BURMESE ROOFED TURTLE (BATAGUR TRIVITTATA) DISEASE SCREENING IN MYANMAR.

The Burmese roofed turtle (Batagur trivittata), a critically endangered freshwater turtle, is endemic to Myanmar. Once thought to be extinct, remnant wild populations were discovered in 2001 and limited captive individuals identified in pagoda ponds or confiscated from fishers in Myanmar. These and their offspring are maintained in five facilities in Myanmar and form the basis of a conservation program (habitat protection, captive breeding, nest protection, egg collection, head-starting, and release). Prerelease health screenings were performed in 2014 and 2018 at Yadanabon Zoological Gardens, a head-starting facility in Limpha Village, and Lawkanandar Wildlife Park. One hundred forty-three turtles were assessed (37 male, 50 female, 56 juveniles [too young to determine sex]; two females were assessed in both years), age range of 1 to 12 y (one unknown age adult founder), and body mass range of 0.111 to 32.72 kg. Health evaluations both years included physical examination and combined choanal/cloacal swab samples for polymerase chain reaction testing of the potential chelonian pathogens intranuclear coccidia, Mycoplasma, Herpesvirus, Ranavirus, and Adenovirus (not all tests performed each year). In 2018, cloacal swabs from 30 and 20 turtles at the Yadanabon Zoological Gardens and Lawkanandar Wildlife Park, respectively, were cultured for Salmonella. All turtles were assessed as healthy based on normal physical examination findings, and all had negative test results. Prerelease health screening, such as performed in this study, is an important component of release, reintroduction, and translocation projects to prevent introduction of novel pathogens into naïve wild populations.

Correction: Adenovirus and Herpesvirus Diversity in Free-Ranging Great Apes in the Sangha Region of the Republic of Congo.

[This corrects the article DOI: 10.1371/journal.pone.0118543.].

HEALTH ASSESSMENT OF FREE-RANGING CHELONIANS IN AN URBAN SECTION OF THE BRONX RIVER, NEW YORK, USA.

The Bronx River in Bronx, New York, US spans an area of significant human development and has been subject to historic and ongoing industrial contamination. We evaluated the health of freeranging native common snapping turtles ( Chelydra serpentina) and nonnative invasive red-eared sliders ( Trachemys scripta) in a segment of the Bronx River between May and July 2012. In 18 snapping turtles and nine sliders, complete physical examinations were performed, ectoparasites collected, and blood was analyzed for contaminants (mercury, thallium, cadmium, arsenic, lead, selenium, oxychlordane, alpha-chlordane, dieldrin, DDD, DDE, polychlorinated biphenyls). Complete blood counts and the presence of hemoparasites were determined in 16 snapping turtles and nine sliders. Swabs of the choana and cloaca were screened for ranavirus, adenovirus, herpesvirus, and Mycoplasma spp. by PCR in 39 snapping turtles and 28 sliders. Both turtle species exhibited bioaccumulation of various environmental contaminants, particularly organochlorines and polychlorinated biphenyls. Molecular screening revealed a unique herpesvirus in each species. A Mycoplasma sp. previously isolated from emydid turtles was detected in red-eared sliders while a unique Mycoplasma sp. was identified in common snapping turtles. Ranaviruses and adenoviruses were not detected. Our study established a baseline health assessment to which future data can be compared. Moreover, it served to expand the knowledge and patterns of health markers, environmental contaminants, and microorganisms of freeranging chelonians.

Post-mortem findings in southern right whales Eubalaena australis at Península Valdés, Argentina, 2003-2012.

Between 2003 and 2012, 605 southern right whales (SRW; Eubalaena australis) were found dead along the shores of Península Valdés (PV), Argentina. These deaths included alarmingly high annual losses between 2007 and 2012, a peak number of deaths (116) in 2012, and a significant number of deaths across years in calves-of-the-year (544 of 605 [89.9%]; average = 60.4 yr(-1)). Post-mortem examination and pathogen testing were performed on 212 whales; 208 (98.1%) were calves-of-the-year and 48.0% of these were newborns or neonates. A known or probable cause of death was established in only a small number (6.6%) of cases. These included ship strike in a juvenile and blunt trauma or lacerations (n = 5), pneumonia (n = 4), myocarditis (n = 2), meningitis (n = 1), or myocarditis and meningitis (n = 1) in calves. Ante-mortem gull parasitism was the most common gross finding. It was associated with systemic disease in a single 1-2 mo old calf. Immunohistochemical labeling for canine distemper virus, Toxoplasma gondii and Brucella spp., and PCR for cetacean morbillivirus (CeMV), influenza A, and apicomplexan protozoa were negative on formalin-fixed, paraffin-embedded lung and brain samples from a subset of whales; PCR for Brucella spp. was positive in a newborn/neonate with pneumonia. Skin samples from whales with gull parasitism were PCR negative for CeMV, poxvirus, and papillomavirus. This is the first long-term study to investigate and summarize notable post-mortem findings in the PV SRW population. Consistent, significant findings within or between years to explain the majority of deaths and those in high-mortality years remain to be identified.

