Lipids in inflammasome activation and autoinflammatory disorders.

Autoinflammatory diseases (AIDs) are a group of rare monogenetic disorders characterized by recurrent episodes of fever and systemic inflammation. A major pathologic hallmark of AIDs is excessive inflammasome assembly and activation, often the result of gain-of-function mutations in genes encoding core inflammasome components, including pyrin and cryopyrin. Recent advances in lipidomics have revealed that dysregulated metabolism of lipids such as cholesterol and fatty acids, especially in innate immune cells, exerts complex effects on inflammasome activation and the pathogenesis of AIDs. In this review, we summarize and discuss the impact of lipids and their metabolism on inflammasome activation and the disease pathogenesis of the most common AIDs, including familial Mediterranean fever, cryopyrin-associated periodic syndromes, and mevalonate kinase deficiency. We postulate that lipids hold diagnostic value in AIDs and that dietary and pharmacologic intervention studies could represent a promising approach to attenuate inflammasome activation and AID progression.

Recent Insights in Pyrin Inflammasome Activation: Identifying Potential Novel Therapeutic Approaches in Pyrin-Associated Autoinflammatory Syndromes.

Pyrin is a cytosolic protein encoded by the MEFV gene, predominantly expressed in innate immune cells. Upon activation, it forms an inflammasome, a multimolecular complex that enables the activation and secretion of IL-1ß and IL-18. In addition, the Pyrin inflammasome activates Gasdermin D leading to pyroptosis, a highly pro-inflammatory cell death. Four autoinflammatory syndromes are associated with Pyrin inflammasome dysregulation: familial Mediterranean fever, hyper IgD syndrome/mevalonate kinase deficiency, pyrin-associated autoinflammation with neutrophilic dermatosis, and pyogenic arthritis, pyoderma gangrenosum, and acne syndrome. In this review, we discuss recent advances in understanding the molecular mechanisms regulating the two-step model of Pyrin inflammasome activation. Based on these insights, we discuss current pharmacological options and identify a series of existing molecules with therapeutic potential for the treatment of pyrin-associated autoinflammatory syndromes.

Extracellular vesicle-associated cholesterol supports the regenerative functions of macrophages in the brain.

Macrophages play major roles in the pathophysiology of various neurological disorders, being involved in seemingly opposing processes such as lesion progression and resolution. Yet, the molecular mechanisms that drive their harmful and benign effector functions remain poorly understood. Here, we demonstrate that extracellular vesicles (EVs) secreted by repair-associated macrophages (RAMs) enhance remyelination ex vivo and in vivo by promoting the differentiation of oligodendrocyte precursor cells (OPCs). Guided by lipidomic analysis and applying cholesterol depletion and enrichment strategies, we find that EVs released by RAMs show markedly elevated cholesterol levels and that cholesterol abundance controls their reparative impact on OPC maturation and remyelination. Mechanistically, EV-associated cholesterol was found to promote OPC differentiation predominantly through direct membrane fusion. Collectively, our findings highlight that EVs are essential for cholesterol trafficking in the brain and that changes in cholesterol abundance support the reparative impact of EVs released by macrophages in the brain, potentially having broad implications for therapeutic strategies aimed at promoting repair in neurodegenerative disorders.

The ubiquitous role of ubiquitination in lipid metabolism.

Lipids are essential molecules that play key roles in cell physiology by serving as structural components, for storage of energy, and in signal transduction. Hence, efficient regulation and maintenance of lipid homeostasis are crucial for normal cellular and tissue function. In the past decade, increasing research has shown the importance of ubiquitination in regulating the stability of key players in different aspects of lipid metabolism. This review describes recent insights into the regulation of lipid metabolism by ubiquitin signaling, discusses how ubiquitination can be targeted in diseases characterized by lipid dysregulation, and identifies areas that require further research.

Fatty acid elongation by ELOVL6 hampers remyelination by promoting inflammatory foam cell formation during demyelination.

