Suppression of inflammatory macrophages reduces atherosclerosis

Atherosclerosis is caused by chronic inflammation of blood vessels due to accumulation of modified low-density lipoprotein (LDL) in the form of plaques. Inflamed vessels may rupture, leading to myocardial infarction, stroke, heart failure and even sudden death. Monocytes enter the region of inflammation and differentiate to macrophages which uptake lipoproteins and gradually become converted to foam cells. The latter are trapped in the intima due to their reduced ability to migrate. These foam cells aggregate and undergo apoptosis. Macrophages in plaque die by various mechanisms such as apoptosis and regulated necrosis (necroptosis, ferroptosis, pyroptosis). Inefficient efferocytosis of foam cells results in the formation of necrotic regions in advanced plaques. These conditions release inflammatory cytokines which elicit an additional immune response, thus perpetuating the inflammation. Macrophages in tissues are of different origin. They develop during embryogenesis, colonize different embryonic tissues and differentiate into long-lived tissue-specific macrophages. An additional postnatal pool of macrophages in tissues is provided by monocyte-derived macrophages (MDMs) which are short-lived and originate from bone marrow. These two macrophage types coexist and maintain homeostasis of tissues under normal conditions, during infection and during inflammation. In atherosclerotic lesions, some additional vascular smooth muscle cells (VSMCs) also differentiate into macrophages and form foam cells. Atherosclerotic lesions have enriched M1 and M2 subtypes of macrophages. In addition to these two extreme subtypes, other subtypes such as Mox, M4, M(Hb) and Mhem have been identified in the plaques. M1, Mox, M4 are proinflammatory macrophages while M(Hb), Mhem and M2 have an atheroprotective effect. Early stages of atherosclerosis have enriched M1-type macrophages and their sustained high level hinders resolution of inflammation (Hou et al., 2023). The presence of increased M2-type macrophages leads to healing of lesions. Macrophages differentiate into different phenotypes based on signalling cues from the environment. Lipoproteins polarize macrophages towards the M1 phenotype by activating toll-like receptors (TLRs) and interferon-gamma (IFNγ) which then secrete high levels of pro-inflammatory cytokines such as IL-6, IL-12 and IL-1β, activate NF-κB (a nuclear transcription factor which increases expression of pro-inflammatory cytokines) and produce reactive oxygen species, all of which induce a condition of chronic inflammation (Fig. 1). The M2 subtype produces anti-inflammatory cytokines such as IL-4, IL-13 and IL-10, and have upregulated expression of scavenger receptors CD206 and CD163 which augment phagocytosis, and promote clearance of cell debris and foam cells. Thus, the presence of M2 macrophages in high numbers at the plaques can help in regression of inflammation and increased efferocytosis (Fig. 1). Excessive lipid uptake changes the metabolic profile of macrophages. It increases glycolysis and superoxide production, and decreases mitochondrial oxidative phosphorylation. The level of pyruvate kinase 2 (PMK2), an important enzyme in glycolysis, is increased. This increases the expression of pro-inflammatory cytokines such as IL-1β and TNFα, causes a surge in lipid uptake, and decreases efflux of cholesterol from cells by interacting with sterol regulatory element-binding proteins (SREBP1). This leads to the conversion of macrophages to foam cells. Increased free cholesterol in foam cells can induce epigenetic modification which triggers inflammatory signalling by TLR4, and NFκB activation pathways, thus enhancing pro-inflammatory cytokine production. Hypoxic conditions form inside the plaques due to increased oxygen demand by inflammatory immune cells. This induces expression of hypoxia-inducible factor (HIF). HIF-1α-induced genes increase glycolysis, decrease cholesterol efflux, accumulate sterol and induce pro-inflammatory protein production in macrophages, thus aggravating atherosclerotic plaques. Proteoglycan 4 (PRG4), also called lubricin, is a ubiquitously produced proteoglycan mainly involved in lubrication of joints and in cartilage repair, and is present at the ocular surface as a boundary lubricant. Hoekstra et al. (2024) identified the positive effect of recombinant human PRG4 (rhPRG4) treatment on atherosclerosis. Female LDL-receptor knockout mice fed an atherogenic western-type diet were treated with rhPRG4 three times per week, which increased expression of the MSR1 receptor on macrophages. The MSR1 receptor helps in uptake of oxidized LDL (oxLDL) by macrophages. A higher level of oxLDL in macrophages leads to their transformation to foam cells. Hoekstra and colleagues demonstrated that PRG4 treatment mediated oxLDL uptake and led to formation of a significantly higher number of foam cells. Subsequently, they observed decreased levels of free cholesterol in the plasma and improvement in atherogenic index. An anti-fibrotic effect of rhPRG4 treatment was evident by the decreased collagen content in the plaque. Total plaque area was also decreased upon treatment which was due to lower macrophage infiltration and reduced collagen content at the plaque site. Analysis of the immune cells in the spleen of rhPRG4-treated mice revealed no apparent change in the distribution profile of T cells, monocytes and macrophages. Interestingly, using M1 and M2 macrophage markers, Hoekstra and colleagues observed a huge