Adenovirus and herpesvirus diversity in free-ranging great apes in the Sangha region of the Republic Of Congo.

Infectious diseases have caused die-offs in both free-ranging gorillas and chimpanzees. Understanding pathogen diversity and disease ecology is therefore critical for conserving these endangered animals. To determine viral diversity in free-ranging, non-habituated gorillas and chimpanzees in the Republic of Congo, genetic testing was performed on great-ape fecal samples collected near Odzala-Kokoua National Park. Samples were analyzed to determine ape species, identify individuals in the population, and to test for the presence of herpesviruses, adenoviruses, poxviruses, bocaviruses, flaviviruses, paramyxoviruses, coronaviruses, filoviruses, and simian immunodeficiency virus (SIV). We identified 19 DNA viruses representing two viral families, Herpesviridae and Adenoviridae, of which three herpesviruses had not been previously described. Co-detections of multiple herpesviruses and/or adenoviruses were present in both gorillas and chimpanzees. Cytomegalovirus (CMV) and lymphocryptovirus (LCV) were found primarily in the context of co-association with each other and adenoviruses. Using viral discovery curves for herpesviruses and adenoviruses, the total viral richness in the sample population of gorillas and chimpanzees was estimated to be a minimum of 23 viruses, corresponding to a detection rate of 83%. These findings represent the first description of DNA viral diversity in feces from free-ranging gorillas and chimpanzees in or near the Odzala-Kokoua National Park and form a basis for understanding the types of viruses circulating among great apes in this region.

Identification of a novel cetacean polyomavirus from a common dolphin (Delphinus delphis) with Tracheobronchitis.

A female short-beaked common dolphin calf was found stranded in San Diego, California in October 2010, presenting with multifocal ulcerative lesions in the trachea and bronchi. Viral particles suggestive of polyomavirus were detected by EM, and subsequently confirmed by PCR and sequencing. Full genome sequencing (Ion Torrent) revealed a circular dsDNA genome of 5,159 bp that was shown to form a distinct lineage within the genus Polyomavirus based on phylogenetic analysis of the early and late transcriptomes. Viral infection and distribution in laryngeal mucosa was characterised using in-situ hybridisation, and apoptosis observed in the virus-infected region. These results demonstrate that polyomaviruses can be associated with respiratory disease in cetaceans, and expand our knowledge of their diversity and clinical significance in marine mammals.

Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response.

The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1Δ strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR.

Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress.

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36⁻(/)⁻ or Tlr2⁻(/)⁻ mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr⁻(/)⁻ mice transplanted with Tlr4⁻(/)⁻Tlr2⁻(/)⁻ bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT →Ldlr⁻(/)⁻ lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis.

Induction of ER stress in macrophages of tuberculosis granulomas.

BACKGROUND: The endoplasmic reticulum (ER) stress pathway known as the Unfolded Protein Response (UPR) is an adaptive survival pathway that protects cells from the buildup of misfolded proteins, but under certain circumstances it can lead to apoptosis. ER stress has been causally associated with macrophage apoptosis in advanced atherosclerosis of mice and humans. Because atherosclerosis shares certain features with tuberculosis (TB) with regard to lesional macrophage accumulation, foam cell formation, and apoptosis, we investigated if the ER stress pathway is activated during TB infection. PRINCIPAL FINDINGS: Here we show that ER stress markers such as C/EBP homologous protein (CHOP; also known as GADD153), phosphorylated inositol-requiring enzyme 1 alpha (Ire1α) and eukaryotic initiation factor 2 alpha (eIF2α), and activating transcription factor 3 (ATF3) are expressed in macrophage-rich areas of granulomas in lungs of mice infected with virulent Mycobacterium tuberculosis (Mtb). These areas were also positive for numerous apoptotic cells as assayed by TUNEL. Microarray analysis of human caseous TB granulomas isolated by laser capture microdissection reveal that 73% of genes involved in the UPR are upregulated at the mRNA transcript level. The expression of two ER stress markers, ATF3 and CHOP, were also increased in macrophages of human TB granulomas when assayed by immunohistochemistry. CHOP has been causally associated with ER stress-induced macrophage apoptosis. We found that apoptosis was more abundant in granulomas as compared to non-granulomatous tissue isolated from patients with pulmonary TB, and apoptosis correlated with CHOP expression in areas surrounding the centralized areas of caseation. CONCLUSIONS: In summary, ER stress is induced in macrophages of TB granulomas in areas where apoptotic cells accumulate in mice and humans. Although macrophage apoptosis is generally thought to be beneficial in initially protecting the host from Mtb infection, death of infected macrophages in advanced granulomas might favor dissemination of the bacteria. Therefore future work is needed to determine if ER-stress is causative for apoptosis and plays a role in the host response to infection.

