Exploring antimicrobial properties of cholesterol esters: a systematic literature review

Abstract

Antibiotic resistance has become an increasingly common problem in treating infectious diseases caused by pathogenic micro-organisms, and this threat is heightened due to the lack of new antibiotic discoveries since the 1980s. This paper aims to summarise the currently available knowledge on the antimicrobial properties of cholesterol esters and to evaluate the use of cholesterol esters as an alternative to antibiotics. A systematic literature search was conducted in three databases namely Pubmed, Embase, and Web of Science to retrieve articles published between 2000 and 2020 where cholesterol esters were the primary or one of the primary examined molecules as part of research targeting immunity and antimicrobial operations. The findings showed antibacterial properties of cholesterol esters, their synergistic activity with antibiotics, antimicrobial role of cholesterol esters in upper respiratory tract and its diseases in humans, and correlation of cholesterol esters with infection protection in physically active individuals. Overall, there is strong evidence that cholesterol esters have antimicrobial properties. However, the currently pooled information available for cholesterol esters’ antimicrobial properties is not expansive enough to draw a conclusion on its ability to replace existing antibiotics as a treatment for infections and this warrants future investigation.

KEYWORDS:

• Antibiotic alternative
• antimicrobial lipids
• antimicrobial resistance
• cholesterol ester
• immunity
• infectious diseases

Introduction

Antimicrobial resistance in bacteria (Chastre 2008; Johnson 2015; Zhou et al. 2016), viruses (Hurt et al. 2012; Li et al. 2015; Lim 2017), and fungi (Arendrup 2013; Mitchell et al. 2013) has risen alarmingly in the twenty-first century, largely due to improper medication usage, particularly its over utilisation and incomplete uptake during infection. For instance, the proportion of methicillin resistant isolates in blood cultures isolates of Staphylococcus aureus rose from 2% in 1990 to 43% in 2001 (Johnson 2015). Similarly, the proportion of ciprofloxacin-resistant and cephalosporin-resistant isolates in blood culture isolates of Escherichia coli rose from approximately 1% in 1993 to 23% (ciprofloxacin) and 12.5% (cephalosporins) in 2006–2007 (Johnson 2015). In the context of antiviral resistance, it has been reported that oseltamivir-resistant strains of the H1N1 influenza virus are an emerging threat, despite their low detected population composition (Hurt et al. 2012; Li et al. 2015). In the context of resistant fungi, through the overexposure to echinocandin-based antifungal drugs, certain Candida spp. have acquired resistance through a similar selection mechanism to how bacterial pathogens become resistant (Arendrup 2013).

Despite the alarming antimicrobial resistance in pathogens, no new classes of antibiotics have been discovered since the 1980s. This has prompted researchers and pharmaceutical companies to investigate alternatives to antibiotics in the field of antimicrobials. One novel alternative is the employment of host-derived antimicrobial lipids. This relatively recent research field has promoted the idea that cholesterol esters – whose main function in the human body is as an inactive form of cholesterol for trafficking the molecule around the body – may possess antimicrobial qualities with regards to certain diseases, such as chronic rhinosinusitis (Lee et al. 2010; Lee et al. 2014), cystic fibrosis (Ma et al. 2015), and as an innate preventative mucosal component of the immune system in physically active individuals (Kiwata et al. 2011, 2014). While current studies focus on the possibility and extent of the intrinsic properties that cholesterol esters have, some findings have also suggested that the incorporation of cholesterol esters into liposomes can be utilised to markedly decrease the presence of antibiotic-resistant bacteria in vitro when compared to the use of antibiotics alone (Lam et al. 2016; Zhang et al. 2020). This suggests a revitalisation of the bacteria’s sensitivity to the drug, which can be explored further with other drugs, including other existing antibiotics.