shift from a pro-inflammatory towards anti-inflammatory phenotype in peritoneal leukocytes of mice treated with rhPRG4. Further, the peritoneal leukocytes of rhPRG4-treated mice showed an increase in anti-inflammatory cytokine IL-10 and decreased expression of pro-inflammatory cytokine TNF-α. The altered cytokine expression levels can be attributed to a macrophage phenotype shift from pro-inflammatory M1 to healing M2 phenotype. The shift was confirmed by checking the expression levels of M2 markers (MRC1, FIZZ1/RETNLA, CHI3L3/YM1) which increased by several fold in rhPRG4-treated mice. A significant 26-fold increase in peritoneal eosinophil count was observed upon treatment, which could be due to overexpression of M2 markers CHI3L3/YM1 which are known to induce infiltration of eosinophils at the inflammation site. The protective effect of rhPRG4 against atherosclerosis could be attributed to the overall transformation of innate immune cells from a chronic inflammatory state towards an anti-inflammatory subtype (Fig. 1). PRG4 is a key player in regulation of the immune response due to its complex interactions with TLRs and significant impact on macrophage behaviour. PRG4 is a structural protein with a central mucin domain that is highly glycosylated and is surrounded by N and C terminals. The biological activity of PRG4 is influenced by its glycosylation pattern, which also affects how it interacts with particular receptors such as L-selectin (CD62L). This gives PRG4 its special lubricating and anti-adhesive qualities (Qadri et al., 2021). Because of its structural framework, PRG4 can bind to TLR2 and TLR4 and functions as an antagonist. PRG4 structurally prevents these receptors from being activated by the ligands that are produced by bacteria and other inflammatory stimuli. This antagonistic function highlights PRG4's strong anti-inflammatory qualities, which are demonstrated by its capacity to lower pro-inflammatory cytokine production and prevent NF-κB from translocating into macrophage nuclei (Qadri et al., 2021). PRG4's antagonistic action to TLRs can reduce the release of inflammatory mediators in physiological contexts such as atherosclerosis, where TLRs on macrophages are essential for identifying danger-associated molecular patterns from damaged tissues and inducing inflammatory responses. Furthermore, PRG4 interacts with TLRs in a concentration-dependent manner that influences downstream inflammatory pathways and promotes macrophage homeostasis. In general, PRG4 is a promising molecule for controlling TLR-mediated inflammatory responses. Additionally, PRG4 influences macrophage phenotype, promoting an anti-inflammatory state that is important for tissue repair and regeneration. On the other hand, the absence of PRG4 can lead to a pro-inflammatory macrophage phenotype, potentially hindering the healing process. Overall, these findings suggest that PRG4 is a multifaceted regulator of immune responses with potential anti-inflammatory benefits. Recent studies have elucidated different pathways involved in atherosclerosis that can be targeted for novel therapeutic interventions. NF-κB activates the inflammatory pathway and blocking this pathway has atheroprotective effects (Gareus et al., 2008). Inhibitors of IκB such as vinpocetine, metformin, natural pentacyclic triterpenoids and synthetic drugs like sulforaphane have emerged as important targets to reduce the release of proinflammatory cytokines and abrogate the chronic nature of the disease. Sirtuins, a class of NAD+-dependent histone deacetylase, alter gene expression epigenetically. Various sirtuins have the ability to reduce NF-κB signalling and sirtuin activators are good targets for the treatment of atherosclerosis. TLRs are able to detect oxLDL in plaques and trigger immune response. Drugs blocking TLRs and other molecules in their inflammatory signalling pathway are promising therapeutics to treat atherosclerosis. Hypoxic conditions in the plaque interior induce expression of HIF-1α in macrophages which contributes to inflammation and further foam cell formation by various molecular mechanisms. Blocking HIF-1α expression is a potential target to treat atherosclerosis. Inhibitors of HIF-1α such as PX-478 have been shown to reduce disease load in mouse models, and drugs targeting HIF-1α can potentially be repurposed for treating atherosclerosis in humans. Factors that reduce M1 macrophage populations and promote the M2 phenotype present another promising therapeutic target for disease resolution. Recently, activation of the calcium ion channel PIEZO1 with its agonist Yoda1 was shown to reduce atherosclerotic plaque severity by altering mitochondrial physiology and macrophage polarization (Pourteymour et al., 2024). Thus, ion channels that are overexpressed in macrophages or foam cells can be selectively targeted to induce macrophage phenotypic change towards a healing subtype. Taken together, strategies that reduce inflammation and promote M2 macrophages hold promise for ameliorating atherosclerosis burden. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare no conflict of interest. S.M.: Conception or design of the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work. A.B.: Conception or design of the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; Agreement to be accountable for all aspects of the work. None. S.M. thanks CSIR India for a PhD fellowship. A.B. thanks IIT Kanpur for a PhD Fellowship. The authors thank Dr Rakesh Kumar Majhi, IIT Kanpur, for valuable suggestions to improve the manuscript.