Vitamin D immune modulation of the anti-inflammatory effects of HDL-associated proteins

Abstract

Vitamin D is a crucial element in bone metabolism and plays a role in innate immunity and inflammation suppression. Deficiency in this vitamin leads to disturbances in many biological functions, including the lipid profile. The decrease in HDL levels is associated with disruptions in its function and dynamic modifications in its components, such as apolipoproteins, including ApoM, ApoA-1, and ApoD. Consequently, the anti-inflammatory potential of HDL and HDL-associated proteins is reduced, resulting in heightened inflammation. However, the relationship between modifications in lipid profile, apolipoproteins, inflammation, and vitamin D remains unclear. This review highlights the connection between vitamin D status and its possible effects on the lipid profile, specifically HDL-associated proteins.

Introduction and background

Vitamin D, a fat-soluble prohormone, plays a crucial role in maintaining bone health by regulating calcium and phosphorus homeostasis. It is obtained from dietary sources (e.g., salmon, egg yolk, mushrooms) or synthesized through exposure to UVB radiation. The native form of vitamin D converts in the liver to 25-hydroxyvitamin D [25(OH)D], a major circulatory vitamer. Subsequently, the active form of vitamin D is produced in the kidney through the conversion of 25(OH)D to 1,25-dihydroxyvitamin D (1,25(OH)2D) via 25-hydroxyvitamin D-1α-hydroxylase [1]. The native form of vitamin D has two analogs: D2 and D3. D2, also known as Ergocalciferol, is derived from plant sources, such as soybeans, while D3 (cholecalciferol) comes from animal sources, like fish. The differences in the structures of both forms affect their binding affinity to various proteins, including their carrier, vitamin D-binding protein (DBP), which impacts their plasma half-life. D2 has a shorter plasma half-life and lower affinity for binding DBP and the vitamin D receptor (VDR) [2].

VDR is a member of a superfamily of nuclear receptors working as a transcription regulator. The VDR gene is located on chromosome 12q13.11 [2]. VDR is usually allocated in the cytoplasm. Specifically, VDR is highly expressed in immune cells, modulating inflammatory responses [3]. Conformational changes occur upon interacting with VDR and 1,25(OH)2D3. Later, VDR dimers bind to retinoid X receptor (RXR) and transfer to the nucleus. The complex binds to the vitamin D response elements (VDREs) in the nucleus of vitamin D-responsive genes. Subsequently, this process activates or suppresses certain genes according to the target genes [4, 5].

Vitamin D: physiology and deficiency

Vitamin D is essential for maintaining bone health by regulating the absorption of calcium and phosphorus in the small intestine and promoting bone mineralization [1]. It also supports muscle function and systemic immune responses. Deficiency in vitamin D impairs calcium homeostasis, leading to conditions such as rickets in children and osteomalacia in adults, as well as an increased risk of osteoporosis due to reduced bone mineral density [1, 6]. Beyond bone health, vitamin D deficiency disrupts immune regulation, increasing susceptibility to infections and exacerbating chronic inflammatory conditions, including metabolic syndrome and dyslipidemia [7].

Vitamin D regulates numerous biological processes, with the vitamin D receptor (VDR) highly expressed in T lymphocytes and macrophages, thereby modulating immunity by reducing T lymphocyte activity and promoting the transcription of TGFβ-1 and IL-4 [8]. Interferon (IFN)-γ is another example; the ligand-bound VDR and VDRE form a complex that binds to the IFN-γ promoter region, resulting in a repressive effect. At the same time, it increases IL-10 production, IL-10 is an anti-inflammatory cytokine [9].

NF-κB is one of 1,25(OH)2D3 targets. Upon the activation of NF-κB, a downstream target nucleotide-binding oligomerization domain 2 (NOD2) will be stimulated. NOD2 is a pathogen-recognizing protein. Subsequently, human β-defensin 2 DEFB4 (skin-antimicrobial peptide 1 (SAP1)) will be activated by NOD2; this process is downregulated by the presence of 1,25(OH)2D3. Another antimicrobial peptide, cathelicidin AMP (CAMP)( LL-37 is another name for the human cathelicidin), is affected by vitamin D, where it has been noticed to be highly induced by 1,25(OH)2D3. The activity of 1,25(OH)2D3 in stimulating the antimicrobial peptides was seen against some pathogens, such as Pseudomonas aeruginosa. This effect has been reported in human immune cells, keratinocytes, monocytes, and neutrophils [10–12].

Additionally, 1,25(OH)2D3 has been demonstrated to activate macrophage autophagy, and autophagy-associated proteins Atg-5 and Beclin-1. Those proteins are also induced through the upregulation of cathelicidin. Furthermore, cathelicidin or LL-37 activation leads to the induction of p38, ERK, and C/EBPβ [13]. Autophagy is a catabolic biological process mediated by a lysosome, aiming to remove unwanted or dysfunctional components [14].

Mature T cells exhibit elevated VDR expression and CYP27B1, the enzyme converting vitamin D to active 1,25(OH)₂D₃, compared to naive T cells, highlighting 1,25(OH)₂D₃’s role in T cell regulation. It suppresses Th1 and Th17 cytokines (IL-2, IFN-γ, TNF-α, IL-17, IL-21, IL-22), promotes Th2 cytokines (IL-4, IL-5, IL-10), and induces FoxP3 for regulatory T cell differentiation [15–17].