Cholesterol esters are part of lipid mixtures in many human body secretions. Antimicrobial effects of lipid mixtures containing cholesterol esters in human secretions such as tears, sebum, saliva and vernix caseosa have been documented (Mudgil 2014; Porter et al. 2015; Fischer 2020). These host-derived lipid mixtures play a role in innate defence of mucosal surfaces but they contain many other antimicrobial lipids such as fatty acids, sphingosines, and phospholipids that have been demonstrated to possess antimicrobial activities (Krogfelt et al. 2000; Drake et al. 2008; Fischer et al. 2012; Wertz and de Szalay 2020), so the specific antimicrobial contribution of cholesterol esters in these mixtures is relatively unclear.

In past studies, some researchers have investigated specific groups of cholesterol esters. Two prominently examined cholesterol esters are cholesterol linoleate and cholesterol arachidonate (Do et al. 2008; Lee et al. 2010). Both types of cholesterol esters have been shown to have a link between their presence in nasal mucosal fluid and the lowered presence of bacterial cells, to such an extent that their removal, as well as the removal of most other nonpolar lipids from the nasal mucosa secretions, resulted in an increase in pathogenic bacteria populations in mucosa in in vitro samples (Do et al. 2008). It is imperative to note that, despite the focus on cholesterol linoleate and arachidonate (Do et al. 2008), the samples tested did include other molecular groups that are known to have antimicrobial properties, particularly polar lipids. These studies focused only on the upper respiratory tract and nasal cavity secretions, and did not indicate possibility of antimicrobial function of these cholesterol esters in other locations where they can be present. In fact, most of the published reports on antimicrobial role of cholesterol esters are limited to upper body cavities (Do et al. 2008; Lee et al. 2010; Ma et al. 2015) and skin of both human and mice (Georgel et al. 2005) and therefore provide an incomplete picture and understanding of the scope of capabilities cholesterol esters can have as a novel innovation in the continuous fight against increasingly resistant pathogenic microbes. However, it does not discount the evidence that there is a basis of connection between cholesterol esters and their use as an antibiotic alternative.

The available literature on antimicrobial properties of cholesterol esters or other nonpolar lipids for that matter is scanty. This is partly due to the fact that limited research has been done in the field of antimicrobial lipids and within this most of the reports have focused on polar lipids. The objective of this systematic review was to collect the currently available information regarding the potential antimicrobial attributes that cholesterol esters have in human hosts, and to determine their suitability as an antibiotic treatment replacement.

Methods

The current review was prepared following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines.

Search strategy

A systematic search of the literature was done in three databases: Pubmed, Web of Science, and Embase via Ovid. The search in Pubmed and Web of Science was performed using the keywords search string: (antimicrobial or antibacterial or antifungal or antiviral) and (‘cholesterol ester’ or ‘cholesteryl ester’ or ‘cholesterol oleate’ or ‘cholesteryl oleate’ or ‘cholesterol linoleate’ or ‘cholesteryl linoleate’ or ‘cholesterol arachidonate’ or ‘cholesteryl arachidonate’). Articles published between 1 January 2000 and 1 August 2020 and only in English language were included for review. The search in the Embase via Ovid database was also filtered for articles with Full Text availability, published between 2000 and 2020, and specifically conducted with the following search string: (antimicrobial or antibacterial or antifungal or antiviral) and ('cholesterol ester' or 'cholesteryl ester' or 'cholesterol oleate' or 'cholesteryl oleate' or 'cholesterol linoleate' or 'cholesteryl linoleate' or 'cholesterol arachidonate' or 'cholesteryl arachidonate'). The Web of Science database search was additionally filtered through the Topic search field, and conducted through the Web of Science Core Collection database and the following Editions: Science Citation Index Expanded (SCI-EXPANDED), Social Sciences Citation Index (SSCI), Arts & Humanities Citation Index (AHCI), Conference Proceedings Citation Index – Science (CPCI–S), Conference Proceedings Citation Index – Social Science & Humanities (CPCI-SSH), Book Citation Index – Science (BCI-S), Book Citation Index – Social Sciences & Humanities (BCI-SSH), Emerging Sources Citation Index (ESCI), Current Chemicals Index (CCI-EXPANDED), and Index Chemicus (IC). All titles and abstracts were imported into RefWorks reference management tool. Titles and abstracts search was conducted followed by the full-text screening by the first author (TJ) and the second author (PM), independently. Any disagreements were resolved by consensus.

