Cardiac and vascular complications are complex multifactorial pathologies and difficult to prevent since are associated with both genetic and environmental factors [1]. Research on car‐ diovascular diseases (CVDs) is constantly evolving and the current focus is directed towards lipid metabolism, molecular and cellular mechanisms, as well as preventive strategies. Most research in the field of lipid metabolism is motivated by an interest to understand normal lipid transport and preventative measures for atherosclerosis abnormalities [2]. Specific genes and apolipoproteins that are involved in lipid metabolism and lipoprotein synthesis have been isolated, sequenced and mapped in the human genome [3]; however, their role in the lipid

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

metabolism and lipid transport can only be inferred by physiological and genetic studies. To determine their overall function, further exploration of genetic alterations must be investigated.

Since the molecular regulation of lipid metabolism and reverse cholesterol transport (RCT) pathway is complex, numerous studies in humans, animals and *in vitro* have been focusing on the protective action of high‐density lipoprotein cholesterol (HDL‐C), RCT and cholesterol efflux, which can also be augmented for potential therapeutic strategies of CVDs [4, 5].

## **2. Overview of RCT and cholesterol efflux**

The RCT pathway represents an important process involving the transfer of excess cholesterol by HDL particles to the liver for excretion. The ability of HDL to remove cholesterol from cells such as macrophages is linked to the anti‐inflammatory and immunosuppressive functions of this lipoprotein [6]. However, the functionality of HDL is impaired in humans with chronic inflammatory diseases and this causes a reduction in the anti‐inflammatory and cholesterol transport properties. Studies have shown that apolipoprotein A‐I (ApoA‐1), lecithin‐choles‐ terol acyltransferase (LCAT), ATP‐binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR‐B1) serve as important cofactors for a number of RCT pathway con‐ stituents [7]. The initial step of the pathway involves ApoA‐1 being produced by the liver and released into the plasma where it is involved in all stages, including the formation of nascent HDL particles, HDL remodelling by LCAT and delivery of HDL cholesterol directly to the liver via SR‐BI or indirectly via CETP‐mediated transfer to apoB‐containing lipoproteins [8]. Through this process, cholesterol efflux is promoted from the macrophages via ABCA1 and also by the ABCG1 transporter using the action of LCAT (**Figure 1**).

**Figure 1.** Reverse cholesterol transport (RCT) pathway. Major components of RCT include apolipoprotein A‐I (apoA‐I), high‐density lipoprotein (HDL), lecithin: cholesterol acyltransferase (LCAT), ATP‐binding cassette transporter (ABCA1/ ABCG1) and cholesterol ester transfer protein (CETP). Free cholesterols (FC) in the HDL are delivered to the liver for excretion through scavenger receptor B1 (SR‐B1). Alternatively, the cholesteryl esters (CE) could also be delivered to the liver through the low‐density‐lipoprotein receptor (LDLR). Figure modified from Rader et al. [4].

## **3. Vervet monkey as an animal model for CVDs**

As with most areas of human biology, studies of human CVDs have been enriched and com‐ plemented by investigations of animal models. Among the nonhuman primates (NHP), the vervet monkey (*Chlorocebus aethiops*) has been validated to be an excellent research model for the study of CVDs [9]. For the purpose of this investigation, we used this NHP model to deter‐ mine the protective action of HDL, its role in the reverse cholesterol transport (RCT) pathway and the expression profile of genes regulating HDL metabolism.

This study was conducted in compliance with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (A5726‐01) and approved by the Ethics Committee of the South African Medical Research Council (*SA*MRC) (REF 11/07). The selected subjects (25) were healthy adult female monkeys with normal plasma HDL. All individuals were kept under identical housing conditions according to the South African National Standard for the Care and Use of Animals for Scientific Purposes (The SANS 10386:2008).

## **3.1. Laboratory analysis**

metabolism and lipid transport can only be inferred by physiological and genetic studies. To determine their overall function, further exploration of genetic alterations must be investigated. Since the molecular regulation of lipid metabolism and reverse cholesterol transport (RCT) pathway is complex, numerous studies in humans, animals and *in vitro* have been focusing on the protective action of high‐density lipoprotein cholesterol (HDL‐C), RCT and cholesterol efflux, which can also be augmented for potential therapeutic strategies of CVDs [4, 5].

The RCT pathway represents an important process involving the transfer of excess cholesterol by HDL particles to the liver for excretion. The ability of HDL to remove cholesterol from cells such as macrophages is linked to the anti‐inflammatory and immunosuppressive functions of this lipoprotein [6]. However, the functionality of HDL is impaired in humans with chronic inflammatory diseases and this causes a reduction in the anti‐inflammatory and cholesterol transport properties. Studies have shown that apolipoprotein A‐I (ApoA‐1), lecithin‐choles‐ terol acyltransferase (LCAT), ATP‐binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR‐B1) serve as important cofactors for a number of RCT pathway con‐ stituents [7]. The initial step of the pathway involves ApoA‐1 being produced by the liver and released into the plasma where it is involved in all stages, including the formation of nascent HDL particles, HDL remodelling by LCAT and delivery of HDL cholesterol directly to the liver via SR‐BI or indirectly via CETP‐mediated transfer to apoB‐containing lipoproteins [8]. Through this process, cholesterol efflux is promoted from the macrophages via ABCA1 and

**Figure 1.** Reverse cholesterol transport (RCT) pathway. Major components of RCT include apolipoprotein A‐I (apoA‐I), high‐density lipoprotein (HDL), lecithin: cholesterol acyltransferase (LCAT), ATP‐binding cassette transporter (ABCA1/ ABCG1) and cholesterol ester transfer protein (CETP). Free cholesterols (FC) in the HDL are delivered to the liver for excretion through scavenger receptor B1 (SR‐B1). Alternatively, the cholesteryl esters (CE) could also be delivered to the

liver through the low‐density‐lipoprotein receptor (LDLR). Figure modified from Rader et al. [4].

**2. Overview of RCT and cholesterol efflux**

44 Advances in Lipoprotein Research

also by the ABCG1 transporter using the action of LCAT (**Figure 1**).

#### *3.1.1. Candidate genes and sequence variants selection*

The genetic variations were evaluated in 10 genes implicated in lipid metabolism (CETP, ABCA1, CYP7A1, apoA‐1, apoB, apoE, SR‐B1, LCAT, apoCI and apoCII). Twenty‐two coin‐ cident single nucleotide polymorphisms (cSNPs) were selected for genotyping. These cSNPs were prioritised based on their function and location within their respective candidate gene and their association with CVD.

#### *3.1.2. Gene expression*

Blood (2 ml) was collected in EDTA‐containing tubes from 25 animals using a femoral vene‐ puncture after ketamine anaesthesia at 10 mg/kg bodyweight. DNA was extracted from whole blood using the Nucleospin Genomic Blood DNA Purification Kit (MACHEREY‐ NAGEL, Germany) and PAXgene Blood RNA Kit (PreAnalytiX, Qiagen) was used for RNA extraction. The extracted DNA was used for Sanger sequencing while RNA was for gene expression experiments. Turbo DNase treatment (Ambion, USA) was used for RNA purification before cDNA conversion (high‐capacity cDNA kit, Applied Biosystems, USA). The effects of niacin treatment on the expression of the 10 prioritised genes were determined using quantitative real‐time PCR (qRT‐PCR). The gene expression data were normalised to the average of phosphoglycerate kinase 2 (PGK2: QT00219023) and glycer‐ aldehyde‐3‐phosphate dehydrogenase (GAPDH: QT01192646), which were used as house‐ keeping genes.

Since the levels of high‐density lipoprotein‐cholesterol (HDL‐C) are a significant determi‐ nant of a cholesterol efflux capacity, a correlation analysis was conducted to determine the relationship between the levels of HDL‐C and mRNA expression of the 10 selected RCT can‐ didate genes.

## **4. Effects of mutations, drug intervention and gene expression on RCT**

A major area in HDL‐based therapeutics is focusing on the development of pharmacological approaches to improve the activity of the RCT pathway. One of these strategies involves the combination of genetic variations and individual responsiveness to drugs [10]. Subsequently, genetic variations in gene encoding transporters contribute to individual differences in drug absorption, elimination and cellular uptake, thereby affecting drug response and toxicity [11].

Among several types of genetic variations, single‐nucleotide polymorphisms (SNPs) are the most abundant throughout the genome [12]. SNPs have lately received much attention as they serve as markers of individual risk for adverse drug reactions or susceptibility to complex diseases [13]. Small‐scale studies have focused on the effects of polymorphisms on physi‐ ological or biochemical factors and have provided useful information on possible mechanistic links between variation at the gene level and risk factors for CVDs [14]. For the purpose of this review, 10 'candidate' genes known to be involved in RCT and HDL metabolism were screened in the vervet model and only six cSNPs (I405V, I883M, A233S, cL96R, ‐62A>C and A350A) were identified in CETP, ABCA1, CYP7A1, apoCII and SR‐B1, respectively (**Table 1**).

**Table 1.** cSNPs identified in vervet monkeys.

For effective changes in lipid metabolism, niacin, as the most potent available lipid‐regulat‐ ing drug [7] was used as a tool to increase HDL levels (**Figure 2**). A strong inverse correlation was observed with CETP, SR‐B1 and CYP7A1 concentrations (*r* = ‐0.14, ‐0.27, ‐0.30; *p* < 0.001). Concurrently, gene expression profile showed that all three genes were down‐regulated when correlated with the three cSNPs (I405V, A350A and A233S) (**Figure 3**). Since I405V is known to lower plasma CETP concentration and elevate HDL‐C concentration [5, 9], the presence of this variant confirmed the same effect in the vervet model. With a similar expression profile observed in SR‐B1 and CYP7A1, the presence of A350A may suggest a possible influence on RCT and HDL‐C synthesis and a plausible involvement of A233S in drug metabolism [6]. The remaining cSNPs (I883M, cL96R and ‐62A>C), however, did not influence the expression of their respective genes (ABCA1 and APOCII) despite being known to alter plasma lipid levels and influence cholesterol efflux [15, 16]. Therefore, these findings suggest that some of these identified sequence variants have significant impact on gene expression which can be corre‐ lated with biochemistry levels (HDL‐C, LDL‐C and triglycerides) following drug intervention.

**Figure 2.** The effect of niacin treatment on HDL‐C, LDL‐C, triglycerides (Trig) and apoA‐1. \*Significant level (*p* < 0.05).

**Figure 3.** The effect of niacin treatment on CETP, SR‐B1 and CYP7A1 mRNA expression. The data were expressed as mean ± SD and mRNA expression in a.u. (arbitrary units). \*Significant level (*p* < 0.05).

## **5. Conclusion**

**4. Effects of mutations, drug intervention and gene expression on RCT**

A major area in HDL‐based therapeutics is focusing on the development of pharmacological approaches to improve the activity of the RCT pathway. One of these strategies involves the combination of genetic variations and individual responsiveness to drugs [10]. Subsequently, genetic variations in gene encoding transporters contribute to individual differences in drug absorption, elimination and cellular uptake, thereby affecting drug response and toxicity [11]. Among several types of genetic variations, single‐nucleotide polymorphisms (SNPs) are the most abundant throughout the genome [12]. SNPs have lately received much attention as they serve as markers of individual risk for adverse drug reactions or susceptibility to complex diseases [13]. Small‐scale studies have focused on the effects of polymorphisms on physi‐ ological or biochemical factors and have provided useful information on possible mechanistic links between variation at the gene level and risk factors for CVDs [14]. For the purpose of this review, 10 'candidate' genes known to be involved in RCT and HDL metabolism were screened in the vervet model and only six cSNPs (I405V, I883M, A233S, cL96R, ‐62A>C and A350A) were identified in CETP, ABCA1, CYP7A1, apoCII and SR‐B1, respectively (**Table 1**).