A hallmark of multiple sclerosis (MS) is the formation of multiple focal demyelinating lesions within the central nervous system (CNS). These lesions mainly consist of phagocytes that play a key role in lesion progression and remyelination, and therefore represent a promising therapeutic target in MS. We recently showed that unsaturated fatty acids produced by stearoyl-CoA desaturase-1 induce inflammatory foam cell formation during demyelination. These fatty acids are elongated by the "elongation of very long chain fatty acids" proteins (ELOVLs), generating a series of functionally distinct lipids. Here, we show that the expression and activity of ELOVLs are altered in myelin-induced foam cells. Especially ELOVL6, an enzyme responsible for converting saturated and monounsaturated C16 fatty acids into C18 species, was found to be up-regulated in myelin phagocytosing phagocytes in vitro and in MS lesions. Depletion of Elovl6 induced a repair-promoting phagocyte phenotype through activation of the S1P/PPARγ pathway. Elovl6-deficient foamy macrophages showed enhanced ABCA1-mediated lipid efflux, increased production of neurotrophic factors, and reduced expression of inflammatory mediators. Moreover, our data show that ELOVL6 hampers CNS repair, as Elovl6 deficiency prevented demyelination and boosted remyelination in organotypic brain slice cultures and the mouse cuprizone model. These findings indicate that targeting ELOVL6 activity may be an effective strategy to stimulate CNS repair in MS and other neurodegenerative diseases.

Fatty acid desaturation by stearoyl-CoA desaturase-1 controls regulatory T cell differentiation and autoimmunity.

The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis (MS). Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity. To date, however, the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood. Here, we report that stearoyl-CoA desaturase-1 (SCD1), an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors, acts as an endogenous brake on regulatory T-cell (Treg) differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner. Guided by RNA sequencing and lipidomics analysis, we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase (ATGL). ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma. Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity, with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS.

Sterol-regulated transmembrane protein TMEM86a couples LXR signaling to regulation of lysoplasmalogens in macrophages.

Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X receptor (LXR) transcription factors are important determinants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid metabolism. However, their role in governing the composition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived macrophages (BMDMs) following LXR activation. We found a marked reduction in the levels of lysoplasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein 86a (TMEM86a), an integral endoplasmic reticulum protein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2+/lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasmalogenase in macrophages that contributes to sterol-dependent membrane remodeling.

Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

The ApoA-I mimetic peptide 5A enhances remyelination by promoting clearance and degradation of myelin debris.

The progressive nature of demyelinating diseases lies in the inability of the central nervous system (CNS) to induce proper remyelination. Recently, we and others demonstrated that a dysregulated innate immune response partially underlies failure of CNS remyelination. Extensive accumulation of myelin-derived lipids and an inability to process these lipids was found to induce a disease-promoting phagocyte phenotype. Hence, restoring the ability of these phagocytes to metabolize and efflux myelin-derived lipids represents a promising strategy to promote remyelination. Here, we show that ApoA-I mimetic peptide 5A, a molecule well known to promote activity of the lipid efflux transporter ABCA1, markedly enhances remyelination. Mechanistically, we find that the repair-inducing properties of 5A are attributable to increased clearance and metabolism of remyelination-inhibiting myelin debris via the fatty acid translocase protein CD36, which is transcriptionally controlled by the ABCA1-JAK2-STAT3 signaling pathway. Altogether, our findings indicate that 5A promotes remyelination by stimulating clearance and degradation of myelin debris.

Phloretin enhances remyelination by stimulating oligodendrocyte precursor cell differentiation.

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Why endogenous repair mechanisms frequently fail in these disorders is poorly understood. However, there is now evidence indicating that this is related to an overly inflammatory microenvironment combined with the intrinsic inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature myelinating cells. Previously, we found that phloretin, a flavonoid abundantly present in apples and strawberries, reduces neuroinflammation by driving macrophages toward an antiinflammatory phenotype. Here, we show that phloretin also markedly stimulates remyelination in ex vivo and in vivo animal models. Improved remyelination was attributed to a direct impact of phloretin on OPC maturation and occurred independently from alterations in microglia function and inflammation. We found, mechanistically, that phloretin acts as a direct ligand for the fatty acid sensing nuclear receptor peroxisome proliferator-activated receptor gamma, thereby promoting the maturation of OPCs. Together, our findings indicate that phloretin has proregenerative properties in central nervous system disorders, with potentially broad implications for the development of therapeutic strategies and dietary interventions aimed at promoting remyelination.