Focused question

The review aimed at answering the questions – Do cholesterol esters have antimicrobial properties? To which extent do cholesterol esters defend against infection? Can cholesterol esters be used as an alternative to existing antibiotics?

Eligibility criteria

The articles that were included in this review were (i) peer-reviewed papers in English language with full text available, (ii) the target molecule was cholesterol ester or the type of cholesterol ester was defined, (iii) the in vitro or in vivo studies were related to cholesterol ester, (iv) cholesterol ester was in the context of antimicrobial properties and innate defence. This review incorporated information that focused on human test subjects or human metabolic analogues. This was chosen to eliminate unnecessary information that derived results based on the metabolism and immune response in animals. The possibility of metabolic and compositional differences between the mucus that harbours cholesterol esters in each species was decided to be too disruptive in determining significance of cholesterol esters in human immune response. Any study that investigated the antimicrobial impact of cholesterols as a general compound group was included in the initial database search but was ultimately removed if there were insufficient data or discussion with reference to cholesterol esters specifically. Studies that focused on Cholesterol Ester Transfer Protein (CEPT) and inhibitors of these proteins (CEPTi) were excluded due to their focus on an unrelated system adjacent to cholesterol esters. Studies that referred to techniques in separating cholesterol esters from other mucosal compounds and different subcategories of cholesterol esters – cholesterol linoleate and arachidonate specifically – were included because they provide information into the future endeavours cholesterol esters research can undertake, especially in the field of antimicrobial use. For human trials, due to the limitation of conducted experiments that focused on cholesterol esters in any regard, there was no age restriction nor a restriction on the production of results. Any article that presented quantitative data that showed statistically significant differences between a control treatment and treatment including cholesterol ester was also considered during the screening process and for the overall review.

Outcomes of interest

The outcomes of interest were to determine the extent by which the current research has investigated the antimicrobial properties of cholesterol esters in human subjects or in vitro, thus profiling where future work can extend to. Specifically, this review was interested in which types of micro-organisms cholesterol esters have been shown to have a response against, and if any particular ester had a prominent role in innate defence. An established baseline of effectiveness was not taken into account in the criteria for accepting an article for review due to the novelty of cholesterol esters.

Results

Database search retrieved a total of 355 articles. Out of these, 339 articles were eligible for screening and analysis after the removal of duplicates. Proceeding the analysis of each article’s title and abstract, 199 articles were excluded due to not meeting the primary inclusion criteria for this review. The full text of 140 articles was assessed, and a further 129 articles were excluded, thus in total, 11 articles were included for the review. Figure 1 illustrates the PRISMA model of included studies and accompanying removal criteria at each stage.

Figure 1. PRISMA flow diagram for selecting articles for ‘Exploring antimicrobial properties of cholesterol esters: A systematic review’.