For effective changes in lipid metabolism, niacin, as the most potent available lipid‐regulat‐ ing drug [7] was used as a tool to increase HDL levels (**Figure 2**). A strong inverse correlation was observed with CETP, SR‐B1 and CYP7A1 concentrations (*r* = ‐0.14, ‐0.27, ‐0.30; *p* < 0.001). Concurrently, gene expression profile showed that all three genes were down‐regulated when correlated with the three cSNPs (I405V, A350A and A233S) (**Figure 3**). Since I405V is known to lower plasma CETP concentration and elevate HDL‐C concentration [5, 9], the presence of this variant confirmed the same effect in the vervet model. With a similar expression profile observed in SR‐B1 and CYP7A1, the presence of A350A may suggest a possible influence on RCT and HDL‐C synthesis and a plausible involvement of A233S in drug metabolism [6]. The remaining cSNPs (I883M, cL96R and ‐62A>C), however, did not influence the expression of their respective genes (ABCA1 and APOCII) despite being known to alter plasma lipid levels and influence cholesterol efflux [15, 16]. Therefore, these findings suggest that some of these identified sequence variants have significant impact on gene expression which can be corre‐ lated with biochemistry levels (HDL‐C, LDL‐C and triglycerides) following drug intervention.

**Gene cSNP Accession number Chr Exon Nucleotide change Amino acid change**

CETP I405V rs5882 16 14 A/G I/V ABCA1 Ile883Met rs4149313 9 18 A/G I/M CYP7A1 Asn233Ser rs8192874 8 3 A/G N/S APOC‐II Leu96Arg rs5167 19 3 T/G L/R

SR‐B1 A350A rs5888 12 8 C/T A/A

‐62A>C rs2288911 Promoter

**Table 1.** cSNPs identified in vervet monkeys.

46 Advances in Lipoprotein Research

It is a fact that characterisation of polymorphisms in lipid metabolism is challenging, however it remains essential for the optimal regulation and functioning of the RCT pathway. This review demonstrates that the genetic determinants of lipid transport and metabolism may provide additional significant benefit in pharmacological therapy for CVDs. Genetic approaches have shown that sequence variants can be correlated with biochemistry levels such as HDL‐C, LDL‐C and triglycerides following drug intervention. Although cholesterol lowering alone may explain the anti‐atherosclerotic effect of niacin on HDL‐C, in this review, gene expression data has shed some light in supporting the hypothesis that genetic variants may influence the expression of genes involved in RCT, which may also play a role in the anti‐atherosclerotic effect of niacin.

It is also noteworthy that this is the first report to provide data of a controlled pharmacologi‐ cal intervention linked to genetic determinants of lipid metabolism in vervet monkeys.

## **Author details**

Chesa G. Chauke\*, Zandisiwe E. Magwebu and Jürgen V. Seier

\*Address all correspondence to<EMAIL_ADDRESS>
South African Medical Research Council, Primate Unit and Delft Animal Centre, Cape Town, South Africa

## **References**

[10] Vergani, C., T. Lucchi, M. Caloni, I. Ceconi, C. Calabresi, S. Scurati and B. Arosio. I405V polymorphism of the cholesteryl ester transfer protein (CETP) gene in young and very old people. *Archives of Gerontology and Geriatrics,* 2006. **43**(2): p. 213–221.

It is also noteworthy that this is the first report to provide data of a controlled pharmacologi‐ cal intervention linked to genetic determinants of lipid metabolism in vervet monkeys.

South African Medical Research Council, Primate Unit and Delft Animal Centre, Cape Town,

[1] Zaragoza, C., C. Gomez‐Guerrero, J.L. Martin‐Ventura, L. Blanco‐Colio, B. Lavin, B. Mallavia, C. Tarin, S. Mas, A. Ortiz and J. Egido. Animal models of cardiovascular dis‐

[2] Cruz, D.E., H.M. Ahmed, S.R. Jones, M.B. Elshazly and S.S. Martin. Discordance in lipid measurements: Can we capitalize to better personalize cardiovascular risk assessment

[3] Breslow, J. Lipoprotein transport gene abnormalities underlying coronary heart disease

[4] Rader, D.J., E.T. Alexander, G.L. Weibel, J. Billheimer and G.H. Rothblat. The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis.

[5] Assmann, G. and A.M. Gotto, Jr. HDL cholesterol and protective factors in atherosclerosis.

[6] Yvan‐Charvet, L., N. Wang and A.R. Tall. Role of HDL, ABCA1 and ABCG1 transport‐ ers in cholesterol efflux and immune responses. *Arteriosclerosis, Thrombosis and Vascular*

[7] Daniels, T.F., X.‐L. Wu, Z. Pan, J.J. Michal, R.W. Wright Jr., K.M. Killinger, M.D. MacNeil and Z. Jiang. The reverse cholesterol transport pathway improves understanding of genetic networks for fat deposition and muscle growth in beef cattle*. PloS One,* 2010.

[8] Rothblat, G.H. and M.C. Phillips. High‐density lipoprotein heterogeneity and function in reverse cholesterol transport. *Current Opinion in Lipidology,* 2010. **21**(3): p. 229.

[9] Moghadasian, M.H., J.J. Frohlich and B.M. McManus. Advances in experimental dyslip‐ idemia and atherosclerosis. *Laboratory Investigation,* 2001. **81**(9): p. 1173–1183.

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\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

48 Advances in Lipoprotein Research

South Africa

**References**

**Inflamatory Conditions and Lipoproteins**

**Provisional chapter**

## **Role of HDL-Associated Proteins and Lipids in the Regulation of Inflammation Regulation of Inflammation**

**Role of HDL-Associated Proteins and Lipids in the**

Roger White, Samantha Giordano and Geeta Datta Geeta Datta

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Roger White, Samantha Giordano and

http://dx.doi.org/10.5772/67141

#### **Abstract**

Lipoproteins are complexes of lipids and proteins that carry water‐insoluble cholesterol in the bloodstream. While cholesterol is required for normal cell function, hypercholes‐ terolemia contributes to the development of cardiovascular disease (CVD). Increased low‐density lipoprotein (LDL) is a major risk factor for CVD. Reduced high‐density lipo‐ protein (HDL) levels are inversely related to CVD risk, suggesting a protective role for HDL. Several diseases, including atherosclerosis, diabetes, chronic kidney disease and rheumatoid arthritis, have been identified where HDL levels are decreased or function is compromised. HDLs are spherical particles with a hydrophobic core of cholesteryl esters surrounded by a monolayer of phospholipids, proteins and unesterified cholesterol. Apolipoprotein (apo) A‐I, the major protein component of HDL, plays an important role in the assembly and function of HDL. One of the major functions of HDL is to mediate cellular cholesterol efflux and the transfer of cholesterol from extrahepatic tissues to the liver for excretion into the bile. In addition to regulating cholesterol metabolism, HDL also exhibits antioxidative, antithrombotic and anti‐inflammatory properties. Under cer‐ tain conditions, however, HDL may undergo biochemical modification resulting in the formation of a particle with pro‐inflammatory properties. This review will focus on the variable properties of HDL under normal physiological conditions and in the context of inflammation.

**Keywords:** HDL, inflammation, lipid composition, protein composition, function, macrophage mitochondria

## **1. Introduction**

Hypercholesterolemia is an important determinant of cardiovascular disease (CVD), the lead‐ ing cause of death globally [1]. Cholesterol, among other lipids, is carried in the bloodstream

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

from the liver to different parts of the body by lipoproteins, complex particles composed of lipids and proteins. There are four major lipoproteins that can be classified on the basis of their density: chylomicrons, very low‐density lipoprotein (VLDL), low‐density lipoprotein (LDL) and high‐density lipoprotein (HDL) [2]. Chylomicrons, VLDL and LDL are larger par‐ ticles with densities ranging from 0.95 to 1.063 g/ml. HDL is a mixture of spherical particles ranging in size from 7 to 12 nm in diameter and 1.063–1.21 g/ml in density. Epidemiological studies have established an inverse relationship between HDL cholesterol and CVD risk [3, 4]. Thus, a reduction in plasma HDL levels represents an important risk factor for CVD. Results of clinical trials demonstrate that lowering LDL levels reduces CVD risk [5, 6]. Evidence sup‐ porting a role for elevated HDL in reducing CVD risk, however, is still forthcoming. Clinical trials have shown that torcetrapib, dalcetrapib and extended‐release niacin significantly increase circulating HDL levels; however, this was not associated with improved outcomes [7–9]. On the other hand, raising plasma HDL by infusion or overexpression of apoA‐I in murine models was shown to reduce atherogenic lesion progression [10]. One hypothesis to explain this disparity proposes that the "quality" or functional status of HDL may be a better indicator of CVD risk than plasma levels of HDL per se [11]. This review will focus on the structure‐function relationship of HDL and how it influences responses to the lipoprotein in the context of inflammation.

HDL particles have a neutral core of cholesteryl ester and triglycerides (TG) surrounded by a monolayer of phospholipids, free cholesterol (FC) and protein. ApoA‐I is the major protein associated with HDL particles and is synthesized in the liver and small intestine. Phospholipids and cholesterol are transferred to apoA‐I by a process mediated by the ATP‐ binding cassette transporter type 1 (ABCA1) [12, 13] resulting in the formation of a lipid poor, dense particle called preβ‐HDL. This particle plays an important role in reverse cholesterol transport, a process by which cholesterol is removed from cells. Although these particles have been predominantly studied under in vitro conditions, little information is available regarding the presence or functional significance of preβ‐HDL in vivo [14]. HDL isolated from plasma by sequential ultracentrifugation yields two major subpopulations: HDL2, a large, light, lipid‐rich particle (d1.063–1.125 g/ml), and HDL3, a smaller, denser protein‐rich particle (d1.125–1.21 g/ml). These two particles can be further subdivided into five distinct populations: HDL2b, HDL2a, HDL3a, HDL3b and HDL3c [15]. These heterogeneous particles vary in their lipid and protein composition, forming particles of varying density, charge, and antigenicity. They also possess discrete functional properties.

## **2. HDL structural components**

*The HDL lipidome***:** Phospholipids (PL) represent the major lipid component of HDL, con‐ stituting about 50% by weight of all the lipids [15]. Phosphatidylcholine (PC), with a carbon backbone of varying length and saturation, is the major PL species. Lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and plasmalogens are also present at lower, but significant, amounts (greater than 1% of total HDL lipids by weight). Other phospholipids (phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA) and cardiolipin) constitute less than 1% of total HDL lipids by weight.

Sphingolipids are also well‐represented in HDL particles. Sphingomyelin (SM) accounts for 5–10% by weight of total HDL lipids [15]. SM is converted to ceramide by sphingomyelin‐ ase [16]. Ceramide constitutes 0.05% by weight of total HDL lipids. Ceraminidase converts ceramide to sphingosine. Finally, the enzyme sphingosine kinase converts sphingosine to sphingosine 1‐phosphate (S1P) [16]. S1P, as well as ceramide‐1‐phosphate, are carried by HDL and are potent signaling molecules that regulate cell growth, survival and differentia‐ tion [17]. S1P plays an important role in the suppression of inflammation [17]. S1P binding to HDL requires its physical interaction with apo M [17, 18]. Sphingosylphosphorylcholine and lysosulfatide are additional, biologically active lysosphingolipids carried by HDL [15]. The principal lipids associated with HDL particles are summarized in **Table 1**.