Vitamin D deficiency represents a significant global public health issue. Reports are pointing to 1 billion people suffering from various degrees of vitamin D deficiency and insufficiency. The Institute of Medicine previously defined vitamin D deficiency as serum 25-hydroxyvitamin D (25(OH)D) concentrations below 12 ng/mL, insufficiency as 12–20 ng/mL, and normal levels above 20 ng/mL; however, current clinical guidelines define deficiency as below 20 ng/mL, insufficiency as 20–30 ng/mL, and sufficiency at or above 30 ng/mL [6, 18]. Vitamin D deficiency disrupts immune homeostasis by enhancing pro-inflammatory pathways, such as NF-κB and MAPK signaling, which are implicated in chronic diseases like metabolic syndrome and dyslipidemia [7]. For example, low 25(OH)D levels (< 20 ng/mL) increase circulating inflammatory markers, such as C-reactive protein (CRP) and interleukin-6 (IL-6), which contribute to insulin resistance and atherogenesis [7, 19]. These inflammatory changes are associated with altered lipid profiles, including reduced high-density lipoprotein (HDL) cholesterol and elevated triglycerides, which exacerbate cardiovascular risk [19]. Vitamin D also influences lipid metabolism, particularly high-density lipoprotein (HDL), a lipoprotein complex that facilitates reverse cholesterol transport and exhibits anti-inflammatory properties [20]. Low 25(OH)D levels are associated with reduced HDL cholesterol, which may exacerbate inflammation and cardiovascular risk [21]. As demonstrated in our previous publication, low 25(OH)D levels (< 20 ng/mL) in young, healthy adults were associated with an increased monocyte-to-HDL ratio (MHR), a biomarker of subclinical inflammation that reflects the balance between pro-inflammatory monocytes and anti-inflammatory HDL. This suggests that vitamin D deficiency may impair HDL’s protective functions, contributing to cardiovascular risk [19].

Vitamin D in cardiovascular health

The risk for cardiovascular diseases is known to be impacted by several lifestyle factors, such as smoking, drinking alcohol, and malnutrition, in addition to metabolic abnormalities, such as diabetes, insulin resistance, and obesity. Those factors are cross-linked with risk factors of vitamin D deficiency [22, 23]. A prospective study by Giovannucci et al. followed 18,225 male US health professionals (aged 40–75 years, predominantly non-obese) over 10 years and found that low 25(OH)D levels (≤ 15 ng/mL) were associated with a 2-fold increased risk of myocardial infarction (adjusted relative risk: 2.09, 95% CI: 1.24–3.54) compared to sufficient levels (≥ 30 ng/mL), after controlling for age, smoking, and other cardiovascular risk factors [24]. Similarly, research on coronary angiography patients over 7.7 years linked reduced 25(OH)D and 1,25(OH)₂D levels to increased cardiovascular mortality [25]. Furthermore, low 25(OH)D significantly elevates risks of heart failure and sudden cardiac death [26].

Vitamin D, in general, protects against cardiovascular diseases through multi-layer mechanisms. The first mechanism by which vitamin D helps relax vessels is by stimulating nitric oxide (NO) production from endothelial cells [27]. Moreover, vitamin D suppresses NFkappaB and p38, which are responsible for activating the inflammatory pathways leading to exacerbation of the atherosclerotic disorder [21].

Similarly, HDL possesses anti-inflammatory effects, and disturbance in HDL and lipid profile would contribute to inflammation.

Restoring the optimal level of serum 25(OH)D above 20ng/mL (50nmol/L)- 30ng/mL (75nmol/L), preferably between 40 and 60 ng/mL (100–150 nmol/L) has documented health benefits including improving the outcomes of chronic disease and reducing the risks of getting the disease in the first place.

In vitro studies have shown that vitamin D supplementation enhances immune response toward infection, including increasing phagocytes, suppressing inflammatory cytokine production, and inducing antimicrobial peptides such as cathelicidin expression [28].

Dyslipidemia and lipoproteins profile

Dyslipidemia involves a range of abnormalities in the metabolism of plasma lipids and lipoproteins, resulting in augmenting or reducing the concentrations of lipoproteins or changing the particle configuration [29]. Dyslipidemia triggers can be related to genetic factors, causing familial dyslipidemia, also called primary dyslipidemia. Conversely, environmental factors such as diseases, diet, and general life regime cause acquired dyslipidemia, also named secondary dyslipidemia [30]. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), or high-density lipoprotein cholesterol (HDL-c) concentrations deviate when suffering from dyslipidemia [31]. Dyslipidemia is defined by elevated total cholesterol (≥ 5.2 mmol/L), LDL-C (≥ 3.0 mmol/L), triglycerides (≥ 1.7 mmol/L), or reduced HDL-C (< 1.0 mmol/L in men, < 1.3 mmol/L in women), with stricter LDL-C targets (< 1.8 mmol/L) for high-risk patients [32, 33]. Variation in lipid metabolites marks dyslipidemia as a significant risk factor in several diseases, for instance, cardiovascular disease, diabetes, and stroke [34]. Research on dyslipidemia and related macromolecules has linked Vitamin D deficiency to increased risk, given that VDRs are present in multiple cell types, such as vascular endothelial cells and cardiomyocytes [29]. Dyslipidemia is a significant element in metabolic syndrome, which is characterized by a combination of several pathological conditions, mainly central obesity, hypertension, low levels of HDL, elevated LDL and TG, endothelial dysfunction, and diabetes [35]. Studies reported a strong correlation between metabolic syndrome and vitamin D deficiency, but the bidirectional nature of this association is still not explicit [36, 37] Lipoproteins, including chylomicrons, low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), and high-density lipoproteins (HDL), are the main players in lipid profile disruption; therefore, CVD [34, 38].

LDL particle

LDL is the primary transporter of cholesterol to peripheral tissues. It drives atherosclerosis plaque formation and cardiovascular disease [39]. LDL is formed via VLDL conversion by lipoproteins [40]. LDL contains apolipoprotein, Apo B-100, and lipid like linoleate [41].