Out of 11 articles included in the review, two articles focused on antimicrobial delivery of lipids using nano-carriers and liposomes. Out of these two articles, one review article described various antimicrobial lipids including cholesterol esters in nano-carriers for antibacterial delivery (Zhang et al. 2020) and one article focused on the synergistic activity of cholesterol linoleate with antibiotics using carrier liposomes against Enterococcus faecalis, Staphylococcus epidermidis, and Pseudomonas aeruginosa (Lam et al. 2016). Five articles focused on the role of cholesterol esters in the upper respiratory tract (Durieu et al. 2007; Do et al. 2008; Lee et al. 2010; Lee et al. 2014; Ma et al. 2015). Out of these five articles, one article illustrated a correlation between the presence of cholesterol esters (cholesterol linoleate and cholesterol arachidonate) in nasal secretion and microbial growth inhibition (Do et al. 2008), two articles showed antimicrobial aspects of cholesterol esters in chronic rhinosinusitis (Lee et al. 2010; Lee et al. 2014), and two articles investigated the role of cholesterol esters in cystic fibrosis (Durieu et al. 2007; Ma et al. 2015). Of the remaining four articles, two articles examined the presence of cholesterol esters in physically active individuals as an innate immunological response (Kiwata et al. 2011, 2014), one article considered cholesterol esters as a biomarker for prion-infections (Pani et al. 2007), and one article provided an improved technique for separation of cholesterol esters from complex biological samples (Jansen et al. 2010). Table 1 is a summary of all the articles except Zhang et al. (2020) which is a review article that cites findings on cholesterol esters from Do et al.(2008) and Lam et al. (2016).

Table 1. Summary of the key findings for ‘Exploring antimicrobial properties of cholesterol esters: A systematic review (2000–2020)’.

Authors	Antimicrobial cholesterol esters	Target organism or Immune aspect	Key findings
Lam et al. (2016)	Cholesterol linoleate	E. faecalis, S. epidermidis, P. aeruginosa	Incorporated in liposomes and paired with antibiotics, it decreased concentrations of pathogens in an in vitro environment
Do et al. (2008)	Cholesterol linoleate, Cholesterol arachidonate	P. aeruginosa, S. epidermidis	Inhibited growth of bacteria in liposomal preparations
Lee et al. (2010, 2014)	Cholesterol esters*	Sinus secretion	Increased levels of cholesteryl esters observed in sinus secretions of chronic rhinosinusitis patients
Ma et al. (2015)	Cholesterol esters*	Bronchoalveolar lavage fluid	Increase in cholesterol ester concentrations in patients with cystic fibrosis, can be used as a biomarker of infections
Durieu et al. (2007)	Cholesterol esters*	NA	The concentrations of cholesterol esters in plasma fluctuated in cystic fibrosis patients when compared to non-cystic fibrosis patients
Kiwata et al. (2011, 2014)	Cholesterol linoleate and cholesterol arachidonate	Nasal fluid	After exercise in active and inactive individuals, the concentration of cholesterol linoleate and cholesterol arachidonate in the nasal fluid of inactive individuals decreased significantly, indicative of a lowered innate immune response
Pani et al. (2007)	Cholesterol esters*	Prion	Biomarker for prion-infection in sheep and humans, due to their increased presence in sheep fibroblasts accompanying prion-infection
Jansen et al. (2010)	Cholesterol esters*	NA	Improved technique for the separation and purification of cholesterol esters from standard mixtures and biological samples

*Type of cholesterol ester not defined.

NA – target organism or immune aspect not mentioned.

Discussion

This review provides a detailed report and analysis of the current collective knowledge of the antimicrobial properties of cholesterol esters in in vivo and in vitro circumstances, as well as describing the involvement of cholesterol esters in innate immune defence. A recent review on nano-carriers for antimicrobial lipids includes some information pertaining to antibacterial activity of cholesterol esters (Zhang et al. 2020); however, our review is more exhaustive and discusses the antimicrobial properties of cholesterol esters in the context of innate immune defence as well as other microbial infections.

Antibacterial findings

In the context of cholesterol esters’ contribution to antibacterial defence, there has been a repeated focus on upper respiratory tract infections. As Do et al. (2008) explain, the nasal mucosa is a major initial site of bacterial invasion from the external environment. As such, the mucosa’s polypeptides have been extensively studied to the point where major antimicrobial polypeptide groups are established with their direct function and mechanism of activity (Posokhova et al. 2008). Do et al. (2008) explored the cholesterol esters found in the nasal mucosa, specifically cholesterol linoleate and cholesterol arachidonate, reporting that there is a correlation between the occurrence of bacterial growth inhibition and death and the presence of cholesterol arachidonate and cholesterol linoleate in the secretions, respectively. They propose that peroxidation of these lipids produces cytotoxic by-products as a mechanism against infection.