**Table 1.** Normal protein and lipid components of HDL.

from the liver to different parts of the body by lipoproteins, complex particles composed of lipids and proteins. There are four major lipoproteins that can be classified on the basis of their density: chylomicrons, very low‐density lipoprotein (VLDL), low‐density lipoprotein (LDL) and high‐density lipoprotein (HDL) [2]. Chylomicrons, VLDL and LDL are larger par‐ ticles with densities ranging from 0.95 to 1.063 g/ml. HDL is a mixture of spherical particles ranging in size from 7 to 12 nm in diameter and 1.063–1.21 g/ml in density. Epidemiological studies have established an inverse relationship between HDL cholesterol and CVD risk [3, 4]. Thus, a reduction in plasma HDL levels represents an important risk factor for CVD. Results of clinical trials demonstrate that lowering LDL levels reduces CVD risk [5, 6]. Evidence sup‐ porting a role for elevated HDL in reducing CVD risk, however, is still forthcoming. Clinical trials have shown that torcetrapib, dalcetrapib and extended‐release niacin significantly increase circulating HDL levels; however, this was not associated with improved outcomes [7–9]. On the other hand, raising plasma HDL by infusion or overexpression of apoA‐I in murine models was shown to reduce atherogenic lesion progression [10]. One hypothesis to explain this disparity proposes that the "quality" or functional status of HDL may be a better indicator of CVD risk than plasma levels of HDL per se [11]. This review will focus on the structure‐function relationship of HDL and how it influences responses to the lipoprotein in

HDL particles have a neutral core of cholesteryl ester and triglycerides (TG) surrounded by a monolayer of phospholipids, free cholesterol (FC) and protein. ApoA‐I is the major protein associated with HDL particles and is synthesized in the liver and small intestine. Phospholipids and cholesterol are transferred to apoA‐I by a process mediated by the ATP‐ binding cassette transporter type 1 (ABCA1) [12, 13] resulting in the formation of a lipid poor, dense particle called preβ‐HDL. This particle plays an important role in reverse cholesterol transport, a process by which cholesterol is removed from cells. Although these particles have been predominantly studied under in vitro conditions, little information is available regarding the presence or functional significance of preβ‐HDL in vivo [14]. HDL isolated from plasma by sequential ultracentrifugation yields two major subpopulations: HDL2, a large, light, lipid‐rich particle (d1.063–1.125 g/ml), and HDL3, a smaller, denser protein‐rich particle (d1.125–1.21 g/ml). These two particles can be further subdivided into five distinct populations: HDL2b, HDL2a, HDL3a, HDL3b and HDL3c [15]. These heterogeneous particles vary in their lipid and protein composition, forming particles of varying density, charge, and

*The HDL lipidome***:** Phospholipids (PL) represent the major lipid component of HDL, con‐ stituting about 50% by weight of all the lipids [15]. Phosphatidylcholine (PC), with a carbon backbone of varying length and saturation, is the major PL species. Lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and plasmalogens are also present at lower, but significant, amounts (greater than 1% of total HDL lipids by weight). Other phospholipids (phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid

(PA) and cardiolipin) constitute less than 1% of total HDL lipids by weight.

antigenicity. They also possess discrete functional properties.

**2. HDL structural components**

the context of inflammation.

54 Advances in Lipoprotein Research

*The HDL proteome:* The HDL proteome has been characterized by several groups over the past 10 years. Using mass spectroscopy, the presence of at least 85 proteins on HDL have been reported [19]. These fall into different regulatory categories: lipid metabolism, acute phase response (APR), hemostasis, immune response, metal binding, vitamin transport, proteinase inhibitor and complement regulation [19, 20]. A representative list of HDL‐associated pro‐ teins is shown in **Table 1**. Among these, the lipid metabolism group is the largest and con‐ tains apoA‐I as well as other apolipoproteins (**Table 1**). As mentioned above, HDL exists as multiple sub‐species. The proteins, lecithin‐cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP), play a major role in converting HDL from one sub‐species to another. APR proteins such as apo A‐IV, SAA1 and SAA2 regulate lipid metabolism and are also present along with Apo J, a protein involved in lipid metabolism and complement regulation. Surprisingly, a variety of other proteins with diverse functions such as hemoglobin, hemopexin and transferrin (iron metabolism), ceru‐ loplasmin (metal binding), and vitamin D binding protein (vitamin binding) are also seen. These are described in detail in the review by Shah et al. [19]. Thus, the protein and lipid cargo on HDL significantly influence particle function.

## **3. Functions of HDL**

*Reverse cholesterol transport:* Under hypercholesterolemic conditions, the accumulation of cholesterol in macrophages leads to the formation of "foam cells" which contribute to ath‐ eroma formation. HDL is commonly referred to as the "good cholesterol". The salutary effect of HDL has been attributed to its ability to transfer cholesterol from extra‐hepatic tissues to the liver for metabolism and excretion into the bile, a process called reverse cholesterol trans‐ port [21]. This is believed to be a critical antiatherogenic function of HDL. Cholesterol from macrophages is transferred to lipid‐poor apoA‐I [22] *via* ABCA1. The cholesterol is converted to cholesterol esters by the action of LCAT present on HDL. Sequestration of cholesterol esters in the hydrophobic core of the particle is associated with the formation of spherical HDL2 and HDL3. These mature HDL particles also incorporate cholesterol *via* an alternate transporter, the ATP‐binding cassette transporter G1 (ABCG1) as well as the scavenger‐receptor class B, type 1 (SR‐BI) pathway [23]. Cholesterol‐enriched HDL is subsequently removed from the circulation by hepatocytes and is excreted by the biliary pathway into bile and feces. In addi‐ tion to mediating reverse cholesterol transport, HDL also possesses antioxidant, anti‐inflam‐ matory and antithrombotic properties. These pleiotropic effects of HDL play a major role in limiting inflammatory injury associated with leukocyte infiltration in the blood vessel wall.

*Antioxidant properties of HDL***:** Chylomicrons, VLDL and LDL are apoB‐containing lipo‐ proteins which deliver cholesterol and TG to cells and are strongly implicated in atheroma formation. The response‐to‐retention hypothesis postulates that [24] LDL is oxidized in the arterial wall by enzymes including myeloperoxidase (MPO), NADPH oxidase, nitric oxide synthase and lipoxygenase, resulting in the accumulation of lipid hydroperoxides (LOOH) [25]. Oxidized LDL (ox‐LDL) is taken up by macrophages leading to the formation of foam cells and fatty plaques. Protein and lipid components of HDL inhibit the accumulation of LOOH in LDL and prevent the formation of ox‐LDL. LOOH and phosphatidyl choline hydro‐ peroxides (PLOOH) are transferred from LDL to HDL. This process is regulated by the lipid composition and rigidity of the HDL surface. Specifically, HDL surface rigidity is determined by the ratios of SM:PC, FC:PL and saturated to polyunsaturated fatty acids (SFA:PUFA) [26]. Zerrad‐Saadi and colleagues have identified the HDL3 particle as a key mediator of LOOH transfer due its optimal surface rigidity and particle content [27].

ApoA‐I is likely the major HDL protein species involved in the removal of LOOH moieties from LDL. The methionine (Met) residues 112 and 148 of apoA‐I can reduce LOOHs to inac‐ tive lipid hydroxides (LOH) [28]. In addition, apoA‐I removes seeding LOOH molecules from LDL [29]. In addition to apoA‐I, other apolipoprotein and enzyme components of HDL, such as, apo E, apo J, apo A‐II, apo L‐1, apo F, apo A‐IV, PON1/3, PLTP and PAF‐AH, play a role in its antioxidant function. Proteomic analyses from the Davidson laboratory [30] demonstrate that HDL3c contains all these proteins along with apo M, apo D, apo A‐II, SAA1,2 and 4 and apo C‐I and apo C‐II. This corroborates earlier studies showing that HDL3c has more potent antioxidant activity than other HDL subspecies [31, 32]. Thus, both lipid and protein compo‐ nents of HDL3c contribute to its antioxidant activity. Kontush et al. [32] have hypothesized that the protein components of HDL3c form a pocket which enables the transfer of LOOH from LDL which is further reduced by the concerted action of apolipoproteins and enzymes in this pocket [26].

lipid metabolism and complement regulation. Surprisingly, a variety of other proteins with diverse functions such as hemoglobin, hemopexin and transferrin (iron metabolism), ceru‐ loplasmin (metal binding), and vitamin D binding protein (vitamin binding) are also seen. These are described in detail in the review by Shah et al. [19]. Thus, the protein and lipid cargo

*Reverse cholesterol transport:* Under hypercholesterolemic conditions, the accumulation of cholesterol in macrophages leads to the formation of "foam cells" which contribute to ath‐ eroma formation. HDL is commonly referred to as the "good cholesterol". The salutary effect of HDL has been attributed to its ability to transfer cholesterol from extra‐hepatic tissues to the liver for metabolism and excretion into the bile, a process called reverse cholesterol trans‐ port [21]. This is believed to be a critical antiatherogenic function of HDL. Cholesterol from macrophages is transferred to lipid‐poor apoA‐I [22] *via* ABCA1. The cholesterol is converted to cholesterol esters by the action of LCAT present on HDL. Sequestration of cholesterol esters in the hydrophobic core of the particle is associated with the formation of spherical HDL2 and HDL3. These mature HDL particles also incorporate cholesterol *via* an alternate transporter, the ATP‐binding cassette transporter G1 (ABCG1) as well as the scavenger‐receptor class B, type 1 (SR‐BI) pathway [23]. Cholesterol‐enriched HDL is subsequently removed from the circulation by hepatocytes and is excreted by the biliary pathway into bile and feces. In addi‐ tion to mediating reverse cholesterol transport, HDL also possesses antioxidant, anti‐inflam‐ matory and antithrombotic properties. These pleiotropic effects of HDL play a major role in limiting inflammatory injury associated with leukocyte infiltration in the blood vessel wall. *Antioxidant properties of HDL***:** Chylomicrons, VLDL and LDL are apoB‐containing lipo‐ proteins which deliver cholesterol and TG to cells and are strongly implicated in atheroma formation. The response‐to‐retention hypothesis postulates that [24] LDL is oxidized in the arterial wall by enzymes including myeloperoxidase (MPO), NADPH oxidase, nitric oxide synthase and lipoxygenase, resulting in the accumulation of lipid hydroperoxides (LOOH) [25]. Oxidized LDL (ox‐LDL) is taken up by macrophages leading to the formation of foam cells and fatty plaques. Protein and lipid components of HDL inhibit the accumulation of LOOH in LDL and prevent the formation of ox‐LDL. LOOH and phosphatidyl choline hydro‐ peroxides (PLOOH) are transferred from LDL to HDL. This process is regulated by the lipid composition and rigidity of the HDL surface. Specifically, HDL surface rigidity is determined by the ratios of SM:PC, FC:PL and saturated to polyunsaturated fatty acids (SFA:PUFA) [26]. Zerrad‐Saadi and colleagues have identified the HDL3 particle as a key mediator of LOOH

on HDL significantly influence particle function.

transfer due its optimal surface rigidity and particle content [27].

ApoA‐I is likely the major HDL protein species involved in the removal of LOOH moieties from LDL. The methionine (Met) residues 112 and 148 of apoA‐I can reduce LOOHs to inac‐ tive lipid hydroxides (LOH) [28]. In addition, apoA‐I removes seeding LOOH molecules from LDL [29]. In addition to apoA‐I, other apolipoprotein and enzyme components of HDL, such as, apo E, apo J, apo A‐II, apo L‐1, apo F, apo A‐IV, PON1/3, PLTP and PAF‐AH, play a role in

**3. Functions of HDL**

56 Advances in Lipoprotein Research

*Anti‐inflammatory properties of HDL***:** The role of inflammation in atherogenesis has been clearly established [33–35]. Acute and chronic inflammations are associated with monocyte adhesion/infiltration and endothelial cell activation [33–35]. HDL is known to suppress the lipopolysaccharide (LPS)‐induced secretion of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and other pro‐inflammatory mediators [36–38]. HDL also reduces inflammation by neutralizing endotoxin, further supporting its anti‐inflammatory role [39]. Thus, HDL exerts its anti‐inflammatory effect in multiple ways.

