 macrophages overexpressing active HIF-1&agr; show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1&agr;–dependent manner. Conclusions—These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1&agr;–induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.