Perilipin-2 limits remyelination by preventing lipid droplet degradation.

Foamy macrophages and microglia containing lipid droplets (LDs) are a pathological hallmark of demyelinating disorders affecting the central nervous system (CNS). We and others showed that excessive accumulation of intracellular lipids drives these phagocytes towards a more inflammatory phenotype, thereby limiting CNS repair. To date, however, the mechanisms underlying LD biogenesis and breakdown in lipid-engorged phagocytes in the CNS, as well as their impact on foamy phagocyte biology and lesion progression, remain poorly understood. Here, we provide evidence that LD-associated protein perilipin-2 (PLIN2) controls LD metabolism in myelin-containing phagocytes. We show that PLIN2 protects LDs from lipolysis-mediated degradation, thereby impairing intracellular processing of myelin-derived lipids in phagocytes. Accordingly, loss of Plin2 stimulates LD turnover in foamy phagocytes, driving them towards a less inflammatory phenotype. Importantly, Plin2-deficiency markedly improves remyelination in the ex vivo brain slice model and in the in vivo cuprizone-induced demyelination model. In summary, we identify PLIN2 as a novel therapeutic target to prevent the pathogenic accumulation of LDs in foamy phagocytes and to stimulate remyelination.

Targeting lipophagy in macrophages improves repair in multiple sclerosis.

Foamy macrophages containing abundant intracellular myelin remnants are an important pathological hallmark of multiple sclerosis. Reducing the intracellular lipid burden in foamy macrophages is considered a promising therapeutic strategy to induce a phagocyte phenotype that promotes central nervous system repair. Recent research from our group showed that sustained intracellular accumulation of myelin-derived lipids skews these phagocytes toward a disease-promoting and more inflammatory phenotype. Our data now demonstrate that disturbed lipophagy, a selective form of autophagy that helps with the degradation of lipid droplets, contributes to the induction of this phenotype. Stimulating autophagy using the natural disaccharide trehalose reduced the lipid load and inflammatory phenotype of myelin-laden macrophages. Importantly, trehalose was able to boost remyelination in the ex vivo brain slice model and the in vivo cuprizone-induced demyelination model. In summary, our results provide a molecular rationale for impaired metabolism of myelin-derived lipids in macrophages, and identify lipophagy induction as a promising treatment strategy to promote remyelination.Abbreviations: Baf: bafilomycin a1; BMDM: bone marrow-derived macrophage; CD68: CD68 antigen; CNS: central nervous system; LD: lipid droplet; LIPE/HSL: lipase, hormone sensitive; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MGLL: monoglyceride lipase; MS: multiple sclerosis; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; ORO: oil red o; PNPLA2: patatin-like phospholipase domain containing 2; PLIN2: perilipin 2; TEM: transmission electron microscopy; TFEB: transcription factor EB; TOH: trehalose.

Protein Lipidation by Palmitate Controls Macrophage Function.

Macrophages are present in all tissues within our body, where they promote tissue homeostasis by responding to microenvironmental triggers, not only through clearance of pathogens and apoptotic cells but also via trophic, regulatory, and repair functions. To accomplish these divergent functions, tremendous dynamic fine-tuning of their physiology is needed. Emerging evidence indicates that S-palmitoylation, a reversible post-translational modification that involves the linkage of the saturated fatty acid palmitate to protein cysteine residues, directs many aspects of macrophage physiology in health and disease. By controlling protein activity, stability, trafficking, and protein-protein interactions, studies identified a key role of S-palmitoylation in endocytosis, inflammatory signaling, chemotaxis, and lysosomal function. Here, we provide an in-depth overview of the impact of S-palmitoylation on these cellular processes in macrophages in health and disease. Findings discussed in this review highlight the therapeutic potential of modulators of S-palmitoylation in immunopathologies, ranging from infectious and chronic inflammatory disorders to metabolic conditions.

