AIBP controls TLR4 inflammarafts and mitochondrial dysfunction in a mouse model of Alzheimer's disease.

Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease (AD). Microglia activation is accompanied by the formation and chronic maintenance of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased ROS and the dilated ER. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/- APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in AD associated oxidative stress and neurodegeneration.

Macrophage inflammarafts in atherosclerosis.

PURPOSE OF REVIEW: Advances in single cell techniques revealed a remarkable diversity in macrophage gene expression profiles in atherosclerosis. However, the diversity of functional processes at the macrophage plasma membrane remains less studied. This review summarizes recent advances in characterization of lipid rafts, where inflammatory receptors assemble, in macrophages that undergo reprogramming in atherosclerotic lesions and in vitro under conditions relevant to the development of atherosclerosis. RECENT FINDINGS: The term inflammarafts refers to enlarged lipid rafts with increased cholesterol content, hosting components of inflammatory receptor complexes assembled in close proximity, including TLR4-TLR4, TLR2-TLR1 and TLR2-CD36 dimers. Macrophages decorated with inflammarafts maintain chronic inflammatory gene expression and are primed to an augmented response to additional inflammatory stimuli. In mouse atherosclerotic lesions, inflammarafts are expressed primarily in nonfoamy macrophages and less in lipid-laden foam cells. This agrees with the reported suppression of inflammatory programs in foam cells. In contrast, nonfoamy macrophages expressing inflammarafts are the major inflammatory population in atherosclerotic lesions. Discussed are emerging reports that help understand formation and persistence of inflammarafts and the potential of inflammarafts as a novel therapeutic target. SUMMARY: Chronic maintenance of inflammarafts in nonfoamy macrophages serves as an effector mechanism of inflammatory macrophage reprogramming in atherosclerosis.

AIBP regulates TRPV1 activation in chemotherapy-induced peripheral neuropathy by controlling lipid raft dynamics and proximity to TLR4 in dorsal root ganglion neurons.

ABSTRACT: Nociceptive afferent signaling evoked by inflammation and nerve injury is mediated by the opening of ligand-gated and voltage-gated receptors or channels localized to cholesterol-rich lipid raft membrane domains. Dorsal root ganglion (DRG) nociceptors express high levels of toll-like receptor 4 (TLR4), which also localize to lipid rafts. Genetic deletion or pharmacologic blocking of TLR4 diminishes pain associated with chemotherapy-induced peripheral neuropathy (CIPN). In DRGs of mice with paclitaxel-induced CIPN, we analyzed DRG neuronal lipid rafts, expression of TLR4, activation of transient receptor potential cation channel subfamily V member 1 (TRPV1), and TLR4-TRPV1 interaction. Using proximity ligation assay, flow cytometry, and whole-mount DRG microscopy, we found that CIPN increased DRG neuronal lipid rafts and TLR4 expression. These effects were reversed by intrathecal injection of apolipoprotein A-I binding protein (AIBP), a protein that binds to TLR4 and specifically targets cholesterol depletion from TLR4-expressing cells. Chemotherapy-induced peripheral neuropathy increased TRPV1 phosphorylation, localization to neuronal lipid rafts, and proximity to TLR4. These effects were also reversed by AIBP treatment. Regulation of TRPV1-TLR4 interactions and their associated lipid rafts by AIBP covaried with the enduring reversal of mechanical allodynia otherwise observed in CIPN. In addition, AIBP reduced intracellular calcium in response to the TRPV1 agonist capsaicin, which was increased in DRG neurons from paclitaxel-treated mice and in the naïve mouse DRG neurons incubated in vitro with paclitaxel. Together, these results suggest that the assembly of nociceptive and inflammatory receptors in the environment of lipid rafts regulates nociceptive signaling in DRG neurons and that AIBP can control lipid raft-associated nociceptive processing.

Differential Expression of Inflammarafts in Macrophage Foam Cells and in Nonfoamy Macrophages in Atherosclerotic Lesions-Brief Report.