ABCA1 and ABCG1 protect against oxidative stress-induced macrophage apoptosis during efferocytosis.

RATIONALE: Antiatherogenic effects of plasma high-density lipoprotein (HDL) include the ability to inhibit apoptosis of macrophage foam cells. The ATP-binding cassette transporters ABCA1 and ABCG1 have a major role in promoting cholesterol efflux from macrophages to apolipoprotein A-1 and HDL and are upregulated during the phagocytosis of apoptotic cells (efferocytosis). OBJECTIVE: The goal of this study was to determine the roles of ABCA1 and ABCG1 in preserving the viability of macrophages during efferocytosis. METHODS AND RESULTS: We show that despite similar clearance of apoptotic cells, peritoneal macrophages from Abca1(-/-)Abcg1(-/-), Abcg1(-/-), and, to a lesser extent, Abca1(-/-) mice are much more prone to apoptosis during efferocytosis compared to wild-type cells. Similar findings were observed following incubations with oxidized phospholipids, and the ability of HDL to protect against oxidized phospholipid-induced apoptosis was markedly reduced in Abca1(-/-)Abcg1(-/-) and Abcg1(-/-) cells. These effects were independent of any role of ABCA1 and ABCG1 in mediating oxidized phospholipid efflux but were reversed by cyclodextrin-mediated cholesterol efflux. The apoptotic response observed in Abca1(-/-)Abcg1(-/-) macrophages after oxidized phospholipid exposure or engulfment of apoptotic cells was dependent on an excessive oxidative burst secondary to enhanced assembly of NADPH oxidase (NOX)2 complexes, leading to sustained Jnk activation which turned on the apoptotic cell death program. Increased NOX2 assembly required Toll-like receptors 2/4 and MyD88 signaling, which are known to be enhanced in transporter deficient cells in a lipid raft-dependent fashion. CONCLUSIONS: We identified a new beneficial role of ABCA1, ABCG1 and HDL in dampening the oxidative burst and preserving viability of macrophages following exposure to oxidized phospholipids and/or apoptotic cells.

Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways.

ER stress-induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress-mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress-induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIgamma (CaMKIIgamma) and JNK. Remarkably, CaMKIIgamma was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress-induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIgamma-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress-induced apoptosis.

Macrophage apoptosis in advanced atherosclerosis.

Plaque necrosis in advanced atheromata, which triggers acute atherothrombotic vascular events, is caused by the apoptosis of lesional macrophages coupled with defective phagocytic clearance of the dead cells. The central enabling event in macrophage apoptosis relevant to advanced atherosclerosis is the unfolded protein response (UPR), an endoplasmic reticulum (ER) stress pathway. The UPR effector CHOP (GADD153) amplifies release of ER Ca(2+) stores, which activates a central integrator of apoptosis signaling, calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII, in turn, leads to activation of pro-apoptotic STAT1, induction of the death receptor Fas, and stimulation of the mitochondria-cytochrome c pathway of apoptosis. While these pathways are necessary for apoptosis, apoptosis occurs only when the cells are also exposed to one or more additional "hits." These hits amplify pro-apoptotic pathways and/or suppress compensatory cell-survival pathways. A second hit relevant to atherosclerosis is activation of pattern recognition receptors (PRRs), such as scavenger and toll-like receptors. In vivo relevance is suggested by the fact that advanced human lesions express markers of UPR activation that correlate closely with the degree of plaque vulnerability and macrophage apoptosis. Moreover, studies with genetically altered mice have shown that ER stress and PRR activation are causative for advanced lesional macrophage apoptosis and plaque necrosis. In summary, a key cellular event in the conversion of benign to vulnerable atherosclerotic plaques is ER stress-induced macrophage apoptosis. Further understanding of the mechanisms and consequences of this event may lead to novel therapies directed at preventing the clinical progression of atheromata.

Macrophage deficiency of p38alpha MAPK promotes apoptosis and plaque necrosis in advanced atherosclerotic lesions in mice.