Elevated LDL promotes atherosclerosis. When LDL enters the endothelial layer, free radicals oxidize the LDL particle, forming ox-LDL [42, 43], stimulating the endothelial cells to produce Monocyte Chemoattractant Protein-1 (MCP-1). This triggers monocytes to infiltrate and differentiate into macrophages. Those macrophages ingest ox-LDL, forming foam cells. The accumulation of foam cells drives plaque formation [31, 44, 45]. Vitamin D deficiency may exacerbate LDL-driven atherosclerosis by reducing VDR-mediated suppression of inflammatory pathways, such as NF-κB, which enhances the formation of ox-LDL and foam cell accumulation [25, 46, 47]. Low 25(OH)D levels (< 20 ng/mL) are associated with increased LDL cholesterol in observational studies [48], suggesting a role in dyslipidemia.

VLDL particle

VLDL, triglyceride-rich particles, are produced in the liver [49]. High concentrations of VLDL-TG were linked to an increased risk of cardiovascular disease [50]. Vitamin D deficiency is associated with elevated VLDL triglycerides, potentially due to impaired VDR signaling in hepatocytes, which disrupts triglyceride metabolism [51]. This contributes to dyslipidemia and heightened cardiovascular risk.

Chylomicrons

Chylomicrons are formed in the intestine from dietary lipids, transport triglycerides to peripheral tissue, and their remnants are cleared by ApoE-mediated endocytosis [52]. Although direct evidence linking vitamin D to chylomicron metabolism is limited, low 25(OH)D levels may indirectly influence triglyceride transport by altering ApoE expression, as VDR regulates lipid metabolism genes [53].

HDL particles

HDL, known for reverse cholesterol transport (RCT), mitigates atherosclerosis by shuttling cholesterol from tissues to the liver. HDL exhibits anti-inflammatory, anti-apoptotic, and antioxidant properties, contributing to its protective effects against CVD [54]. HDL-cholesterol (HDL-c) is a good indicator of the functionality of HDL and the ability to carry cholesterol from tissues more than HDL particle (HDL-P). However, HDL-P has been more studied in clinical studies in cardiovascular disorders in isolation from HDL-c [55]. Low HDL-c has a significant impact on disease prognosis or survival rate. For instance, in severe sepsis, it has been observed that patients who have low values of HDL upon the beginning of sepsis deteriorate very rapidly, and have the worst mortality and clinical outcomes [56]. High values of HDL-c in patients with reduced ejection fraction were associated with a lower risk of heart failure and a better prognosis and survival. In a similar study, patients with high HDL-c recorded a decline in inflammation biomarker C-reactive protein (CRP) values [52].

HDL is a very condensed, heterogeneous complex in comparison to other lipoproteins. HDL size ranges from 1.063 to 1.210 g/mL. It contains a high composition of proteins in comparison with other lipoproteins [57]. Proteins form up to 60% of HDL mass, and ApoA-I is the main component of those proteins [58]. The biogenesis of HDL starts mainly in the liver upon the interaction between ApoA-I and the cell membrane protein ATP-binding cassette transporter A1 (ABCA1) [59]. ABCA1 is found in the basolateral surface of the hepatocytes [60]. The mutation in ABCA1 proteins could result in severe malfunctioning in HDL. As a result, a large amount of cholesterol is deposited in tissue macrophages, leading to atherosclerotic formation [61]. Later, this lipidated ApoA-I is catalyzed by an enzyme called lecithin/cholesterol acyltransferase (LCAT) [62]. The first step of transforming the immature HDL into mature HDL occurs when scavenger receptor class B, type I (SR-BI) mediates the cholesterol efflux from macrophages and tissues into discoidal HDL [63].

The second step in transforming the discoidal hydrophobic core of ABCA1-derived HDL into spherical lipid-rich HDL is the interaction with LCAT for the second time [64]. A mutation in LCAT is also associated with cardiovascular diseases. Two disorders are associated with LCAT mutation: familial LCAT deficiency (FLD), which affects the esterification of cholesterol on HDL and LDL, resulting in the accumulation of discoidal HDL in the plasma. Fish eye disease (FED) affects the esterification of cholesterol on HDL only [65].

The cholesteryl ester transfer protein (CETP) pathway follows the RCT process. In this step, CETP facilitates the exchange of cholesteryl esters from HDL particles with the triglycerides in low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and Chylomicrons. The liver takes up the cholesterol via the LDL receptor, which is present in VLDL and LDL [66] (Fig. 1).

Fig. 1.

The biogenesis of HDL and RCT. Created by Hanaa Mousa using the BioRender website. Upon the interaction of ApoA-1 and ABCA1, the resulting complex is catalyzed by the LCAT enzyme, forming a discoidal HDL. SR-B1 mediates cholesterol efflux from macrophages and tissues into discoidal high-density lipoprotein (HDL). Another interaction with LCAT formed the mature HDL. CETP facilitates the exchange of cholesteryl esters from HDL particles with the triglycerides in LDL, VLDL, and Chylomicrons. The liver takes the cholesterol via the LDL receptor. Note: Figures 1, and 3 Created with BioRender.com

HDL is a dynamic particle, that undergoes continuous remodeling due to the instant interaction of cell receptors and other proteins and cofactors particularly serum amyloid A (SAA), hepatic lipase (HL), endothelial lipase (EL), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and apolipoprotein M (ApoM) [67, 68]. Furthermore, the HDL-associated apolipoproteins, which are bound to HDL, interact with numerous cellular receptors, e.g., ATP-binding cassette A1 or G1. This interaction enables cholesterol efflux from cells to the HDL particle [69]. Consequently, HDL particles continuously remodel their physical structure and metabolite composition [70]. Inflammation impairs the reverse cholesterol transport (RCT) pathway, hindering cholesterol efflux from macrophage foam cells to the liver and resulting in dysfunctional high-density lipoprotein (HDL) particles [71].