*Regulation of endotoxicity***:** In the context of infection, Gram‐negative bacteria release LPS in the circulation which binds CD14 located in membrane rafts on cell surfaces. CD14 engagement facilitates the activation of toll‐like receptor 4 (TLR4) binding, resulting in the release of pro‐ inflammatory cytokines such as IL‐6 and TNF‐α. HDL is able to inhibit this initial activation step *via* binding to lipid A, a glycolipid component of LPS, thus preventing TLR4 activation. Gram‐positive bacteria release lipoteichoic acid (LTA) which, similar to LPS, binds CD14 and activates pro‐inflammatory signaling *via* the TLR2/6 pathway [40, 41]. HDL additionally con‐ tributes to the inactivation of LPS and LTA by disrupting membrane rafts. In this manner, HDL mediates cholesterol and phospholipid efflux which destabilizes rafts and prevents the assembly of receptor complexes for LPS and LTA [14, 40].

*Regulation of macrophage function*: Macrophages are a versatile group of cells that play a criti‐ cal role in regulating immunity, inflammation and lipid metabolism. Macrophage phenotype and function are regulated, in large part, by their environmental milieu [42–45]. On the basis of cell morphology and function, two populations of activated macrophages have been identi‐ fied [46]. The classically activated M1 macrophage is induced by LPS and Th1 cytokines such as IFN‐γ, interleukin‐2 (IL‐2) and TNF‐α [43, 44]. These cells are pro‐inflammatory and secrete inflammatory mediators (TNF‐α, IL‐1, IL‐6, IL‐15, IL‐18, IL‐23, IFN‐γ), stimulate inducible nitric oxide synthase (iNOS) and promote the formation of reactive oxygen and nitrogen spe‐ cies [47]. The second macrophage phenotype, the alternatively activated M2 macrophage, is induced by IL‐4, IL‐10, IL‐13 and glucocorticoid hormones [42–45]. M2 macrophages play an important role in the resolution of inflammation by inhibiting inflammatory cytokine expression and promoting wound healing [42–45]. HDL and apo A‐I have been shown to promote the formation of anti‐inflammatory M2 macrophages in human monocyte‐derived macrophages [48] and mice [49]. As mentioned in the previous section, HDL3 is a key media‐ tor of reverse cholesterol transport and possesses potent antioxidant properties. Reports from several laboratories suggest that HDL‐associated S1P inhibits inflammation *via* activa‐ tion of the PI3‐kinase/*Akt* signaling pathway [50–52]. Pretreatment of bone marrow‐derived  macrophages (BMDMs) with S1P suppressed LPS‐induced secretion of TNF‐α, monocyte chemoattractant protein (MCP) and IL‐12 [53]. Additionally, Hughes and colleagues reported that S1P enhanced the activity of Arg1 and suppressed the NF‐kB‐mediated induction of iNOS [53]. These responses to S1P are associated with M2 macrophage polarization.

*Regulation of mitochondrial function:* The mitochondrion is a double‐membraned, energy‐pro‐ ducing organelle, which contains its own maternally inherited mitochondrial DNA [54–56]. Under normal conditions, the mitochondrial respiratory chain shuttles electrons through the respiratory complexes, consumes oxygen at Complex IV and pumps hydrogen ions from inside the mitochondria to the intermembrane space at Complexes I, III and IV. This allows ATP production to proceed at the level of Complex V (ATP synthase). Under normal condi‐ tions, oxidative phosphorylation is a tightly regulated process with heat and reactive oxygen species (ROS) being produced as byproducts.

In the presence of ox‐LDL and other oxidized lipids, the mitochondrion increases the for‐ mation of ROS, which can damage the mitochondria and other organelles causing cellular dysfunction and death. HDL, by virtue of its antioxidant properties, can decrease the cellu‐ lar damage caused by oxidized lipids. The HDL protein PON1 hydrolyzes cholesterol esters and phospholipids in oxidized lipoproteins [52, 57, 58] thus inhibiting mitochondrial damage in the presence of oxidized lipids [58]. Further, HDL‐associated apoA‐I has been implicated in electron transport chain maintenance and repair [59]. In apoA‐I null mice (apoA‐I‐/‐), an increase in coronary ischemia‐reperfusion injury is observed compared to wild‐type mice [59] and is associated with a decrease in the content of the mitochondrial protein Coenzyme Q (CoQ) in cardiomyocytes. CoQ normally supports oxidative phosphorylation by shuttling electrons from Complex II to Complex III. Exogenous administration of CoQ to apo‐A‐I‐/‐ mice attenuated myocardial infarct size compared to the injury response in untreated mice. These data indicate the importance of HDL, and specifically, apoA‐I in preserving mitochondrial structure and function.

Potential mechanisms by which HDL preserves mitochondrial function include activation of the Reperfusion Injury Salvage Kinase (RISK) pathway and the Survivor Activating Factor Enhancement (SAFE) cascade. These are cell survival pathways which are known to prevent mitochondrial damage in models of ischemic pre‐ and postconditioning [60]. Activation of STAT3 is an important component of the SAFE pathway and results in the downregulation of pro‐apoptotic factors Bax and Bad and upregulation of antiapoptotic factor Bcl‐2 and the antioxidants manganese superoxide dismutase and metallothionein [60, 61]. Further, STAT3 is transported to the mitochondrion by the GRIM‐19 chaperone where it inhibits the release of cytochrome c and reduces cell death [62–64]. In a rodent model of coronary artery occlusion, the administration of apoA‐I was shown to decrease infarct size and inhibit mitochondrial morphological changes seen in the heart [60]. Further analyses showed that apoA‐I increased the phosphorylation of *Akt* and glycogen synthase kinase 3 beta (GSK3β), known mediators of the RISK and SAFE survival pathways.

The S1P component of HDL is also able to activate the RISK And SAFE pathways [51, 52, 65]. Interestingly, studies conducted in neonatal rat cardiomyocytes showed that S1P is criti‐ cally required for the phosphorylation of STAT3. In contrast, STAT3 phosphorylation was absent in cells treated with HDL that was deficient in S1P [65]. In addition, S1P stimulates the phosphorylation of the transcription factor, forkhead box O‐1 (FOXO‐1), which inhibits ROS formation and apoptosis in the phosphorylated form [66, 67]. These data suggest that HDL activates RISK and SAFE pathways and inhibits ROS, mitochondrial dysfunction and cell death.

 macrophages (BMDMs) with S1P suppressed LPS‐induced secretion of TNF‐α, monocyte chemoattractant protein (MCP) and IL‐12 [53]. Additionally, Hughes and colleagues reported that S1P enhanced the activity of Arg1 and suppressed the NF‐kB‐mediated induction of

*Regulation of mitochondrial function:* The mitochondrion is a double‐membraned, energy‐pro‐ ducing organelle, which contains its own maternally inherited mitochondrial DNA [54–56]. Under normal conditions, the mitochondrial respiratory chain shuttles electrons through the respiratory complexes, consumes oxygen at Complex IV and pumps hydrogen ions from inside the mitochondria to the intermembrane space at Complexes I, III and IV. This allows ATP production to proceed at the level of Complex V (ATP synthase). Under normal condi‐ tions, oxidative phosphorylation is a tightly regulated process with heat and reactive oxygen

In the presence of ox‐LDL and other oxidized lipids, the mitochondrion increases the for‐ mation of ROS, which can damage the mitochondria and other organelles causing cellular dysfunction and death. HDL, by virtue of its antioxidant properties, can decrease the cellu‐ lar damage caused by oxidized lipids. The HDL protein PON1 hydrolyzes cholesterol esters and phospholipids in oxidized lipoproteins [52, 57, 58] thus inhibiting mitochondrial damage in the presence of oxidized lipids [58]. Further, HDL‐associated apoA‐I has been implicated in electron transport chain maintenance and repair [59]. In apoA‐I null mice (apoA‐I‐/‐), an increase in coronary ischemia‐reperfusion injury is observed compared to wild‐type mice [59] and is associated with a decrease in the content of the mitochondrial protein Coenzyme Q (CoQ) in cardiomyocytes. CoQ normally supports oxidative phosphorylation by shuttling electrons from Complex II to Complex III. Exogenous administration of CoQ to apo‐A‐I‐/‐ mice attenuated myocardial infarct size compared to the injury response in untreated mice. These data indicate the importance of HDL, and specifically, apoA‐I in preserving mitochondrial

Potential mechanisms by which HDL preserves mitochondrial function include activation of the Reperfusion Injury Salvage Kinase (RISK) pathway and the Survivor Activating Factor Enhancement (SAFE) cascade. These are cell survival pathways which are known to prevent mitochondrial damage in models of ischemic pre‐ and postconditioning [60]. Activation of STAT3 is an important component of the SAFE pathway and results in the downregulation of pro‐apoptotic factors Bax and Bad and upregulation of antiapoptotic factor Bcl‐2 and the antioxidants manganese superoxide dismutase and metallothionein [60, 61]. Further, STAT3 is transported to the mitochondrion by the GRIM‐19 chaperone where it inhibits the release of cytochrome c and reduces cell death [62–64]. In a rodent model of coronary artery occlusion, the administration of apoA‐I was shown to decrease infarct size and inhibit mitochondrial morphological changes seen in the heart [60]. Further analyses showed that apoA‐I increased the phosphorylation of *Akt* and glycogen synthase kinase 3 beta (GSK3β), known mediators

The S1P component of HDL is also able to activate the RISK And SAFE pathways [51, 52, 65]. Interestingly, studies conducted in neonatal rat cardiomyocytes showed that S1P is criti‐ cally required for the phosphorylation of STAT3. In contrast, STAT3 phosphorylation was

iNOS [53]. These responses to S1P are associated with M2 macrophage polarization.

species (ROS) being produced as byproducts.

structure and function.

58 Advances in Lipoprotein Research

of the RISK and SAFE survival pathways.

Interestingly, S1P has also been shown to regulate mitochondrial Complex IV assembly and cellular respiration by interacting with mitochondrial prohibitin‐2 (PBH‐2) [68]. PBH‐2 acts as a scaffolding protein for mitochondria and its interaction with S1P during ischemic pre‐ conditioning of cardiomyocytes is essential for cardioprotection [68–70]. These data suggest that S1P can stabilize mitochondrial complexes and inhibit ROS formation, suggesting an alternate cardioprotective mechanism of S1P action.

Recent studies have suggested that other HDL‐associated apolipoproteins play a role in pre‐ serving mitochondrial structure and function. ApoJ is expressed ubiquitously and is present on small dense HDL3 particles [71–73]. It is considered to be an antioxidant due to the pres‐ ence of disulfide bonds that inhibit ROS‐induced injury and preserve mitochondrial function [74]. Further, apoJ has been implicated in activating *Akt* and GSK3β and the RISK survival pathway [71]. ApoM is found in association with approximately 5% of HDL particles where it confers several cytoprotective properties that include stimulating preβ‐HDL formation, facilitating reverse cholesterol transport and inhibiting LDL oxidation [75–78]. ApoM also plays an important role in the cytoprotective response to S1P by binding the sphingolipid and facilitating its incorporation into HDL particles [75, 79, 80]. It follows that overexpression of apoM in mice reduces infarct size in response to ischemia‐reperfusion injury and preserves mitochondrial function by increasing the HDL content of S1P.

## **4. Inflammation‐induced alterations in HDL structure**

Changes in HDL sub‐species and their function have been reported in several disease states, including atherosclerosis [4], rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) [81, 82], diabetes [83], hypertension [84] and psoriasis [85–87]. Inflammation/infection triggers an APR that causes a reduction in HDL quantity and alterations in both its lipid and protein composition. Van Lenten and colleagues [88] first reported that HDL loses its ability to inhibit LDL oxidation during the APR, demonstrating that inflammation affects the structure and function of HDL.