BACKGROUND: Reprogramming of monocytes and macrophage manifests in hyperinflammatory responses and chronification of inflammation in atherosclerosis. Recent studies focused on epigenetic, transcriptional, and metabolic alterations that characterize trained immunity. However, the underlying effector mechanisms driving the hyperinflammatory response of reprogrammed macrophages remain unclear. We hypothesized that the plasma membrane of atherosclerotic lesion macrophages undergoes reprogramming to maintain inflammarafts, enlarged lipid rafts (LR) serving as a platform for assembly of inflammatory receptor complexes. METHODS: Single-cell suspensions from the aortae of Western diet-fed Ldlr-/- mice were gated for BODIPY-high foamy and BODIPY-low nonfoamy F4/80 macrophages by flow cytometry. Inflammarafts were characterized by increased levels of LR, TLR4 (toll-like receptor-4) localization to LR, TLR4 dimers, and the proximity between TLR2, TLR1, and CD36. In a cellular model of trained immunity, LR, TLR4 dimers, and the inflammatory response were measured in bone marrow-derived macrophages subjected to a 24-hour treatment with LPS (lipopolysaccharide) or OxLDL (oxidized low-density lipoprotein), followed by a 6-day wash-out period. RESULTS: Nonfoamy macrophages, which constituted ≈40% of macrophages in atherosclerotic lesions, expressed significantly higher levels of LR and TLR4 dimers, as well as proximity ligation signals for TLR4-LR, TLR2-CD36, and TLR2-TLR1 complexes, compared with foamy macrophages. These inflammaraft measures associated, to a different degree, with plasma cholesterol and inflammatory cytokines, as well as the size of the atherosclerotic lesions and necrotic cores. The bone marrow-derived macrophages trained with LPS simulated nonfoamy atherosclerotic lesion macrophages and continued to express inflammarafts and inflammatory genes for 6 days after LPS removal and displayed a hyperinflammatory response to Pam3CSK4, a TLR2/TLR1 agonist. OxLDL-exposed, lipid-laden macrophages did not express inflammarafts. CONCLUSIONS: Our data support the hypothesis that persistent inflammarafts in nonfoamy macrophages in atherosclerotic lesions serve as effectors of macrophage reprogramming into a hyperinflammatory phenotype.

Macrophages and glial cells: Innate immune drivers of inflammatory arthritic pain perception from peripheral joints to the central nervous system.

Millions of people suffer from arthritis worldwide, consistently struggling with daily activities due to debilitating pain evoked by this disease. Perhaps the most intensively investigated type of inflammatory arthritis is rheumatoid arthritis (RA), where, despite considerable advances in research and clinical management, gaps regarding the neuroimmune interactions that guide inflammation and chronic pain in this disease remain to be clarified. The pain and inflammation associated with arthritis are not isolated to the joints, and inflammatory mechanisms induced by different immune and glial cells in other tissues may affect the development of chronic pain that results from the disease. This review aims to provide an overview of the state-of-the-art research on the roles that innate immune, and glial cells play in the onset and maintenance of arthritis-associated pain, reviewing nociceptive pathways from the joint through the dorsal root ganglion, spinal circuits, and different structures in the brain. We will focus on the cellular mechanisms related to neuroinflammation and pain, and treatments targeting these mechanisms from the periphery and the CNS. A comprehensive understanding of the role these cells play in peripheral inflammation and initiation of pain and the central pathways in the spinal cord and brain will facilitate identifying new targets and pathways to aide in developing therapeutic strategies to treat joint pain associated with RA.

Normalization of cholesterol metabolism in spinal microglia alleviates neuropathic pain.

Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naive mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia or reverse CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling the TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.

Lipid rafts in glial cells: role in neuroinflammation and pain processing.

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

Modulation of nNOSser852 phosphorylation and translocation by PKA/PP1 pathway in endothelial cells.

Neuronal nitric oxide synthase (nNOS) is now considered an important player in vascular function. It has a protective role in atherosclerosis and hypertension. However, despite its importance, little is known about the mechanisms that regulate its activity in vascular cells. Here we explore the mechanisms by which nNOS is activated in endothelium. We evaluated aorta relaxation response and phosphorylation of nNOS during protein phosphatases 1 and 2 (PP1 and PP2) inhibition, in eNOS silenced mice. PP1 translocation and interaction between the nuclear inhibitor of PP1 (NIPP1) and PP1 was evaluated in endothelial EA.hy926 cells. We demonstrate here that acetylcholine (Ach)-induced relaxation is completely abolished by nNOS inhibition in eNOS silenced mice aorta which also decreased NO and H2O2 concentrations. ACh induced dephosphorylation of nNOSser852 in aorta after 20 min stimulation. Endothelial cells also showed a decrease in nNOSser852 phosphorylation during 20 min of ACh stimulation. PP2 inhibition had no effect on Ach-induced nNOSSer852 dephosphorylation in endothelial cells and did not modify Ach-induced vasodilation in aorta from eNOS silenced mice. Non-selective PP1/PP2 inhibition prevented nNOSSer852 dephosphorylation in endothelial cells and prevented Ach-induced vasodilation in eNOS silenced mice. ACh induced time-dependent PP1 and NIPP1 dissociation and PP1 translocation to cytoplasm. Protein kinase A (PKA) inhibition abolished PP1 translocation and further nNOSser852 dephosphorylation. In addition, 8-Br-cAMP reduced NIPP1/PP1 interaction, stimulated PP1 translocation and nNOSser852 dephosphorylation. Moreover, PKA Inhibition led to a decreased nNOS translocation to perinuclear region. Taken together, our results elucidate a mechanism whereby PP1 is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of nNOSser852 and subsequent NO and possible H2O2 production resulting in endothelium-dependent vascular relaxation.