HDL anti-inflammatory properties

The apolipoproteins on the HDL surface, such as ApoA-1, ApoM, and ApoD, which are mainly associated with HDL particles, more than the other lipoproteins [72, 73], enabling HDL to exhibit anti-inflammatory and anti-oxidative properties [58]. HDL suppresses the sphingosine kinase in the endothelial cell, an enzyme derived from sphingomyelins and considered essential in the activation of nuclear factor κB (NF-κB), which induces adhesion molecules [57]. In addition, HDL can suppress those adhesion molecules directly. An in vitro study in human umbilical vein endothelial cells (HUVECs) demonstrated that HDL suppressed cytokine-induced adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, in a concentration-dependent manner [74]. Therefore, HDL suppresses the adhesion molecules produced by endothelial cells, which is crucial for the cellular adhesion of circulating leukocytes during atherogenesis formation [75].

Van der Vorst et al. study. In vitro experiments using murine and human primary macrophages demonstrated that HDL triggers a pro-inflammatory response through passive cholesterol depletion. The result was confirmed using peritoneal macrophages from ApoA-1 transgenic mice, which have elevated HDL levels. Pseudomonas aeruginosa bacteria were used to induce infectious inflammatory conditions. HDL activated the TLR in general, particularly TLR4, through the PKC-NF-kB/STAT1-IRF1 pathway, followed by an elevation of inflammatory cytokine expression IL-12 and TNF-α and down-regulation in the anti-inflammatory cytokine IL-10 [76].

HDL possesses some Antioxidant properties due to its association with antioxidant molecules, e.g., paraoxonase/arylesterase 1 (PON1). PON1 enables the HDL to prevent LDL glycation and lipid peroxidation, thus protecting the cardiovascular system from the bad effects of glycated LDL particles [77].

Vitamin D enhances HDL’s anti-inflammatory properties by upregulating ApoA-1 and ApoM expression in some contexts, though 1,25(OH)₂D₃ may suppress ApoA-1 transcription in hepatocytes [78, 79]. For example, supplementation with vitamin D (1000 mg capsule) daily for one month in healthy children increased HDL cholesterol level, potentially improving its anti-inflammatory capacity [80].

Clinical aspects of HDL

A low serum concentration of HDL, Less than 40 mg/dL (1.0 mmol/L) in men and Less than 50 mg/dL (1.3 mmol/L) in women, is associated with a high risk of developing cardiovascular diseases [66]. Hyperalphalipoproteinemia is a genetic disorder characterized by a deficiency in HDL levels in the blood, accompanied by a mild elevation in triglycerides. Hyperalphalipoproteinemia affects components responsible for HDL biogenesis, ApoA-1, ABCA1, and LCAT. The Functional mutation in ABCA1 (Tangier disease) impairs the ABCA1’s ability to secrete the newly released ApoA-1 from the liver and intestines. This causes rapid catabolism and clearance of ApoA-1, resulting in low HDL levels. In in vivo trials, ABCA1 knockout in the liver induced an 80% reduction in HDL concentration while ABCA1 knockout in the intestines caused a 30% reduction in HDL. When HDL cholesterol and ApoA-1 levels are low, as is the case in Fish Eye Disease, more small HDL particles are produced, resulting in decreased overall HDL levels. The opposite scenario occurs in CETP deficiency, where high levels of HDL cholesterol are associated with large HDL particles. Here, the deficient CETP will be unable to exchange triglycerides for cholesterol between HDL, VLDL, and LDL, resulting in the accumulation of large, malformed HDL particles [81, 82].

Other than the hereditary causes of low HDL, factors like smoking, obesity particularly central obesity, alcohol, poor diet habits, lack of physical activity, metabolic disorders e.g., insulin resistance, and medications e.g. steroids, niacin, statins, etc. can lead to a decreased level of HDL [83, 84]. Low HDL was linked to several illnesses. A recent study of stable angina patients revealed that low HDL-c and high triglyceride levels were correlated with a higher prevalence of coronary atherosclerotic disease events. Accordingly, the ratio of HDL-c to triglycerides could predict the outcome of coronary atherosclerotic disease independently of LDL-c [85]. Nevertheless, a meta-analysis study of 108 randomized trials, including 299,310 participants, demonstrated that raising HDL alone could not modify the progression of cardiovascular diseases or the resulting mortality. However, the study suggested that lowering the LDL as a primary goal could be a good alternative [86]. The LDL-c/HDL-c ratio was found to be a more accurate indicator of the severity of coronary artery disease in STEMI patients than HDL or LDL [87]. Overall, both LDL and HDL are important in reflecting cardiovascular health.

The role of HDL in reducing inflammation by endotoxin neutralization

Inflammation is induced by pathogen-derived molecular patterns (PAMPs), such as endotoxin, or by tissue damage and the release of danger-associated molecular patterns (DAMPs). Sepsis is a severe inflammatory response that usually occurs due to bacterial endotoxin and overproduction of cytokines released from inflammatory cells, particularly Tumor necrosis factor-alpha (TNF-α) [88]. Sepsis incidence has increased over the past decade, where a study conducted from 1979 to 2016 estimated 31.5 million sepsis cases with 5.3 million annual deaths [89].

Toll-like receptor 4 (TLR4), a transmembrane glycoprotein highly expressed on macrophages and other immune cells, recognizes approximately 5% of circulating LPS [90]. TLR4 recognizes endotoxin (LPS) via its co-receptor MD-2. The binding of LPS in the calyx of MD-2 leads to TLR4 dimerization and the initiation of a signaling cascade that activates the NF-κB complex (nuclear factor kappa-light-chain-enhancer of activated B cells) and other signaling pathways, ultimately resulting in inflammation and cytokine production [91–93]. NF-κB was found to have an adverse effect on ApoA-1 and HDL-C by suppressing peroxisome proliferator-activated receptor alpha (PPARα), a key regulator of lipid metabolism in the liver [94]. Experimental evidence revealed the downregulation of TLR4 following an increase in HDL-C after transferring ApoA-1 genes in mice. The decrease of TLR4 expression occurred after 2 weeks of transferring the ApoA-1 gene, and the fold change was about 8.4. The decrease of TLR4 continues even after the administration of LPS in those mice, and the survival rate was noticed to be improved [95]. Another study on mice demonstrated that the infusion of reconstituted HDL reduced inflammation, improved survival, decreased bacterial count, and prevented the organ damage that occurs usually as a consequence of sepsis [96].