*Lipidome alterations***:** The phospholipid content of HDL is altered during the APR [89]. This may be due to an increase in the activity of secretory phospholipase 2 (sPLA2 ) [90, 91]. Acute phase HDL also contains lower amounts of PE and PI along with several species of LPC with different levels of saturation. An important feature of acute phase HDL is that it contains oxidized phospholipids generated by the actions of transition metal ions, free radicals and hypochlorous acid (HOCl) [92, 93]. Formation of acute phase HDL in patients with coronary heart disease is also associated with a reduction in SM content [94]. An increase in triglycer‐ ides with a decrease in cholesteryl esters is also commonly observed in acute phase HDL [89].

*Proteome alterations:* Several changes in HDL‐associated proteins arise in response to inflam‐ mation (**Table 2**). While a reduction in apo A‐I represents perhaps the most prominent change in HDL composition, data suggest that the lipoprotein content of SAA may increase up to 1000‐ fold [85]. Endotoxin and inflammatory cytokines (TNF‐α, IL‐1β and IL‐6) decrease the expres‐ sion of apoA‐I which leads to a decrease in circulating HDL concentration [95, 96]. In addition, an increase in the synthesis of SAA results in the displacement of apoA‐I from acute phase HDL [85]. Inflammation further decreases HDL levels by inducing the upregulation of sPLA<sup>2</sup> which degrades phospholipid components of the lipoprotein particle [89]. Loss of LCAT activity [97, 98] reduces the cholesterol carrying capacity of HDL by preventing the formation of cholesterol esters. Finally, PON1 activity is reduced by inflammation in patients with RA, SLE and psoriasis and infections and is associated with a reduction in the antioxidant capacity of HDL [99–102].

**Table 2.** Inflammation‐induced changes in HDL composition.

The presence of apoM in HDL particles is thought to contribute to atheroprotection [103]. LPS and inflammatory cytokines, however, attenuate apoM mRNA levels and protein expression in Hep3B cells [104]. A decrease in serum apoM is also observed in patients with sepsis and HIV infections [104]. Further, a reduction in apoM reduces the association of S1P with HDL resulting in degradation of anti‐inflammatory function [103].

The association of other apolipoproteins with HDL may impair the function of the lipopro‐ tein. ApoO is incorporated by HDL, LDL and VLDL particles [105]. Data suggest that apoO provides structural stability for mitochondria by stabilizing the inner mitochondrial mem‐ brane and cristae [105]. Other data, however, show that overexpression of apoO degrades mitochondrial protein and increases cardiac dysfunction in hypercholesterolemic mice [106]. In cardiomyocyte cultures, upregulation of apoO was associated with an increase in ROS and apoptosis compared to control cells that were apoO‐deficient [106]. ApoC is an additional, exchangeable apolipoprotein associated with HDL and apoB‐containing lipoproteins. In iso‐

lated rat liver mitochondria, addition of the apoC‐III isoform was shown to inhibit mitochon‐ drial oxygen consumption and attenuate ATP formation [107]. Another study showed that enrichment of HDL with apoC‐I stimulates cytochrome c release, caspase 3 cleavage and cell death in human aortic smooth muscle cells [108]. Finally, apoC‐I enrichment of HDL is associ‐ ated with a reduction in HDL‐associated apoA‐I, suggesting that loss of apoA‐I and its cyto‐ protective effects is a component of apoC‐I‐mediated cell injury [107, 108]. Clearly, additional in vitro and in vivo studies are required to define the mechanistic role of specific apolipopro‐ tein species in the development of inflammatory injury.

## **5. Functional consequences of acute phase HDL formation**

Changes in HDL lipid and protein composition induced by the APR impair normal HDL function resulting in the formation of "dysfunctional" HDL.

*Loss of cholesterol efflux ability:* Since cholesterol efflux involves the participation of apoA‐I, phospholipids, LCAT and CETP, several aspects of dysfunctional HDL inhibit normal reverse cholesterol transport. The reduction in apoA‐I and increase in HDL‐associated SAA impair cholesterol efflux capacity [109, 110]. The presence of SAA on HDL increases foam cell forma‐ tion by facilitating the uptake of cholesterol esters by macrophages. At the level of the hepa‐ tocyte, this acute phase HDL impairs cholesterol uptake and degradation [111]. Decreased content of LCAT, PL and CETP on HDL also contribute to a loss of efflux activity as does the oxidative modification of apoA‐I [112, 113].

*Impairment of antioxidative activity:* An increase in TG and decrease in cholesterol ester content in dysfunctional HDL leads to a change in conformation of the HDL particle. The formation of a TG‐rich HDL particle induces structural changes in apoA‐I and decreases its stability [114]. Additionally, an increase in SAA and loss of PON1 result in a reduced antioxi‐ dant capacity of the HDL particle.

*Attenuation of anti‐inflammatory activity:* Dysfunctional HDL has an impaired capacity to counteract the action of LPS and inflammatory cytokines. The ability to regulate membrane raft cholesterol content is reduced and can thus enhance TLR activation in response to pro‐ inflammatory mediators [115]. Oxidation of apoA‐I also results in a loss of functionality with respect to its ability to efflux cholesterol. The protein and lipid alterations observed (reduced apoA‐I, cholesterol ester, PON1 and LCAT levels and increased TG and SAA levels) are also responsible for the attenuated anti‐inflammatory activity observed with dysfunctional HDL.

## **6. Conclusions**

*Proteome alterations:* Several changes in HDL‐associated proteins arise in response to inflam‐ mation (**Table 2**). While a reduction in apo A‐I represents perhaps the most prominent change in HDL composition, data suggest that the lipoprotein content of SAA may increase up to 1000‐ fold [85]. Endotoxin and inflammatory cytokines (TNF‐α, IL‐1β and IL‐6) decrease the expres‐ sion of apoA‐I which leads to a decrease in circulating HDL concentration [95, 96]. In addition, an increase in the synthesis of SAA results in the displacement of apoA‐I from acute phase HDL

[85]. Inflammation further decreases HDL levels by inducing the upregulation of sPLA<sup>2</sup>

degrades phospholipid components of the lipoprotein particle [89]. Loss of LCAT activity [97, 98] reduces the cholesterol carrying capacity of HDL by preventing the formation of cholesterol esters. Finally, PON1 activity is reduced by inflammation in patients with RA, SLE and psoriasis and infections and is associated with a reduction in the antioxidant capacity of HDL [99–102].

**Increased Decreased Increased Decreased** Serum Amyloid A (SAA) Apo A‐I Triglycerides Total lipid Apo J Apo A‐II FC Phospholipids

sPLA2 Apo C LPC CE Apo E Apo M FFA SM

The presence of apoM in HDL particles is thought to contribute to atheroprotection [103]. LPS and inflammatory cytokines, however, attenuate apoM mRNA levels and protein expression in Hep3B cells [104]. A decrease in serum apoM is also observed in patients with sepsis and HIV infections [104]. Further, a reduction in apoM reduces the association of S1P with HDL

The association of other apolipoproteins with HDL may impair the function of the lipopro‐ tein. ApoO is incorporated by HDL, LDL and VLDL particles [105]. Data suggest that apoO provides structural stability for mitochondria by stabilizing the inner mitochondrial mem‐ brane and cristae [105]. Other data, however, show that overexpression of apoO degrades mitochondrial protein and increases cardiac dysfunction in hypercholesterolemic mice [106]. In cardiomyocyte cultures, upregulation of apoO was associated with an increase in ROS and apoptosis compared to control cells that were apoO‐deficient [106]. ApoC is an additional, exchangeable apolipoprotein associated with HDL and apoB‐containing lipoproteins. In iso‐

resulting in degradation of anti‐inflammatory function [103].

**Table 2.** Inflammation‐induced changes in HDL composition.

**Proteinsa Lipidsb**

Ceruloplasmin LCAT PAF‐AH CETP LBP Transferrin Apo A‐IV Hepatic lipase Apo A‐V Paraoxanase I

Adapted from Refs. [87, 96, 97, 104].

Adapted from Refs. [15, 89, 94].

60 Advances in Lipoprotein Research

a

b

which

HDL plays an important role in regulating atherogenesis *via* its ability to mediate reverse cholesterol transport. The ability of HDL to reduce inflammatory injury and oxidant stress has also been shown to reduce CVD risk. As discussed in this review, both protein and lipid components of the lipoprotein particle play critical roles in attenuating inflammation. Identification of these cytoprotective HDL components has been facilitated by recent pro‐ teomic analyses. Under pathological conditions, HDL levels may be reduced and the lipo‐ protein may undergo biochemical and structural modification resulting in the formation of dysfunctional HDL with pro‐inflammatory properties. It has been suggested that the anti‐ inflammatory status of HDL may be of greater predictive value for CVD risk than HDL levels per se [116, 117]. Therapeutic approaches that increase the functional properties of HDL may thus be superior to simply raising circulating HDL. Unfortunately, specific and reliable bio‐ markers for anti‐inflammatory HDL have not been identified. Under ex vivo conditions, the quality of HDL can be assessed by studying lipoprotein effects of processes such as mono‐ cyte chemotaxis and endothelial inflammation. These assays, however, are cumbersome and time‐consuming. Despite these drawbacks, there is significant interest in developing new pharmacotherapies that positively impact circulating lipoproteins. Randomized clinical tri‐ als have assessed effects of several classes of drugs on plasma cholesterol levels in patients at risk. Niacin and statins significantly lower LDL and were shown to induce modest increases in HDL [8]. Residual risk, however, may be present in patients with persistently low HDL despite a reduction in LDL. CETP inhibitors have been shown to increase HDL levels in ani‐ mal models and in human subjects with low HDL [118, 119]. The ILLUMINATE trial tested effects of the CETP inhibitor torcetrapib on HDL and outcomes in high risk patients but was terminated early due to an increase in mortality due to off‐target effects [7]. In ongoing stud‐ ies, the antiatherogenic and anti‐inflammatory effects of reconstituted HDL therapy as well as apolipoprotein mimetics are being evaluated. Recent exciting data also show that HDL serves as a carrier for functional miRNAs that suppress inflammation at the level of the endothelial cell [120]. miRNAs have also been identified that regulate HDL biogenesis [121]. These recent observations may lay the foundation for a new field of miRNA‐based HDL therapeutics.

## **Author details**

Roger White1 , Samantha Giordano<sup>1</sup> and Geeta Datta<sup>2</sup> \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Medicine, University of Alabama at Birmingham, USA

2 Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

## **References**

[3] Gordon DJ, Rifkind BM. High‐density lipoprotein—the clinical implications of recent studies. The New England Journal of Medicine. 1989;321:1311–6.

Identification of these cytoprotective HDL components has been facilitated by recent pro‐ teomic analyses. Under pathological conditions, HDL levels may be reduced and the lipo‐ protein may undergo biochemical and structural modification resulting in the formation of dysfunctional HDL with pro‐inflammatory properties. It has been suggested that the anti‐ inflammatory status of HDL may be of greater predictive value for CVD risk than HDL levels per se [116, 117]. Therapeutic approaches that increase the functional properties of HDL may thus be superior to simply raising circulating HDL. Unfortunately, specific and reliable bio‐ markers for anti‐inflammatory HDL have not been identified. Under ex vivo conditions, the quality of HDL can be assessed by studying lipoprotein effects of processes such as mono‐ cyte chemotaxis and endothelial inflammation. These assays, however, are cumbersome and time‐consuming. Despite these drawbacks, there is significant interest in developing new pharmacotherapies that positively impact circulating lipoproteins. Randomized clinical tri‐ als have assessed effects of several classes of drugs on plasma cholesterol levels in patients at risk. Niacin and statins significantly lower LDL and were shown to induce modest increases in HDL [8]. Residual risk, however, may be present in patients with persistently low HDL despite a reduction in LDL. CETP inhibitors have been shown to increase HDL levels in ani‐ mal models and in human subjects with low HDL [118, 119]. The ILLUMINATE trial tested effects of the CETP inhibitor torcetrapib on HDL and outcomes in high risk patients but was terminated early due to an increase in mortality due to off‐target effects [7]. In ongoing stud‐ ies, the antiatherogenic and anti‐inflammatory effects of reconstituted HDL therapy as well as apolipoprotein mimetics are being evaluated. Recent exciting data also show that HDL serves as a carrier for functional miRNAs that suppress inflammation at the level of the endothelial cell [120]. miRNAs have also been identified that regulate HDL biogenesis [121]. These recent observations may lay the foundation for a new field of miRNA‐based HDL therapeutics.