ER stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress-induced apoptosis may affect advanced atherosclerosis. Here we placed Apoe-/- mice deficient in macrophage p38alpha MAPK on a Western diet and found that they had a marked increase in macrophage apoptosis and plaque necrosis. The macrophage p38alpha-deficient lesions also exhibited a significant reduction in collagen content and a marked thinning of the fibrous cap, which suggests that plaque progression was advanced in these mice. Consistent with our in vivo data, we found that ER stress-induced apoptosis in cultured primary mouse macrophages was markedly accelerated under conditions of p38 inhibition. Pharmacological inhibition or genetic ablation of p38 suppressed activation of Akt in cultured macrophages and in atherosclerotic lesions. In addition, inhibition of Akt enhanced ER stress-induced macrophage apoptosis, and expression of a constitutively active myristoylated Akt blocked the enhancement of ER stress-induced apoptosis that occurred with p38 inhibition in cultured cells. Our results demonstrate that p38alpha MAPK may play a critical role in suppressing ER stress-induced macrophage apoptosis in vitro and advanced lesional macrophage apoptosis in vivo.

Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K.

The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.

Mechanisms and consequences of macrophage apoptosis in atherosclerosis.

Macrophage apoptosis is an important feature of atherosclerotic plaque development. Research directed at understanding the functional consequences of macrophage death in atherosclerosis has revealed opposing roles for apoptosis in atherosclerotic plaque progression. In early lesions, macrophage apoptosis limits lesion cellularity and suppresses plaque progression. In advanced lesions, macrophages apoptosis promotes the development of the necrotic core, a key factor in rendering plaques vulnerable to disruption and in acute lumenal thrombosis. The first section of this review will examine the role of phagocytic clearance of apoptotic macrophages, a process known as efferocytosis, in the dichotomous roles of macrophage apoptosis in early vs. advanced lesions. The second section will focus on the molecular and cellular mechanisms that are thought to govern macrophage death during atherosclerosis. Of particular interest is the complex and coordinated role that the endoplasmic reticulum (ER) stress pathway and pattern recognition receptors (PRRs) may play in triggering macrophage apoptosis.

Forkhead transcription factors (FoxOs) promote apoptosis of insulin-resistant macrophages during cholesterol-induced endoplasmic reticulum stress.

OBJECTIVE: Endoplasmic reticulum stress increases macrophage apoptosis, contributing to the complications of atherosclerosis. Insulin-resistant macrophages are more susceptible to endoplasmic reticulum stress-associated apoptosis probably contributing to macrophage death and necrotic core formation in atherosclerotic plaques in type 2 diabetes. However, the molecular mechanisms of increased apoptosis in insulin-resistant macrophages remain unclear. RESEARCH DESIGN AND METHODS: The studies were performed in insulin-resistant macrophages isolated from insulin receptor knockout or ob/ob mice. Gain- or loss-of-function approaches were used to evaluate the roles of forkhead transcription factors (FoxOs) in endoplasmic reticulum stress-associated macrophage apoptosis. RESULTS: Insulin-resistant macrophages showed attenuated Akt activation and increased nuclear localization of FoxO1 during endoplasmic reticulum stress induced by free cholesterol loading. Overexpression of active FoxO1 or FoxO3 failed to induce apoptosis in unchallenged macrophages but exacerbated apoptosis in macrophages with an active endoplasmic reticulum stress response. Conversely, macrophages with genetic knockouts of FoxO1, -3, and -4 were resistant to apoptosis in response to endoplasmic reticulum stress. FoxO1 was shown by chromatin immunoprecipitation and promoter expression analysis to induce inhibitor of kappaBepsilon gene expression and thereby to attenuate the increase of nuclear p65 and nuclear factor-kappaB activity during endoplasmic reticulum stress, with proapoptotic and anti-inflammatory consequences. CONCLUSIONS: Decreased Akt and increased FoxO transcription factor activity during the endoplasmic reticulum stress response leads to increased apoptosis of insulin-resistant macrophages. FoxOs may have a dual cellular function, resulting in either proapoptotic or anti-inflammatory effects in an endoplasmic reticulum stress-modulated manner. In the complex plaque milieu, the ultimate effect is likely to be an increase in macrophage apoptosis, plaque inflammation, and destabilization.

Signal transducer and activator of transcription-1 is critical for apoptosis in macrophages subjected to endoplasmic reticulum stress in vitro and in advanced atherosclerotic lesions in vivo.