More studies have also shown that structural and functional modifications occur in HDL particles during sepsis. For instance, serum amyloid A (SAA1) is one of the acute-phase proteins associated with high-density lipoprotein (HDL). During sepsis, SAA-1 is overexpressed, replacing ApoA-1 in HDL. SAA-1 binds directly to LPS and shuttles it with HDL to promote clearance. Clinical evidence has shown that the displacement of HDL contents leads to HDL particle remodeling, which affects the anti-inflammatory and antioxidant functions [97, 98]. SAA-1-enriched HDL enlarges the HDL size and becomes denser; as a consequence, the mobility of HDL becomes slower [99]. Moreover, the concentration of total HDL in serum is decreased due to the pathophysiological changes associated with sepsis. Several mechanisms contribute to the decrease in HDL, including human secretory phospholipase A2 (sPLA2). This sPLA2 is an acute-phase protein that is upregulated in the presence of LPS, which stimulates SAA1, leading to modifications in HDL particle size and composition [100]. The interaction between LPS and HDL is mediated by several proteins, including CETP, PLTP, and LPS-binding protein (LBP). It is worth noting that HDL and associated proteins have a high affinity for binding and eliminating LPS compared to other lipoproteins [101]. HDL evolved to become an important component of the innate immune system, protecting against endotoxins and immune system overstimulation [102]. However, the exact mechanism by which HDL neutralizes endotoxin and confers anti-inflammatory activity remains unclear. These anti-inflammatory and endotoxin-neutralizing functions of HDL are mediated by its associated apolipoproteins, which are explored in detail in the following section. Vitamin D enhances HDL’s endotoxin-neutralizing capacity by inducing cathelicidin (LL-37), a peptide associated with HDL that inhibits LPS-TLR4 interactions and reduces TNF-α release [103, 104]. The LL-37 gene contains a VDRE, making it highly responsive to 1,25(OH)₂D₃ [105].

Apolipoproteins

Apolipoproteins are a group of proteins considered key elements in lipoproteins composition. They are responsible mainly for lipid binding and transportation and are classified into apolipoprotein A (ApoA-1, ApoA-2, ApoA-4, and apolipoprotein A-V (ApoA5), apolipoprotein B (Apo B48 and Apo B100), apolipoprotein C (Apo C-I, Apo C-II, Apo C-III, and Apo C-IV), apolipoprotein D, E, F, H, L, and M [106]. The four classes of apolipoproteins, ApoA-I, ApoAII, ApoCs, and ApoE, are found in all lipoproteins [107].

HDL-associated apolipoproteins

Apolipoproteins facilitate lipid transport in HDL, with ApoA-I, ApoM, and ApoD being central to its anti-inflammatory roles. The table below summarizes their functions (Table 1).

Table 1.

Apolipoproteins associated with HDL and their functions, and the reference paper

Apolipoprotein	Function	Reference
ApoA-1	Reverse cholesterol transportation	[108]
ApoA-II	cholesterol binding	[109]
ApoC-I	Inhibitors of lipoprotein binding to the LDL receptor	[110]
ApoC-II	Triglyceride hydrolysis of VLDL and CM for energy regulation	[111]
ApoC3-III	Antiatherogenic properties and Triglyceride homeostasis.	[112, 113]
ApoD	Molecule transportation, e.g., Retinoic acid, sphingomyelins.	[114]
ApoE	Lipid transportation	[115]
ApoM	A ligand of S1P, anti-inflammatory, antioxidant effects	[116, 117]
ApoL 1	Lipid transportation	[118]
ApoJ	Extracellular chaperone	[119, 120]

Apolipoproteins A-1 (ApoA-1)

Apolipoprotein A-1 is the major component of HDL, located on chromosome 11 and composed of 3 helical chains with a 45 kDa molecular weight, produced in the liver and intestine [121]. ApoA-1 production and regulation depend on the following factors: peroxisome proliferator-activated receptor-γ (PPARγ), the hepatocyte nuclear factor 4 (HNF4), Liver Receptor Homologue 1 (LRH1), and the ApoA-I Regulatory Protein 1 (ARP1/NR2F2) [122]. The construction of HDL starts with the assembly of two to four molecules of ApoA-I with ABCA1. ApoA-1 helps stabilize the ABCA1 protein in hepatocytes and enterocytes, allowing for the synthesis of lipid-poor apoA-1 HDL particles by mediating the efflux of cellular phospholipids and free cholesterol [123]. Most of the ApoA-1 in serum was found associated with HDL, and about 8% of the total ApoA-1 was found lipid-free [108]. In addition, ApoA-1 mediated the cholesterol that effluxes from macrophages in ABCA1-dependent and -independent mechanisms [124].