Role of ERK1/2 activation and nNOS uncoupling on endothelial dysfunction induced by lysophosphatidylcholine.

BACKGROUND AND AIMS: Lysophosphatidylcholine (LPC) - a main component of oxidized LDL - is involved in endothelial dysfunction that precedes atherosclerosis, with an increased superoxide anions and a reduced NO production via endothelial NO synthase (eNOS) uncoupling. However, there is no evidence about the mechanisms involved in neuronal NOS (nNOS) uncoupling. Extracellular signal-regulated kinase (ERK) is related to the control of NO production and inflammatory gene transcription activation in atherosclerosis. Our aim was to investigate the role of nNOS/ERK1/2 pathway on endothelial dysfunction induced by LPC, in mouse aorta and human endothelial cells. METHODS: Thoracic aorta from wild type mice was used to perform vascular reactivity studies in the presence or absence of LPC. Human endothelial cells were used to investigate the effect of LPC on expression of nNOS and his products NO and H2O2. RESULTS: LPC reduced acetylcholine (ACh)-induced vasodilation in mouse aorta (EmaxCT/LPC = âˆ¼95 ± 2/62 ± 3%, p = 0.0004) and increased phenylephrine-induced vasoconstriction (EmaxCT/LPC = âˆ¼4 ± 0,1/6 ± 0,1 mN/mm, p = 0.0002), with a reduction in NO (fluorescence intensityCT/LPC = 91 ± 3/62±2 × 103, p = 0.0002) and H2O2 (fluorescence intensityCT/LPC = âˆ¼16 ± 0,8/10 ± 0,7 × 103, p = 0.0041) production evocated by ACh. An inhibition of nNOS by TRIM (EmaxCT/CT+TRIM = âˆ¼93 ± 1/43 ± 3%, p = 0,0048; EmaxLPC/LPC+TRIM = âˆ¼62 ± 3/65 ± 3%) or H2O2 degradation by catalase (EmaxCT/CT+cat = âˆ¼93 ± 1/46 ± 2%, p < 0,001; EmaxLPC/LPC+cat = âˆ¼62,8 ± 3,2/60,5 ± 4,7%) reduced the relaxation in the control but not in LPC group. PD98059, an ERK1/2 inhibitor, abolished the increase in vasoconstriction in LPC-treated vessels (EmaxLPC/LPC+PD = âˆ¼6 ± 0,1/3 ± 0,1 mN/mm, p = 0.0001). LPC also reduced the dimer/monomer proportion and increased nNOSser852 phosphorylation. CONCLUSIONS: LPC induced nNOS uncoupling and nNOSSer852 phosphorylation, reduced NO and H2O2 production and improved superoxide production by modulating ERK1/2 activity in human and murine endothelial cells.

Oral butyrate reduces oxidative stress in atherosclerotic lesion sites by a mechanism involving NADPH oxidase down-regulation in endothelial cells.

Butyrate is a 4-carbon fatty acid that has antiinflammatory and antioxidative properties. It has been demonstrated that butyrate is able to reduce atherosclerotic development in animal models by reducing inflammatory factors. However, the contribution of its antioxidative effects of butyrate on atherogenesis has not yet been studied. We investigated the influence of butyrate on oxidative status, reactive oxygen species (ROS) release and oxidative enzymes (NADPH oxidase and iNOS) in atherosclerotic lesions of ApoE(-/-) mice and in oxLDL-stimulated peritoneal macrophages and endothelial cells (EA.hy926). The lesion area in aorta was reduced while in the aortic valve, although lesion area was unaltered, superoxide production and protein nitrosylation were reduced in butyrate-supplemented mice. Peritoneal macrophages from the butyrate group presented a lower free radical release after zymosan stimulus. When endothelial cells were pretreated with butyrate before oxLDL stimulus, the CCL-2 and superoxide ion productions and NADPH oxidase subunit p22phox were reduced. In macrophage cultures, in addition to a reduction in ROS release, nitric oxide and iNOS expression were down-regulated. The data suggest that one mechanism related to the effect of butyrate on atherosclerotic development is the reduction of oxidative stress in the lesion site. The reduction of oxidative stress related to NADPH oxidase and iNOS expression levels associated to butyrate supplementation attenuates endothelium dysfunction and macrophage migration and activation in the lesion site.