Two articles with the same lead and senior researchers focused on the nasal mucosa in the context of chronic rhinosinusitis (CR) infection (Lee et al. 2010, 2014). In the initial study, Lee et al. (2010) reported that the concentrations of cholesterol esters – specifically cholesterol linoleate – found in CR-positive patients were equivalent to the concentrations that were previously shown to have bactericidal activity against Pseudomonas aeruginosa at physiological levels (Do et al. 2008). This increase in cholesterol esters’ presence was also documented in their later study (Lee et al. 2014). The initial study hypothesised and discovered that, under the assumption of cholesterol esters partaking in the innate immune response to infection, its concentration would increase in nasal mucosa, which can be extrapolated to infer a connection to antimicrobial properties. Do et al. (2008) removed nonpolar lipids including cholesterol esters from the nasal fluid samples and observed that the infection rate of P. aeruginosa increased in comparison to when the lipids were included. For validation that cholesterol esters are a contributor to innate immunity in mucosal secretions, they re-introduced nonpolar lipids including cholesterol esters into the samples. Subsequently, the infection rate decreased, however, not to the extent when lipids were inherent to the mucus (Do et al. 2008), suggesting that cholesterol esters alone do not completely counteract bacterial infections; rather they work in conjunction with other lipids and antimicrobial peptides to deter infection.

The current knowledge of antibacterial properties of cholesterol esters provides evidence that these properties exist, however, all published reports retain the same sentiment that further research is required to develop an understanding of the mechanism and standalone nature, if any, of cholesterol esters in innate immunity.

Synergistic utility

The approach of cholesterol esters working in tandem with existing antimicrobial peptides has been explored in a paper by Lam et al. (2016). They investigated the use of cholesterol esters incorporated into liposomes, the hydrophilic molecular vehicles used for drug delivery, for targeted delivery of cholesterol linoleate paired with vancomycin against an Enterococcus faecalis strain resistant to the antibiotic vancomycin. Results from this study strongly suggested that cholesterol linoleate reinvigorated the susceptibility of E. faecalis towards vancomycin; the relative fluorescence units reflected a significant decrease in the bacteria’s concentration, as well as warranting a less concentrated dose of vancomycin. They also reported antibacterial effects of cholesterol linoleate against strains of S. epidermidis and P. aeruginosa. However, the mechanism of action of cholesterol ester was not discussed in their paper.

Upper respiratory relations

As previously discussed, many of the currently published articles pertaining to the antimicrobial properties of cholesterol esters focus on the upper respiratory tract. It is important to consider whether the presence of infection or an abnormal biophysiological state contributes to an altered composition of mucosal secretions with regards to cholesterol esters levels. One alternate condition that has been researched into is with patients suffering from cystic fibrosis (CF). Ma et al. (2015) produced results where the ratio of lipid to protein concentration shifted to a decreased lipid proportion in patients with CF; however, the percentage of cholesterol esters in lipid secretions increased, which the researchers suggested was a result of inflammation. While this could be a coincidence, the evidence suggested that the increase in the concentration of cholesterol esters reflected an innate immune response that contributed to inflammation; however, this requires further investigation, as Ma et al. (2015) concluded. A different CF-centric study performed by Durieu et al. (2007) found that the concentration of cholesterol esters in plasma of CF patients was either similar to non-CF patients, or decreased depending upon the ester-linked fatty acids of the cholesterol ester. The comparison between these two research articles that examined the same compounds under the same biomedical limitation, yet in different secretions do not only reflect the ambiguity of cholesterol esters’ antimicrobial properties; it also reflects the limitation of the extent of currently published research.