ApoA-1 modulates innate immunity and adaptive immunity. For instance, ApoA-1 represses both NF-κB and phosphatidylinositol 3-kinase (PI3K). Add to that stimulating the Activator of Transcription Factor 3 (ATF3) (Toll-like receptor down-regulator), which prevents of activation the pro-inflammatory chemokines release e.g. CCL2, CCL5, and CX [3]CL1 [125, 126]. In vivo, Iqbal et al. demonstrated that acute ApoA-I administration reduces monocyte chemotaxis by suppressing the expression of CCL2 and CCL5 [127]. The aforementioned action of ApoA-1 protects against atherosclerosis, and for this reason, some studies consider ApoA-1 a biomarker for predicting cardiovascular diseases [128]. However, a recent meta-analysis of 15 randomized controlled trials has shown that HDL/apoA-1 mimetics are ineffective in reducing atheroma size in the arteries of patients with acute coronary syndrome. Vitamin D modulates ApoA-1 expression, with in vitro studies showing that 1,25(OH)₂D₃ suppresses ApoA-1 gene transcription in HepG2 cells via VDR-VDRE interactions, resulting in a reduction of ApoA-1 mRNA by up to 50% [78]. However, human supplementation studies (e.g., 50,000 IU/week for 12 weeks) suggest vitamin D increases ApoA-1 and HDL cholesterol levels, indicating context-dependent effects [80, 129].

Apolipoprotein M (ApoM)

ApoM is a 23–25 kDa protein, with its human gene located in the major histocompatibility complex (MHC) class III locus on chromosome 6 [130]. ApoM belongs to the lipocalin family (a group of proteins that transport small hydrophobic molecules) and is produced mainly by the liver and kidneys (Fig. 2). Several important proteins belong to this group, such as retinol-binding protein (RBP), apolipoprotein D, and MD-2, the co-receptor for toll-like receptor 4 (TLR4) [131].

Fig. 3.

illustrates the effect of vitamin D on the different HDL-associated proteins, eventually suppressing inflammation. Note: Figures 1, and 3 Created with BioRender.com

Fig. 2.

3D structure of ApoM PDB file 2WEW as a lipid carrier generated using Chimera. The Lipocalin fold, consisting of eight β-strands forming a β-barrel and an α-helix with the calyix carrying myristic acid ligand, is shown. A: 3D structure with Ribon presentation. B: 3D structure with surface presentation. The signaling peptide tail is not cleaved, as shown at the bottom of the 3D figure. (Figure 2 generated using Chimera)

ApoM is required for HDL biogenesis, thereby accounting for approximately 95% of plasma HDL. ApoM is found in association with HDL, where it functions as the primary carrier of sphingosine-1-phosphate (S1P). This bioactive lipid mediator plays a key role in modulating vascular inflammation, and research indicates that ApoM-bound S1P is essential for HDL’s documented antiatherogenic and anti-inflammatory properties [116]. Several transcription factors regulate ApoM expression, such as liver receptor homolog (LRH)-1, hepatic nuclear factor (HNF)-1α, and Forkhead box A2 (Foxa2). Any mutation in those factors could lead to ApoM dysregulation, thereby causing HDL malfunction [132, 133]. However, it is unknown whether ApoM is a vitamin D-responsive gene, i.e., whether it contains a VDRE binding site. Structurally, ApoM has a typical lipocalin fold that consists of eight β-strands forming a β-barrel (pocket-like or calyx) and an α-helix [134]. The distinctive structure of ApoM bears some resemblance to the MD2 unit within TLR4. MD2 itself is characterized by two β-strands that collectively form a cup-like architecture [135]. The substantial similarity in the structure of ApoM and MD2 suggests an analogous function for both, which may result in an affinity for binding endotoxins.

ApoM is found to be associated mainly with HDL and, to a lesser extent, with LDL [136]. The terminal signal peptide of ApoM doesn’t cleave from prior release to plasma, as is the case in the other proteins. This character helps ApoM anchor HDL particles and prevents dissociation between the two [137].

As demonstrated in our previous publication, ApoM is more than a vehicle for S1P1 and other lipids. It was observed that ApoM bound to LPS with high affinity using Isothermal Titration Calorimetry (ITC) and computational molecular dynamics modeling. A poM exhibited a high affinity for E. coli LPS, forming 1:1 complexes with dissociation constants (Kd) below 1 µM. The binding process is strongly exothermic and driven by enthalpy (ΔrH = − 36.5 kJ/mol), suggesting the formed complex of ApoM and LPS is highly stable. Computational simulations also predicted high-affinity binding between ApoM and E. coli LPS, showing that E. coli LPS docks near the calyx of ApoM without obstructing the pocket. In vitro studies confirmed the neutralizing capability of HDL for E. coli LPS, as evidenced by the suppression of TNF-α release from human THP-1 cells. This direct interaction facilitates endotoxin neutralization and clearance by HDL, which involves shuttling LPS to the liver for removal [138]. This process of endotoxin neutralization reduces the exacerbation of the immune response towards bacterial endotoxins, aiming to suppress inflammation and restore hemostasis in the body.

Functionally, ApoM is known as a carrier of sphingosine-1-phosphate (S1P) [139]. Some studies have documented that the complex of ApoM/S1P is important for the antiatherogenic and anti-inflammatory effects of HDL [116]. Ruiz et al. reported that the ApoM/S1P complex plays a crucial role in suppressing the surface abundance of vascular adhesion molecule-1 (VCAM-1) and E-selectin during inflammation. Whereas ApoM alone and HDL alone were not successful in conferring this adhesion suppression [116]. The association of HDL/ApoM/S1P may also contribute to HDL’s vasoprotective and antiatherogenic effects [140, 141].

ApoM/S1P was also observed to be upregulated during the inflammation [142]. However, A study by Winkler et al. observed a decline in S1P levels in septic shock patients. They proposed that this significant decrease in S1P was attributable to the drastic reduction in HDL levels commonly seen during septic shock [143]. S1P also interacts with the endothelial scavenger receptor class B type 1 (SR-BI). This SR-BI is also known as the cholesterol-sensing receptor. The importance of S1P is due to its role in facilitating the uptake of cholesterol from tissues by HDL particles [144]. Briefly, the associated S1P with HDL interacts with S1P1 to generate some proximity with SR-BI. Later, intracellular calcium is released, and cholesterol efflux and downstream signaling events occur [145].