BACKGROUND: Macrophage apoptosis is a critical process in the formation of necrotic cores in vulnerable atherosclerotic plaques. In vitro and in vivo data suggest that macrophage apoptosis in advanced atheromata may be triggered by a combination of endoplasmic reticulum stress and engagement of the type A scavenger receptor, which together induce death through a rise in cytosolic calcium and activation of toll-like receptor-4. METHODS AND RESULTS: Using both primary peritoneal macrophages and studies in advanced atheromata in vivo, we introduce signal transducer and activator of transcription-1 (STAT1) as a critical and necessary component of endoplasmic reticulum stress/type A scavenger receptor-induced macrophage apoptosis. We show that STAT1 is serine phosphorylated in macrophages subjected to type A scavenger receptor ligands and endoplasmic reticulum stress in a manner requiring cytosolic calcium, calcium/calmodulin-dependent protein kinase II, and toll-like receptor-4. Remarkably, apoptosis was inhibited by approximately 80% to 90% (P<0.05) by STAT1 deficiency or calcium/calmodulin-dependent protein kinase II inhibition. In vivo, nuclear Ser-P-STAT1 was found in macrophage-rich regions of advanced murine and human atheromata. Most important, macrophage apoptosis was decreased by 61% (P=0.034) and plaque necrosis by 34% (P=0.02) in the plaques of fat-fed low density lipoprotein receptor null Ldlr-/- mice transplanted with Stat1-/- bone marrow. CONCLUSIONS: STAT1 is critical for endoplasmic reticulum stress/type A scavenger receptor-induced apoptosis in primary tissue macrophages and in macrophage apoptosis in advanced atheromata. These findings suggest a potentially important role for STAT1-mediated macrophage apoptosis in atherosclerotic plaque progression.

The impact of insulin resistance on macrophage death pathways in advanced atherosclerosis.

Macrophage death in advanced atherosclerosis causes plaque necrosis, which promotes plaque rupture and acute atherothrombotic vascular events. Of interest, plaque necrosis and atherothrombotic disease are markedly increased in diabetes and metabolic syndrome. We discovered a novel 'multi-hit' macrophage apoptosis pathway that appears to be highly relevant to advanced atherosclerosis. The elements of the pathway include: (a) activation of the unfolded protein response (UPR) by cholesterol overloading of the endoplasmic reticulum or by other UPR activators known to exist in atheromata; and (b) pro-apoptotic signalling involving the type A scavenger receptor (SRA). The downstream apoptosis effectors include CHOP (GADD153) for the UPR and JNK for SRA signalling. Remarkably, components of this pathway are enhanced in macrophages with defective insulin signalling, including UPR activation and SRA expression. As a result, insulin-resistant macrophages show increased susceptibility to apoptosis when exposed to UPR activators and SRA ligands. Moreover, the advanced lesions of atherosclerosis-prone mice reconstituted with insulin-resistant macrophages show increased macrophage apoptosis and plaque necrosis. Based on these findings, we propose that one mechanism of increased plaque necrosis and atherothrombotic vascular disease in insulin resistant syndromes is up-regulation of a two-hit signal transduction pathway involved in advanced lesional macrophage death.

Combinatorial pattern recognition receptor signaling alters the balance of life and death in macrophages.

Macrophage pattern recognition receptors (PRRs) play key roles in innate immunity, but they also may contribute to disease processes under certain pathological conditions. We recently showed that engagement of the type A scavenger receptor (SRA), a PRR, triggers JNK-dependent apoptosis in endoplasmic reticulum (ER)-stressed macrophages. In advanced atherosclerotic lesions, the SRA, activated JNK, and ER stress are observed in macrophages, and macrophage death in advanced atheromata leads to plaque necrosis. Herein, we show that SRA ligands trigger apoptosis in ER-stressed macrophages by cooperating with another PRR, Toll-like receptor 4 (TLR4), to redirect TLR4 signaling from prosurvival to proapoptotic. Common SRA ligands activate both TLR4 signaling and engage the SRA. The TLR4 effect results in activation of the proapoptotic MyD88-JNK branch of TLR4, whereas the SRA effect silences the prosurvival IRF-3-IFN-beta branch of TLR4. The normal cell-survival effect of LPS-induced TLR4 activation is converted into an apoptosis response by immunoneutralization of IFN-beta, and the apoptosis effect of SRA ligands is converted into a cell-survival response by reconstitution with IFN-beta. Thus, combinatorial signaling between two distinct PRRs results in a functional outcome-macrophage apoptosis that does not occur with either PRR alone. PRR-induced macrophage death may play important roles in advanced atherosclerosis and in other innate immunity-related processes in which the balance between macrophage survival and death is critical.