Carnosine quenches the reactive carbonyl acrolein in the central nervous system and attenuates autoimmune neuroinflammation.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease driven by sustained inflammation in the central nervous system. One of the pathological hallmarks of MS is extensive free radical production. However, the subsequent generation, potential pathological role, and detoxification of different lipid peroxidation-derived reactive carbonyl species during neuroinflammation are unclear, as are the therapeutic benefits of carbonyl quenchers. Here, we investigated the reactive carbonyl acrolein and (the therapeutic effect of) acrolein quenching by carnosine during neuroinflammation. METHODS: The abundance and localization of acrolein was investigated in inflammatory lesions of MS patients and experimental autoimmune encephalomyelitis (EAE) mice. In addition, we analysed carnosine levels and acrolein quenching by endogenous and exogenous carnosine in EAE. Finally, the therapeutic effect of exogenous carnosine was assessed in vivo (EAE) and in vitro (primary mouse microglia, macrophages, astrocytes). RESULTS: Acrolein was substantially increased in inflammatory lesions of MS patients and EAE mice. Levels of the dipeptide carnosine (ß-alanyl-L-histidine), an endogenous carbonyl quencher particularly reactive towards acrolein, and the carnosine-acrolein adduct (carnosine-propanal) were ~ twofold lower within EAE spinal cord tissue. Oral carnosine treatment augmented spinal cord carnosine levels (up to > tenfold), increased carnosine-acrolein quenching, reduced acrolein-protein adduct formation, suppressed inflammatory activity, and alleviated clinical disease severity in EAE. In vivo and in vitro studies indicate that pro-inflammatory microglia/macrophages generate acrolein, which can be efficiently quenched by increasing carnosine availability, resulting in suppressed inflammatory activity. Other properties of carnosine (antioxidant, nitric oxide scavenging) may also contribute to the therapeutic effects. CONCLUSIONS: Our results identify carbonyl (particularly acrolein) quenching by carnosine as a therapeutic strategy to counter inflammation and macromolecular damage in MS.

Liver X receptor beta deficiency attenuates autoimmune-associated neuroinflammation in a T cell-dependent manner.

The initiation and progression of autoimmune disorders such as multiple sclerosis (MS) is linked to aberrant cholesterol metabolism and overt inflammation. Liver X receptors (LXR) are nuclear receptors that function at the crossroads of cholesterol metabolism and immunity, and their activation is considered a promising therapeutic strategy to attenuate autoimmunity. However, despite clear functional heterogeneity and cell-specific expression profiles, the impact of the individual LXR isoforms on autoimmunity remains poorly understood. Here, we show that LXRα and LXRß have an opposite impact on immune cell function and disease severity in the experimental autoimmune encephalomyelitis model, an experimental MS model. While Lxrα deficiency aggravated disease pathology and severity, absence of Lxrß was protective. Guided by flow cytometry and by using cell-specific knockout models, reduced disease severity in Lxrß-deficient mice was primarily attributed to changes in peripheral T cell physiology and occurred independent from alterations in microglia function. Collectively, our findings indicate that LXR isoforms play functionally non-redundant roles in autoimmunity, potentially having broad implications for the development of LXR-based therapeutic strategies aimed at dampening autoimmunity and neuroinflammation.

Lipophagy: a new player in CNS disorders.

Lipophagy is the process of selective degradation of lipid droplets (LDs) by autophagy. Several studies have highlighted the importance of lipophagy in regulating cellular lipid levels in various tissues and disease conditions. In recent years, disruption of autophagy and accumulation of LDs have been reported as pathological hallmarks in several neurodegenerative and neuroinflammatory diseases, raising the question whether lipophagy is a process that is important in the progression of these disorders. This supports the growing interest in lipid metabolism as a major player in neurodegeneration, and the emerging understanding of several neurological pathologies as not only proteinopathies but also lipidopathies. In this review we discuss the importance of lipophagy in the most common central nervous system diseases. We examine the latest evidence for the reported interplay between abnormalities in lipid accumulation and autophagy, and propose lipophagy as a potentially important mechanism in neurodegeneration.

Fatty Acid Synthesis in Glial Cells of the CNS.

Fatty acids (FAs) are of crucial importance for brain homeostasis and neural function. Glia cells support the high demand of FAs that the central nervous system (CNS) needs for its proper functioning. Additionally, FAs can modulate inflammation and direct CNS repair, thereby contributing to brain pathologies such Alzheimer's disease or multiple sclerosis. Intervention strategies targeting FA synthesis in glia represents a potential therapeutic opportunity for several CNS diseases.

Phloretin suppresses neuroinflammation by autophagy-mediated Nrf2 activation in macrophages.