**Author details**

62 Advances in Lipoprotein Research

, Samantha Giordano<sup>1</sup>

Birmingham, Birmingham, AL, USA

\*Address all correspondence to<EMAIL_ADDRESS>
Climatological Association. 2011;122:256–89.

Pathology Supplement. 1973;5:26–31.

and Geeta Datta<sup>2</sup>

2 Division of Cardiovascular Disease, Department of Medicine, University of Alabama at

[1] Gotto AM, Jr. Jeremiah Metzger Lecture: cholesterol, inflammation and atheroscle‐ rotic cardiovascular disease: is it all LDL? Transactions of the American Clinical and

[2] Lewis B. Classification of lipoproteins and lipoprotein disorders. Journal of Clinical

1 Department of Medicine, University of Alabama at Birmingham, USA

\*

Roger White1

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**Provisional chapter**

## **Plasma Lipoproteins as Crucial Components of Host Defence Against Infections Host Defence Against Infections**

**Plasma Lipoproteins as Crucial Components of**

Kaustubh Bora and Probodh Borah Kaustubh Bora and Probodh Borah Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67601

#### **Abstract**

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Communications. 2014;5:3292.

72 Advances in Lipoprotein Research

function. Circulation Research. 2014;114:183–92.

Interactions between lipoproteins and infectious microorganisms are diverse and often multifaceted. There is a growing body of evidence which suggests that circulating plasma lipoproteins play an important role in warding off various infections. They are increasingly recognized as vital components of the host immune system. The purpose of this chapter is to provide the reader with an overview of this emerging domain. We review the anti-infective role of different lipoprotein particles and their components and further highlight the known molecular mechanisms involved therein. Instances where lipoproteins facilitate infections instead of protecting against them are also summarized. Finally, broad implications for the future in this active line of research are discussed.

**Keywords:** lipoproteins, apolipoproteins, lipids, infection, immune system

## **1. Introduction**

Circulating lipoproteins are macromolecular complexes of lipids and specific proteins (known as apolipoproteins). They facilitate the transport and distribution of various lipids (such as cholesterol, cholesteryl esters, triglycerides, and phospholipids) via blood throughout the body. Owing to their hydrophobicity, they are otherwise sparingly soluble in the predominantly aqueous plasma [1]. Scientific work on plasma lipoproteins has historically focused on their role in atherosclerotic changes and cardiovascular health. Much of the impetus in this line of enquiry was provided by the Framingham Heart Study (FHS) that was started in 1948 by the National Heart Institute (NHI). The FHS and a number of large clinico-epidemiological studies thereafter have been instrumental in advancing our knowledge about the link between circulating lipoproteins and cardiovascular health [1–5]. There is a growing body

of evidence suggesting that plasma lipoproteins are crucial players in a host of other conditions as well, viz. neurodegeneration [6], psychiatric ailments [7], and various cancers [8, 9], to name a few.

Although the earliest reports about the relationship of lipids and lipoproteins with various infections date back to 1940s and 1950s [10–12], yet the interest on lipoproteins for a long time was mostly revolved around noncommunicable disorders. However, in a marked departure from this conventional outlook, the importance of circulating lipoproteins in relation to infectious diseases is now widely recognized. Perhaps the best example in this regard is the study of the role of high-density lipoproteins (HDL) particles in conferring immunity against *Trypanosoma brucei brucei* in humans [13]. This change in the outlook is probably due to the fact that derangements (quantitative as well as qualitative) in plasma lipoproteins that were earlier documented in a variety of infections, viz. bacterial, viral, and parasitic [14–16] have now been corroborated by experimental evidence as well [17–19], such that there is an improved understanding of the underlying mechanisms at a molecular level.

Lipoproteins represent structurally and functionally a very diverse species of complex particles with dynamic interactions that travel throughout the body through circulation. Thus, they are increasingly appreciated as components of the innate immune system [15, 17, 20]. Recent evidence suggests that lipoproteins are also involved in adaptive immune responses [21]. On the basis of difference in hydrated densities (as determined by their rate in sedimentation on ultracentrifugation in salt solutions), human plasma proteins have been traditionally divided into four major groups—chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) [1, 4, 5]. Apart from these four major groups, sometimes, other lipoprotein classes are also described, such as intermediate-density lipoproteins (IDL)—produced by catabolism of VLDL, and lipoprotein(a) [Lp(a)]—which is structurally similar to LDL and has a density range that overlaps that of HDL [1, 4, 5]. Many of these lipoprotein particles or their components (e.g., lipids or apolipoproteins) have been found to exert anti-infective role.

This chapter aims to review this emerging domain where plasma lipoproteins are now widely recognized as important players of the host immune system. We have summarized the different types of lipoprotein derangements during various infections, the anti-infective role of lipoproteins in conferring protection against pathogens, and the known molecular mechanisms involved therein.

## **2. Lipoproteins in relation to various infections**

Lipoprotein derangements and infections appear to have a bidirectional relationship. This means that alterations in circulatory lipoproteins can modulate or predispose to infections, and conversely, alterations in circulating lipoproteins can be an outcome of the infections themselves. In other words, lipoprotein derangements can both be a contributory cause and a resultant effect of infections. The focus of this chapter is predominantly on the former relationship which underscores the role of lipoproteins in modulating susceptibility to infections. The latter relationship is linked to the active phase of the infection and has been recorded in relation to several kinds of infectious agents [10–12, 22–24]. Such lipoprotein derangements are a part of the acute-phase responses (APR) mounted by the body and are beyond the scope of this chapter. Similarly, lipoprotein derangements can occur as a result of drug therapy against infections (e.g., dyslipidemia in HIV-AIDS patients due to anti-retroviral therapy) [25–28] and are outside the purview of this chapter too.

Generally speaking, most of the experimental and clinical evidences suggest that high levels of lipoproteins and lipids are protective against respiratory and gastrointestinal infections [29–31]. Case studies in homogenous populations residing in high-infection environments affirm this view. For instance, the Tsimane people of Bolivian Amazon have very high burden of infection, and this is often attributed to the low levels of lipids and lipoproteins [32]. Likewise, in the Shipibo people, another indigenous group in the Amazon, the density of parasitic infection correlates inversely to the HDL levels [33]. Further, reduced levels of apolipoproteins in hospital-based studies have been reported to be associated with increased susceptibility to nosocomial infections following severe trauma [34]. However, generalizations are difficult and exceptions to these trends have also been reported [28], and the mechanism involved is not clear.

In the account that follows, we give an overview of different infections where lipoproteins provide protection.

### **2.1. Viral infections**

of evidence suggesting that plasma lipoproteins are crucial players in a host of other conditions as well, viz. neurodegeneration [6], psychiatric ailments [7], and various cancers [8, 9],

Although the earliest reports about the relationship of lipids and lipoproteins with various infections date back to 1940s and 1950s [10–12], yet the interest on lipoproteins for a long time was mostly revolved around noncommunicable disorders. However, in a marked departure from this conventional outlook, the importance of circulating lipoproteins in relation to infectious diseases is now widely recognized. Perhaps the best example in this regard is the study of the role of high-density lipoproteins (HDL) particles in conferring immunity against *Trypanosoma brucei brucei* in humans [13]. This change in the outlook is probably due to the fact that derangements (quantitative as well as qualitative) in plasma lipoproteins that were earlier documented in a variety of infections, viz. bacterial, viral, and parasitic [14–16] have now been corroborated by experimental evidence as well [17–19], such that there is an

Lipoproteins represent structurally and functionally a very diverse species of complex particles with dynamic interactions that travel throughout the body through circulation. Thus, they are increasingly appreciated as components of the innate immune system [15, 17, 20]. Recent evidence suggests that lipoproteins are also involved in adaptive immune responses [21]. On the basis of difference in hydrated densities (as determined by their rate in sedimentation on ultracentrifugation in salt solutions), human plasma proteins have been traditionally divided into four major groups—chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) [1, 4, 5]. Apart from these four major groups, sometimes, other lipoprotein classes are also described, such as intermediate-density lipoproteins (IDL)—produced by catabolism of VLDL, and lipoprotein(a) [Lp(a)]—which is structurally similar to LDL and has a density range that overlaps that of HDL [1, 4, 5]. Many of these lipoprotein particles or their components (e.g., lipids or apolipoproteins) have been

This chapter aims to review this emerging domain where plasma lipoproteins are now widely recognized as important players of the host immune system. We have summarized the different types of lipoprotein derangements during various infections, the anti-infective role of lipoproteins in conferring protection against pathogens, and the known molecular mecha-

Lipoprotein derangements and infections appear to have a bidirectional relationship. This means that alterations in circulatory lipoproteins can modulate or predispose to infections, and conversely, alterations in circulating lipoproteins can be an outcome of the infections themselves. In other words, lipoprotein derangements can both be a contributory cause and a resultant effect of infections. The focus of this chapter is predominantly on the former relationship which underscores the role of lipoproteins in modulating susceptibility to infections.

improved understanding of the underlying mechanisms at a molecular level.

to name a few.

74 Advances in Lipoprotein Research

found to exert anti-infective role.

**2. Lipoproteins in relation to various infections**

nisms involved therein.

Lipoproteins, particularly HDL particles, have been found to account for part of the broad nonspecific antiviral activity of human serum [35, 36]. Such antiviral activity of lipoproteins has been detected across a wide spectrum of enveloped as well as nonenveloped DNA and RNA viruses. Examples include Rabies virus, Rubella virus, Japanese encephalitis virus (JEV), Poliovirus, Epstein-Barr virus (EBV), Herpes simplex virus (HSV), Vaccinia virus, New Castle disease virus, and Vesicular stomatitis virus (VSV), to name a few [35, 37–41]. This is in tune with the protection conferred by other components of the innate immune system, which are often nonspecific and broad-based. However, some lipoproteins (e.g., LDL and VLDL) have been found to be particularly active against certain viruses (e.g., JEV, Rubella, Rabies, and VSV) [35, 38, 40].

#### **2.2. Bacterial infections**

Lipoproteins are protective against several toxins produced by pathogenic bacteria. Lipoproteins can neutralize lipopolysaccharides (LPS) from Gram-negative bacteria [30, 36, 42]. LPS are implicated in complications of Gram-negative bacteraemia such as endotoxic shock and disseminated intravascular coagulation. Several classes of lipoproteins, such as LDL, VLDL, HDL, Lp(a), and chylomicrons, can potentially help in neutralizing LPS [30, 36, 43–45]. In fact, infusion of reconstituted HDL particles (rHDL) has been shown to protect against Gram-negative bacteraemia and endotoxic shock and to further blunt the LPS-induced unregulated activation of the coagulation cascade [46–48].

Lipoproteins are protective against Gram-positive organisms too. Lipoproteins have been shown to inactivate lipotechoic acid (LTA) and alpha-toxin from Gram-positive bacteria such as *Staphylococcus aureus* [30, 36, 49].

In addition to these toxin-neutralizing effects, lipoproteins can directly interact with cell surface virulence factors in bacteria and help in limiting their pathogenicity. Such interactions have been noted in infections by *Yersinia pestis* [50] and many Group A *Streptococcus* (GAS) strains [30, 51]. Besides, experiments in knockout animals have revealed that apoE-/- mice are susceptible to infection by *Listeria monocytogenes* and *Mycobacterium tuberculosis* [52, 53].