Certain disorders have been associated with the disturbance in ApoM levels and function; for example, plasma ApoM concentration was noticed to be decreased in type 2 diabetes patients [146] and heart failure [147]. Furthermore, the complications of diseases were found to be positively associated with changes in ApoM level and, consequently, HDL function, such as diabetes, e.g., diabetic nephropathy [148]. Similarly, changes in ApoM activity due to genetic polymorphisms, for example, are associated with increased susceptibility to impaired HDL and cholesterol metabolism, and a higher risk of developing coronary artery disease [149].

However, most studies have linked ApoM and S1P to a positive outcome in human health. Christoffersen et. al.‘s research linked the overstimulation of the S1P1 receptor to the decrease in triglyceride uptake and brown adipose formation. In this study, it was revealed that ApoM-deficient mice exhibited an increase in stimulating brown adipose tissue formation, which in turn increased triglyceride uptake, thereby protecting against diet-induced obesity [150]. Hajny et al.‘s study also supports this claim; they examined the triglyceride turnover rate in the human ApoM transgenic mouse model. This model is designed to simulate higher concentrations of ApoM and S1P levels compared to normal. Upon comparing ApoM transgenic with wild-type mice, researchers noticed a reduction in overall triglyceride turnover rate. Additionally, the rate of fatty acid uptake in subcutaneous adipocytes was lower than usual. This could be explained by the decrease in both the plasma level of lipase and the plasma level of fibroblast growth factor 21 (FGF21), as a response to the dramatic increase of ApoM and S1P levels. Both lipase and FGF21 are very crucial for triglyceride clearance from plasma. The disturbance in their level impacts causes an elevation in plasma triglyceride levels after administering a high-fat diet to ApoM transgenic mice [151]. Vitamin D may influence ApoM expression indirectly, as ApoM overexpression in colorectal cancer cells upregulates VDR mRNA, suggesting a bidirectional regulatory relationship [152]. Proteomic analyses also show reduced ApoM levels in vitamin D-deficient individuals with dyslipidemia, correlating with enhanced inflammatory pathways like MAPK and JAK-STAT [79].

Apolipoprotein D (ApoD)

ApoD is another member of the lipocalin family associated with HDL and with the enzyme lecithin-cholesterol acyltransferase (LCAT) that converts cholesterol to cholesteryl ester. ApoD is secreted into the plasma and enriched in the brain, testes, breast, and B cells. ApoD has a molecular weight of 33 kDa and is structurally similar to ApoM [153]. In contrast to other HDL-associated proteins like ApoA-1, which adopt a helix structure, ApoM and ApoD are lipocalins characterized by the typical β-sheet fold with calyx, and act as carrier proteins. ApoD binds hydrophobic ligands such as progesterone, retinoic acid, sphingomyelin, pregnenolone, and arachidonic acid [114]. ApoD plays a significant role in the neurological system, especially in the regeneration and healing of nerves. In vivo studies, ApoD was noticed to elevate tremendously during the healing process of sciatic nerve injury [53]. An increase in ApoD expression has been shown in some neurological diseases such as schizophrenia, bipolar disorder, and Multiple sclerosis [154]. Alzheimer’s disease has been linked to the elevation of ApoD level in CSF and cortex [155, 156].

Moreover, in vivo studies exhibit suppression of the innate immunity system by ApoD during the acute encephalitis induced by coronavirus OC43 in mice with restricted phospholipase A2 activity. Additionally, it has been demonstrated that overexpressing human ApoD in a mouse model of viral infection decreases T-cell infiltration into the CNS, lowers the production of pro-inflammatory cytokines, including IL-1ß and TNF, and downregulates the activity of phospholipase A2 (PLA2) [157]. Vitamin D upregulates ApoD expression, with 1,25(OH)₂D₃ increasing ApoD mRNA up to 5-fold in breast and prostate cancer cells, potentially enhancing HDL’s anti-inflammatory capacity [158, 159]. Proteomic data confirm reduced ApoD in vitamin D-deficient, dyslipidemic individuals, linked to heightened inflammation [79].

Vitamin D impact on HDL-associated proteins ApoA-1, apom, apod, and LL-37

In vitro studies on HepG2 cells (hepatocellular carcinoma) demonstrated that 1,25(OH)₂D₃ suppresses ApoA-1 gene expression at the transcriptional level through VDR-mediated binding to vitamin D response elements (VDREs) in the ApoA-1 promoter [78]. This suppression reduces ApoA-1 mRNA and protein secretion in a dose-dependent manner, potentially impacting HDL biogenesis [78, 160]. For example, Wehmeier et al. [78]showed that 1,25(OH)₂D₃ at concentrations of 10–100 nM inhibited ApoA-1 promoter activity by up to 50%, suggesting a direct regulatory role of vitamin D in HDL composition.

Upon comparing the wild-type mice with VDR knockout ones, experiments illustrated increased HDL-c in general and ApoA-1 expression in the liver. In addition to an increase in total cholesterol levels. However, some differences in the serum concentration of HDL-c between female and male VDR knockout mice have been reported, with a greater increase in males. Consequently, ApoA-1 mRNA was also increased by 49.2% in males [160]. This indicates a more substantial effect of vitamin D in suppressing HDL and ApoA-1 in both genders, particularly in males. Due to species-specific differences, this result could be debated, as rodents and humans do not share similar regulatory mechanisms for HDL-c, ApoA-1, and VDR [161]. From another perspective, Vitamin D supplementation in human studies has been shown to positively affect HDL-c levels [80]. Radkhah et al.‘s systematic review suggested that vitamin D supplementation for ≤ 12 weeks increases Apo-A1 levels; however, the data were not conclusive, and the link was not well elucidated [129].