BACKGROUND: Macrophages play a dual role in neuroinflammatory disorders such as multiple sclerosis (MS). They are involved in lesion onset and progression but can also promote the resolution of inflammation and repair of damaged tissue. In this study, we investigate if and how phloretin, a flavonoid abundantly present in apples and strawberries, lowers the inflammatory phenotype of macrophages and suppresses neuroinflammation. METHODS: Transcriptional changes in mouse bone marrow-derived macrophages upon phloretin exposure were assessed by bulk RNA sequencing. Underlying pathways related to inflammation, oxidative stress response and autophagy were validated by quantitative PCR, fluorescent and absorbance assays, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice, western blot, and immunofluorescence. The experimental autoimmune encephalomyelitis (EAE) model was used to study the impact of phloretin on neuroinflammation in vivo and confirm underlying mechanisms. RESULTS: We show that phloretin reduces the inflammatory phenotype of macrophages and markedly suppresses neuroinflammation in EAE. Phloretin mediates its effect by activating the Nrf2 signaling pathway. Nrf2 activation was attributed to 5' AMP-activated protein kinase (AMPK)-dependent activation of autophagy and subsequent kelch-like ECH-associated protein 1 (Keap1) degradation. CONCLUSIONS: This study opens future perspectives for phloretin as a therapeutic strategy for neuroinflammatory disorders such as MS. TRIAL REGISTRATION: Not applicable.

Altered PPARγ Expression Promotes Myelin-Induced Foam Cell Formation in Macrophages in Multiple Sclerosis.

Macrophages play a crucial role during the pathogenesis of multiple sclerosis (MS), a neuroinflammatory autoimmune disorder of the central nervous system. Important regulators of the metabolic and inflammatory phenotype of macrophages are liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs). Previously, it has been reported that PPARγ expression is decreased in peripheral blood mononuclear cells of MS patients. The goal of the present study was to determine to what extent PPARγ, as well as the closely related nuclear receptors PPARα and ß and LXRα and ß, are differentially expressed in monocytes from MS patients and how this change in expression affects the function of monocyte-derived macrophages. We demonstrate that monocytes of relapsing-remitting MS patients display a marked decrease in PPARγ expression, while the expression of PPARα and LXRα/ß is not altered. Interestingly, exposure of monocyte-derived macrophages from healthy donors to MS-associated proinflammatory cytokines mimicked this reduction in PPARγ expression. While a reduced PPARγ expression did not affect the inflammatory and phagocytic properties of myelin-loaded macrophages, it did impact myelin processing by increasing the intracellular cholesterol load of myelin-phagocytosing macrophages. Collectively, our findings indicate that an inflammation-induced reduction in PPARγ expression promotes myelin-induced foam cell formation in macrophages in MS.

CNS delivery of anti-CD52 antibodies modestly reduces disease severity in an animal model for multiple sclerosis.

BACKGROUND AND AIMS: Alemtuzumab is a humanized monoclonal antibody that depletes CD52-bearing B and T lymphocytes. Clinical trials defined that systemic administration of alemtuzumab reduces disease severity in the relapsing-remitting phase of multiple sclerosis (MS). However, its efficacy in progressive MS patients is limited, which may reflect the inability of alemtuzumab to cross the reconstituted BBB in these patients. Objective: to study whether central nervous system (CNS) delivery of anti-CD52 antibodies reduces disease severity and the neuroinflammatory burden in the experimental autoimmune encephalomyelitis (EAE) model. METHODS: Anti-CD52 antibodies were administered intrathecally during the acute and chronic phases of EAE. Flow cytometry and immunohistochemistry were utilized to define immunological and pathological parameters. RESULTS: We show that subcutaneously administrated anti-CD52 antibodies completely abolish EAE disease severity. CNS delivery of anti-CD52 antibodies during both the acute and chronic phases of EAE moderately reduces disease severity and the neuroinflammatory burden. Our findings further suggest that CNS delivery of anti-CD52 antibodies impacts both the peripheral and CNS immune cell compartments in the EAE model but not in healthy mice. CONCLUSION: Collectively, our findings highlight the therapeutic potential of CNS delivery of alemtuzumab for the treatment of progressive as well as early MS.