#### **2.3. Parasitic infections**

Humans are immune to infection by the parasite *Trypanosoma brucei brucei*. This protection is attributed to a subset of HDL particles called trypanosome lytic factors (TLFs), present in human serum [13]. TLFs have also been shown to ameliorate infection by *Leishmania* sp. [15]. However, this protection does not extend against other trypanosomes such as *Trypanosoma cruzi, Trypanosoma brucei rhodesiense*, and *Trypanosoma brucei gambiense* [13]. Lipoproteins are suggested to modulate the infectivity of malaria parasite and *Schistosoma* as well [36, 54–56].

## **3. Anti-infective mechanisms of lipoproteins**

The biological mechanisms for the anti-infective role of lipoproteins are diverse. While, in some instances, the complete ensemble of a lipoprotein particle has been found to contribute to the immunological defenses, in some other occasions the individual constituents (such as apoproteins or lipid moieties such as phospholipids and cholesterol) are credited to be involved [36, 41, 57]. A broad scheme of the anti-infective mechanisms with respect to circulating lipoproteins is provided below. Experimental evidences from in vivo and in vitro studies suggest that these schemes are actually recurring themes. These strategies are common to a variety of antimicrobial defenses mobilized by circulating lipoproteins against a plethora of infectious agents.

#### **3.1. Inhibiting the entry of intracellular pathogens into host cells**

Lipoproteins can inhibit the attachment and subsequent entry of pathogens into their target cells. This defensive mechanism of lipoproteins has been particularly well described in relation to viruses. The presence of HDL is capable of retaining viruses on the cell surface, lending credibility to this idea. Apoproteins (such as apoA-I) in host circulatory lipoproteins contain stretches of amphipathic residues that have been proposed to interact with amphipathic counterparts in alpha-helices of viral envelope glycoproteins. These interactions interfere with membrane fusion and entry of viruses into host cells. Synthetic amphipathic peptide analogues of apoA-I can also exert similar effects [36, 58, 59]. In fact, such analogues have been found to inhibit HIV-induced syncytium formation [60]. Inhibition of viral penetration inside host cells is also supported by VLDL. Recent in vivo studies have revealed that VLDL in serum effectively blocks hepatitis C virus (HCV) cell attachment, thereby acting as a restriction factor against HCV infection [18]. Clinical studies have earlier revealed that serum level of apoC-III (an integral apolipoprotein in VLDL) was a significant predictor of chronic hepatitis C infection and associated hepatic fibrosis [16].

Alternatively, viral infection stimulates production of interferons, which in turn induce secretion of some soluble forms of lipoprotein receptors. These soluble receptors can modulate viral pathogenesis. For instance, a soluble LDL receptor shed during hepatitis and rhinovirus infections is used by the viruses for gaining entry into their respective host target cells [36]. However, endogenous LDL competes with these viruses or such similar viruses for cellular uptake, protecting the host against infection. Such receptors are also implicated in virus assembly and budding [61, 62]. Likewise, a VLDL receptor fragment that binds rhinoviruses has also been described in cell culture studies [63].

In addition to viruses, circulating lipoproteins have been found to prevent the entry of nonviral intracellular pathogens. For example, lipoproteins can interfere with the adhesion of *Salmonella typhimurium* to host cells and subsequent organ invasion [64].

## **3.2. Inactivating the effect of microbial toxins**

Lipoproteins are protective against Gram-positive organisms too. Lipoproteins have been shown to inactivate lipotechoic acid (LTA) and alpha-toxin from Gram-positive bacteria such

In addition to these toxin-neutralizing effects, lipoproteins can directly interact with cell surface virulence factors in bacteria and help in limiting their pathogenicity. Such interactions have been noted in infections by *Yersinia pestis* [50] and many Group A *Streptococcus* (GAS) strains [30, 51]. Besides, experiments in knockout animals have revealed that apoE-/- mice are susceptible to infection by *Listeria monocytogenes* and *Mycobacterium tuberculosis* [52, 53].

Humans are immune to infection by the parasite *Trypanosoma brucei brucei*. This protection is attributed to a subset of HDL particles called trypanosome lytic factors (TLFs), present in human serum [13]. TLFs have also been shown to ameliorate infection by *Leishmania* sp. [15]. However, this protection does not extend against other trypanosomes such as *Trypanosoma cruzi, Trypanosoma brucei rhodesiense*, and *Trypanosoma brucei gambiense* [13]. Lipoproteins are suggested to modulate the infectivity of malaria parasite and *Schistosoma* as well [36, 54–56].

The biological mechanisms for the anti-infective role of lipoproteins are diverse. While, in some instances, the complete ensemble of a lipoprotein particle has been found to contribute to the immunological defenses, in some other occasions the individual constituents (such as apoproteins or lipid moieties such as phospholipids and cholesterol) are credited to be involved [36, 41, 57]. A broad scheme of the anti-infective mechanisms with respect to circulating lipoproteins is provided below. Experimental evidences from in vivo and in vitro studies suggest that these schemes are actually recurring themes. These strategies are common to a variety of antimicrobial defenses mobilized by circulating lipoproteins against a plethora of infectious agents.

Lipoproteins can inhibit the attachment and subsequent entry of pathogens into their target cells. This defensive mechanism of lipoproteins has been particularly well described in relation to viruses. The presence of HDL is capable of retaining viruses on the cell surface, lending credibility to this idea. Apoproteins (such as apoA-I) in host circulatory lipoproteins contain stretches of amphipathic residues that have been proposed to interact with amphipathic counterparts in alpha-helices of viral envelope glycoproteins. These interactions interfere with membrane fusion and entry of viruses into host cells. Synthetic amphipathic peptide analogues of apoA-I can also exert similar effects [36, 58, 59]. In fact, such analogues have been found to inhibit HIV-induced syncytium formation [60]. Inhibition of viral penetration inside host cells is also supported by VLDL. Recent in vivo studies have revealed that VLDL in serum effectively blocks hepatitis C virus (HCV) cell attachment, thereby acting as a restriction factor

as *Staphylococcus aureus* [30, 36, 49].

**3. Anti-infective mechanisms of lipoproteins**

**3.1. Inhibiting the entry of intracellular pathogens into host cells**

**2.3. Parasitic infections**

76 Advances in Lipoprotein Research

Lipoproteins effectively neutralize bacterial toxins such as LPS (from Gram-negative bacteria) and LTA (from Gram-positive bacteria) and enhance their clearance. The mechanisms involved in inactivating LPS are particularly well established [30, 36, 42–45, 65, 66]. The lipid components of lipoproteins are vital in this regard. Ultrastructural studies have shown that LPS binding with LDL causes fatty acyl chain of crucial lipid moieties in LPS to be incorporated into the phospholipid surface of lipoproteins. This masks the active sites of LPS and attenuates their toxic action [36, 67].

Binding with lipoproteins also enhances the clearance of LPS. During Gram-negative bacteremia, LPS released in the circulation is primarily taken up by macrophages in liver (Kupffer cells). The macrophages thus activated cause a splurge of pro-inflammatory cytokines, which are responsible for the LPS-induced septic shock. However, binding of LPS with lipoproteins prevents this and causes two-pronged benefits. Firstly, on binding with lipoproteins, the uptake of LPS by hepatic macrophages decreases, which prevents their activation and cytokine release [36, 68–70]. Lipoproteins can prevent the LPS-mediated activation and release of cytokines from peripheral monocytes/macrophages too. Lipoproteins have been found to promote the release of LPS from the cell surface of monocytes to which they were bound, further dampening the cellular response [36, 71]. Secondly, the lipoprotein bound LPS are instead taken up by hepatocytes that lead to their rapid secretion into bile [36, 68–70]. Triglyceriderich lipoproteins such as chylomicrons and VLDL are especially active in accelerating the clearance of LPS in this fashion [36, 68].

In a somewhat analogous manner, lipoproteins are believed to neutralize the toxic effects of LTA [72]. Further, potent peptide toxins such as phenol-soluble modulins (PSM) secreted by bacteria such as *Staphylococcus aureus* can also be inactivated by lipoproteins such as HDL, LDL, and VLDL. Highest binding and neutralizing potentials of *Staphylococcal* PSMs are displayed by HDL [17].

## **3.3. Lysis of pathogens**

Certain pathogens are directly lysed by plasma lipoproteins or their components. A good example of this is the lysis of the parasite *Trypanosoma brucei brucei* [13, 36]. This lipoproteinmediated lysis is attributed to two distinct trypanosome lytic factors (TLFs), namely TLF1 and TLF2. TLF1 is actually a lipid-rich subset of HDL that contains mostly apoA-I and haptoglobulin-related protein (HRP) with some amount of other proteins such as apoA-II, apoL-I, and paraoxonase. On the other hand, TLF2 is lipid-poor lipoprotein complex that contains apoA-I, HRP, and immunoglobulin M [73–75]. It is believed that apoL-I and HRP in TLFs target the parasites within the acidic parasitophorous vacuoles of macrophages and damage them directly without taking recourse to macrophage activation [15]. It is noteworthy that *Trypanosoma cruzi*, a trypanosome to which humans are susceptible, cleaves apoA-I, the chief protein constituent of HDL using cruzipain, a cysteine protease present in the cell membrane as well as internal lysosomal structure of the parasite [76]. Such targeted breakdown of vital lipoprotein constituents may aid the *Trypanosoma cruzi* parasite in evading the anti-parasitic action of TLFs.

#### **3.4. Promoting opsonization**

Experiments involving in vitro and ex vivo systems have suggested that some lipoproteins such as LDL may act as opsonins and enhance phagocytosis of several types of Group A *Streptococcus* (GAS) bacteria by monocytes. Interaction of LDL with CD36 scavenger receptor expressed in monocytes and streptococcal collagen such as protein 1 (Scl1) present on the cell surface of GAS is believed to underlie this phagocytosis promoting activity [19].

#### **3.5. Activation of complement system**

Lipid-free and HDL-associated apoA-I can activate the host complement pathways which is effective in killing the gastrointestinal pathogen, *Yersinia enterocolitica*. The C-terminal domain of apoA-I is the primary effector site responsible for this bactericidal property [77].

#### **3.6. Inhibition of plasminogen recruitment**

Many pathogens recruit human plasminogen (which is an integral part of the fibrinolytic system) in the course of their pathogenesis. This helps them in penetrating tissue barriers and facilitate invasion. Some pathogens even secrete plasminogen activators to amplify the effect. For example, streptokinase produced by GAS is a highly specific activator for plasminogen. Thus, it is believed that many infections can be inhibited and prevented considerably if recruitment and activation of host plasminogen by pathogens can be blocked. Lp(a) is believed to be a vital component of the host defense system in this context. Apo(a) present in Lp(a) shares a high degree of homology with plasminogen. Thus, it competes for the binding of plasminogen to pathogens. It reduces the amount of plasminogen immobilized on the pathogen surface and further inhibits the activation of plasminogen by activators such as streptokinase. In vitro studies have demonstrated the inhibition of streptokinase to catalyze the activation of plasminogen. Thus Lp(a) can help in preventing infections and promoting wound healing and repair of tissue injuries [29, 51, 78–81].

## **3.7. Chemical modification of lipoproteins**

**3.3. Lysis of pathogens**

78 Advances in Lipoprotein Research

**3.4. Promoting opsonization**

**3.5. Activation of complement system**

**3.6. Inhibition of plasminogen recruitment**

wound healing and repair of tissue injuries [29, 51, 78–81].

Certain pathogens are directly lysed by plasma lipoproteins or their components. A good example of this is the lysis of the parasite *Trypanosoma brucei brucei* [13, 36]. This lipoproteinmediated lysis is attributed to two distinct trypanosome lytic factors (TLFs), namely TLF1 and TLF2. TLF1 is actually a lipid-rich subset of HDL that contains mostly apoA-I and haptoglobulin-related protein (HRP) with some amount of other proteins such as apoA-II, apoL-I, and paraoxonase. On the other hand, TLF2 is lipid-poor lipoprotein complex that contains apoA-I, HRP, and immunoglobulin M [73–75]. It is believed that apoL-I and HRP in TLFs target the parasites within the acidic parasitophorous vacuoles of macrophages and damage them directly without taking recourse to macrophage activation [15]. It is noteworthy that *Trypanosoma cruzi*, a trypanosome to which humans are susceptible, cleaves apoA-I, the chief protein constituent of HDL using cruzipain, a cysteine protease present in the cell membrane as well as internal lysosomal structure of the parasite [76]. Such targeted breakdown of vital lipoprotein constituents may aid the *Trypanosoma cruzi* parasite in evading the anti-parasitic action of TLFs.