An in vitro study using colorectal cancer cell lines demonstrated that ApoM overexpression increases VDR mRNA expression, indicating a regulatory relationship [152]. However, the effect of vitamin D on ApoM expression remains unknown. The study by Yu et al. on ApoM in Hepatocellular carcinoma cell lines, using the CRISPR/Cas9 technique, revealed an interesting result. Knocking out ApoM genes in SMMC7721 cell lines was observed to inhibit tumor cell death and induce tumorigenic characteristics, such as increased proliferation rates. A considerable reduction in VDR expression followed this. Further investigations demonstrated that VDR overexpression could reverse the previous effect and suppress the tumorigenicity. Therefore, ApoM can suppress the tumorigenicity characteristics in hepatocellular carcinoma cell lines via enhancing the expression of VDR [162]. In some diseases, such as Systemic lupus erythematosus (SLE), ApoM is inversely correlated with disease activity [163]. Similarly, ApoM was inversely related to the risk of type 2 diabetes [164]. ApoD expression is affected in 1,25(OH)2 D3 presence. ApoD increased up to 5-fold in the presence of D3, more than the increase from other steroid hormones (ApoD ligands). This increase is associated with an inhibitory effect on certain cancers, including breast and prostate cancer cells [158, 165]. These data indicate a direct relationship between ApoA-1, ApoM, ApoD, and vitamin D, although the nature of this association is unclear.

Mousa et al. proteomic analysis of 274 participants revealed reduced HDL-associated ApoM and ApoD in individuals with vitamin D deficiency and dyslipidemia. Conversely, those participants had increased levels of proteins (SAA1 and SOD1), which are associated with the activation of inflammatory pathways. Furthermore, pathway enrichment analysis revealed increased inflammatory activity in individuals with vitamin D deficiency and dyslipidemia, with significant enrichment in pathways such as MAPK, JAK-STAT, Ras signaling, cytokine-cytokine receptor interaction, AGE-RAGE, ErbB signaling, and cancer pathways. Meanwhile, participants with vitamin D deficiency and normal lipid profiles had enrichment in inflammation pathways, such as cytokine signaling pathways [79].

Vitamin D is a potent inducer of cathelicidins (LL-37/ hCAP18), small cationic peptides with antimicrobial properties towards gram-negative and gram-positive bacteria [103]. Cathelicidin (LL-37/hCAP18) plays a crucial role in innate immunity by inhibiting the interaction between LPS and LPS-binding protein, thereby preventing TLR4 activation and resulting in decreased TNF-α release [104, 166, 167]. Furthermore, LL-37 suppresses inflammation and cell death by inhibiting IL-1β expression and caspase-1 activation [168]. Proteomics studies have documented that LL-37 is associated with lipoproteins, including HDL, VLDL, and LDL [169]. Further, the LL-37 gene has a vitamin D response element (VDRE) in its promoter. Thus, it is highly induced by vitamin D [105].

Recent studies have elucidated the association between vitamin D deficiency and dyslipidemia. Sharba et al. found that low vitamin D levels have impacted the lipid profile, including cholesterol, triglycerides, HDL, and LDL [170]. Consistent with our prior findings, the combination of dyslipidemia and vitamin D deficiency modifies the proteomic profiles of healthy individuals. In addition to enhancing inflammatory pathways, a decrease in HDL-associated apolipoproteins, particularly ApoM and ApoD, has been noticed [79]. Vitamin D supplementation has been found to impact dyslipidemia and help alleviate inflammation [171] (Fig. 3).

Strengths and limitations

This review offers a focused and comprehensive analysis of how Vitamin D modulates immune function through its effects on HDL and associated proteins. It provides mechanistic insights into these complex interactions, covering Vitamin D’s influence on lipid metabolism, HDL biogenesis mediated by ApoM, and anti-inflammatory pathways. The detailed discussion of key apolipoproteins (ApoA-1, ApoD, and ApoM) and their roles in lipid transport and anti-inflammatory processes establishes a robust scientific framework. Furthermore, the review effectively highlights the clinical relevance of these molecular interactions in cardiovascular health and chronic inflammatory conditions.

As a comprehensive narrative review, this work synthesizes existing literature without presenting new experimental findings. While it integrates diverse observations, the review could further reconcile seemingly contradictory data on Vitamin D’s impact on apolipoprotein expression and function. Additionally, it highlights a broader limitation in the current research, where studies comprehensively elucidating Vitamin D’s relationship with the full spectrum of apolipoproteins remain limited.

Conclusion and future directions

Vitamin D modulates HDL functionality through its effects on apolipoproteins (ApoA-1, ApoM, ApoD) and cathelicidin (LL-37), influencing lipid metabolism and inflammation [78–80, 103, 158]. While in vitro studies show that 1,25(OH)₂D₃ suppresses ApoA-1 transcription [78], human supplementation increases HDL cholesterol and ApoA-1 [80, 129], suggesting context-dependent effects. ApoM and ApoD levels are reduced in vitamin D-deficient, dyslipidemic individuals, correlating with the activation of enhanced inflammatory pathways [79]. LL-37, induced by vitamin D via VDRE, enhances HDL’s endotoxin-neutralizing capacity [105].

Longitudinal studies and preclinical models (e.g., VDR-knockout mice) are needed to establish causality between vitamin D status, HDL protein composition, and cardiovascular outcomes. Clinical trials evaluating the impact of vitamin D supplementation on ApoM, ApoD, and LL-37 levels could inform therapeutic strategies for dyslipidemia and inflammation-related diseases.

Author contributions

H.M. wrote the main manuscript text of the review as part of her thesis work, which has not been published previously. S.Z. edited and revised the manuscript for clarity and scientific accuracy. Both H.M. and S.Z. served as corresponding authors, managing communication and submission processes. All authors reviewed and approved the final manuscript.

Funding

This review was not supported by any grant funding; however, the Article Processing Charges (APC) were covered by Qatar University.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Not applicable.

Conflict of interest

The authors declare no conflicts of interest.