Experiments involving in vitro and ex vivo systems have suggested that some lipoproteins such as LDL may act as opsonins and enhance phagocytosis of several types of Group A *Streptococcus* (GAS) bacteria by monocytes. Interaction of LDL with CD36 scavenger receptor expressed in monocytes and streptococcal collagen such as protein 1 (Scl1) present on the cell

Lipid-free and HDL-associated apoA-I can activate the host complement pathways which is effective in killing the gastrointestinal pathogen, *Yersinia enterocolitica*. The C-terminal domain

Many pathogens recruit human plasminogen (which is an integral part of the fibrinolytic system) in the course of their pathogenesis. This helps them in penetrating tissue barriers and facilitate invasion. Some pathogens even secrete plasminogen activators to amplify the effect. For example, streptokinase produced by GAS is a highly specific activator for plasminogen. Thus, it is believed that many infections can be inhibited and prevented considerably if recruitment and activation of host plasminogen by pathogens can be blocked. Lp(a) is believed to be a vital component of the host defense system in this context. Apo(a) present in Lp(a) shares a high degree of homology with plasminogen. Thus, it competes for the binding of plasminogen to pathogens. It reduces the amount of plasminogen immobilized on the pathogen surface and further inhibits the activation of plasminogen by activators such as streptokinase. In vitro studies have demonstrated the inhibition of streptokinase to catalyze the activation of plasminogen. Thus Lp(a) can help in preventing infections and promoting

surface of GAS is believed to underlie this phagocytosis promoting activity [19].

of apoA-I is the primary effector site responsible for this bactericidal property [77].

Infections and the associated inflammatory responses lead to oxidative stress and generation of reaction oxygen species (ROS). ROS induces chemical modifications in several lipoprotein species, most notable of which is oxidative changes in LDL [82]. Oxidized LDL (oxLDL) contributes to immune responses against invading pathogens in several ways. OxLDL upregulates scavenger receptor expression in macrophages, which facilitates their ingestion of Gram-positive and Gram-negative bacteria by phagocytosis. One of the oxidized components in oxLDL, namely oxidized 1-palmitoyl-2-arachidonyl-*sn*-glycero-3-phosphorylcholine (oxPAPC), modulates LPS-mediated signaling pathways in favor of the host. It inhibits LPS-induced adhesion of neutrophils to endothelial cells (thereby limiting LPS-induced tissue damage) and checks unregulated pro-inflammatory pathways [30, 82–86]. Besides, oxLDL has been shown to block cellular entry by several HCV strains [87] and malarial sporozoites [88].

Further, oxLDL elicits the production of natural antibodies against the membrane phospholipid, phosphorylcholine (PC). These anti-PC antibodies may target PC epitopes present in a broad spectrum of pathogens and provide protection against them. These include Grampositive bacteria, Gram-negative bacteria, trematodes, nematodes, and even fungi [30, 89–93].

#### **3.8. Acting in concert with acute-phase responses**

The acute-phase response (APR), characterized by acute specific changes in concentration of plasma proteins, in response to noxious stimuli (such as infection) serves to protect the host from further injury. It helps in neutralizing the invading microbes, limits the extent of tissue damage, and promotes tissue repair and regeneration. In many instances, lipoproteins work with players of the APR in tandem and help in projecting antimicrobial defenses of the body.

For example, lipoprotein-binding protein (LBP) is an acute-phase protein carried on lipoproteins [36, 94]. It is associated with HDL, LDL, VLDL, and chylomicrons. LBP catalyzes the detoxification of bacterial toxins such as LPS and LTA by lipoproteins. LBP can modulate the effects of LPS by binding to the lipid A moiety of the latter. During infections, very high concentrations of LBP are attained, which helps in transferring LPS (and LTA) to lipoproteins for inactivation. LBP is also produced in the intestine and in the lungs where it is believed to play important roles in mobilizing local immune responses against bacterial LPS [36, 72, 94–96].

C-reactive protein (CRP) is another acute-phase protein that is associated with LDL and VLDL. Infection by the parasite *Schistosoma* leads to increase in serum CRP. CRP can activate platelets, which have cytotoxic effects against schistosomes. Such cytotoxic effects are exerted by activated monocytes as well. However, LDL binds to the surface of schistosomes, which masks them from activated monocytes. This is circumvented by oxidative changes in the parasite-bound LDL brought about by ROS from activated monocytes. OxLDL is endocytosed by the monocytes through scavenger receptors, which exposes the parasite to attack by monocytes and other immune cells [54, 55].

#### **3.9. Redistribution of lipids to immune cells**

During infection, there are quantitative and qualitative changes in plasma lipoproteins due to redistribution of lipids to the immune cells and areas of cellular injury. These changes are believed to potentiate the immune system and enhance healing in the host that helps to tide over the infective crisis [36]. For instance, there is an increase in triglyceride-rich VLDL particles, which provide lipid substrates to macrophages of the activated immune system. Similarly, there is a decrease in HDL levels. Since HDL is the central component of reverse cholesterol transport (RCT) pathway, such decrements in its level help in conserving cholesterol in peripheral sites. It has been found that during the acute phase of infection, there is an increase in apolipoprotein serum amyloid A (apoSAA) and concurrent decrease in apoA-I. ApoSAA redirects cholesterol away from catabolism in hepatocytes and delivers cholesterol to other cells. Cholesterol is required for new membrane synthesis in areas of cellular injury that accompany infections. Cholesterol may also be used for activation and proliferation of lymphocytes [97–101].

## **4. Lipoproteins as double-edged sword of the immune system**

The immune system is a double-edged sword. Autoimmune diseases and hypersensitivity reactions are classic examples in this regard. The lipoproteins (as components of the immune system) have no exception. Lipoproteins may facilitate invasion and spread of infection by certain pathogens to the detriment of the host. Besides, lipoproteins are important risk factors for some other disorders. The following are certain examples:

However, the opportunistic fungal pathogen *Candida glabrata* can circumvent such ergosterol-deprived killing by utilizing host sterols instead. It can take up cholesterol from host circulating lipoproteins and use it for its survival in the presence of azole antifungals [120].

## **5. Conclusion and future directions**

**3.9. Redistribution of lipids to immune cells**

lymphocytes [97–101].

80 Advances in Lipoprotein Research

endocytosis [102].

ols [103–108].

During infection, there are quantitative and qualitative changes in plasma lipoproteins due to redistribution of lipids to the immune cells and areas of cellular injury. These changes are believed to potentiate the immune system and enhance healing in the host that helps to tide over the infective crisis [36]. For instance, there is an increase in triglyceride-rich VLDL particles, which provide lipid substrates to macrophages of the activated immune system. Similarly, there is a decrease in HDL levels. Since HDL is the central component of reverse cholesterol transport (RCT) pathway, such decrements in its level help in conserving cholesterol in peripheral sites. It has been found that during the acute phase of infection, there is an increase in apolipoprotein serum amyloid A (apoSAA) and concurrent decrease in apoA-I. ApoSAA redirects cholesterol away from catabolism in hepatocytes and delivers cholesterol to other cells. Cholesterol is required for new membrane synthesis in areas of cellular injury that accompany infections. Cholesterol may also be used for activation and proliferation of

**4. Lipoproteins as double-edged sword of the immune system**

for some other disorders. The following are certain examples:

tion [109, 114, 115], and assembly [116–119].

The immune system is a double-edged sword. Autoimmune diseases and hypersensitivity reactions are classic examples in this regard. The lipoproteins (as components of the immune system) have no exception. Lipoproteins may facilitate invasion and spread of infection by certain pathogens to the detriment of the host. Besides, lipoproteins are important risk factors

• The obligate intracellular parasite, *Toxoplasma gondii*, is dependent on host cholesterol from extracellular LDL for growth and replication. The parasite resides in a special parasitophorous vacuole to which cholesterol is delivered by uptake of LDL through receptor-mediated

• There are tremendous requirements of various lipids for successful replication of the malaria parasite in the host. These requirements are met by the parasite by scavenging and modifying lipids from the host itself. Lipids such as phospholipids and free fatty acids (FFA) can be obtained from circulating lipoproteins or directly from the serum and used without further modification. Or else, the scavenged lipids are modified by elongation and desaturation reactions and subsequently incorporated as diacylglycerols and triacylglycer-

• Similarly, a large number of viruses can hijack the host lipid and lipoprotein machinery to their benefit [109, 110]. It is increasingly appreciated that viruses can modulate lipid metabolism, composition, and signaling in the host to facilitate their entry [111–113], replica-

• Fungal pathogens require ergosterol to grow and thrive in the host tissues. The supply of ergosterol is maintained by the endogenous sterol synthesis pathway present in the fungus. The azole group of antifungal drugs inhibits this fungal sterol synthesizing pathway. As our knowledge about the role of lipoproteins as crucial components of the immune system continues to advance, two types of implications for the future have emerged. First, there is the possibility of characterizing the lipoprotein-pathogen interactions in greater detail. This will lead to an improved understanding of the pathophysiological significance of these interactions and may help in elucidating novel anti-infective mechanisms. For instance, a very recent study has described serum lipoproteins as critical components for pulmonary innate defense against quorum-sensing-based pathogenesis by *Staphylococcus aureus* [125]. Second is the potential use of drug therapies to modulate lipoprotein-pathogen interactions with the aim of controlling infections. As discussed earlier, reconstituted HDL and apoA-I mimetic peptides have shown promise in this regard [46–48, 60]. Further, drugs targeting lipid metabolism have also been suggested. For example, plant extracts modulating lipoprotein metabolism have shown promising antimalarial properties [126]. Similarly, there is potential for developing therapeutics targeting fatty acid synthesis (which is required by many viruses) as broadspectrum antiviral agents [110, 118].

To conclude, lipoproteins are increasingly recognized as important players of the host immune system. They offer a multitude of strategies to ward off infections and limit their detrimental effects in the body of the host. At times, many of these strategies act together in a complementary manner, rather than being mutually exclusive. On the other hand, an anti-infective mechanism resulting from a particular lipoprotein-pathogen interaction that may be beneficial for one specific infection may not be applicable sometimes in another infection [127]. Instead, such an interaction may promote infection and lead to untoward effects (as the previous examples show). As seen from the examples in the text, the interactions between host lipoproteins and invading pathogens are complex and multifaceted. This warrants further studies and very detailed knowledge of the different lipoprotein-pathogen interactions to design effective therapeutic options.

## **Conflict of interest**

None.

## **Author details**

Kaustubh Bora¹\* and Probodh Borah²

\*Address all correspondence to<EMAIL_ADDRESS>
1 Regional Medical Research Centre, N.E. Region (ICMR), Dibrugarh, Assam, India

2 Department of Animal Biotechnology, Bioinformatics Infrastructure Facility (BIF) and State Biotech Hub (SBT-Hub), College of Veterinary Sciences, Guwahati, Assam, India

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infection [127]. Instead, such an interaction may promote infection and lead to untoward effects (as the previous examples show). As seen from the examples in the text, the interactions between host lipoproteins and invading pathogens are complex and multifaceted. This warrants further studies and very detailed knowledge of the different lipoprotein-pathogen

interactions to design effective therapeutic options.

**Conflict of interest**

82 Advances in Lipoprotein Research

**Author details**

**References**

Kaustubh Bora¹\* and Probodh Borah²

\*Address all correspondence to<EMAIL_ADDRESS>
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