Other avenues of research

Of the remaining articles included in this review, not all directly research cholesterol esters’ antimicrobial properties in contexts that overlap enough for their own sections. Hence, they will be discussed here. Two studies conducted largely by the same research group (Kiwata et al. 2011, 2014) contextualised cholesterol esters’ involvement in physically active individuals and presented the hypothesis that cholesterol esters act as a countermeasure against a potential infection through the correlation of cholesterol esters’ observed increased concentrations after vigorous exercise in physically active individuals. In their more recent study (Kiwata et al. 2014), the group observed post-exercise changes in both active and sedentary patients. The concentration of cholesterol esters rose significantly in the active group, whereas it decreased in the sedentary group, despite basal levels between the groups being comparably in line with existing knowledge of concentrations of cholesterol esters pre-exercise. The researchers’ proposed explanation for this decrease stems from the known observation that various other body fluids increase in secretion rate during and post-exercise, thus diverting energy away from the production of cholesterol esters. These two studies contribute to the overall idea of this review in that they explore realistic, temporarily immunocompromising scenarios where cholesterol esters play an important comparative reference point by which the examination of this avenue can derive the importance of cholesterol esters in the innate immunity.

It must be considered that despite mounting evidence to prove that cholesterol esters have antimicrobial properties, they may not be as effective, if not more effective, than existing medication. Thus, utilising these molecules for different purposes could lead to different innovations in the field. Among the articles included for review, Pani et al. (2007) suggest the use of cholesterol esters as a biomarker for prion-infection susceptibility and protein-based degenerative pathology in livestock and humans. This idea is based on their results illustrating abnormal accumulation of cholesterol esters in sheep fibroblasts which reflected one of the symptoms of scrapie infection. While the context of this study was in sheep, the article expresses that much of the findings can be translated into a human context.

In a similar vein, innovation needs to occur to determine the extent of antimicrobial properties of various types of cholesterol esters. This requires better techniques for separation and characterisation of cholesterol esters in biological fluids. Currently, research conducted by Jansen et al. (2010) was the only published research included under this review’s criteria that defined and utilised a technique for chromatography-based separation of cholesterol esters from both standard mixtures and biological samples in a timely manner. Their method was built upon a previous work (Do et al. 2008) that involved a one-step protocol for total lipids extracts but this protocol incompletely resolved cholesterol esters. Jansen et al. (2010)’s technique used artificial neural networks to optimise the analytical protocol and with this approach cholesterol esters were fully separated in standard mixtures and better resolved in a biological fluid. This technique is a step in the direction of exploring biological functions of cholesterol esters that will further contribute to our understanding of antimicrobial properties of cholesterol esters.

Limitations of the review

The main limitation of this review stems from the limited number of articles published for research into antimicrobial properties of cholesterol esters. The oldest included article in this review is from 2007 (Durieu et al. 2007), and the recent article that directly comments on antimicrobial use of cholesterol esters is from 2016 (Lam et al. 2016). In comparison to the history of antibiotics being over a century old, the scientific community has not explored antimicrobial properties of cholesterol esters. Similarly, other lipid types have been researched for their antimicrobial properties – for instance, fatty acids (Desbois and Smith 2010) – thus taking attention away from cholesterol esters. This is highlighted prominently by the lack of mechanism of any of cholesterol esters’ hypothesised actions in innate immunity. With a lack of mechanism, scientists cannot develop techniques to control the application of cholesterol esters in immunity and cannot produce cholesterol ester-based medication that either works in tandem with antibiotics or replaces them outright. The limited number of databases examined for relevant articles for this review can also be an explanation for the lack of information in the specifics of cholesterol ester as antimicrobial.

Conclusion

In conclusion, the current state of cholesterol esters’ known antimicrobial properties is at an initial stage where evidence is highly suggestive of its presence. There has been a foray into its use as a complementary molecule with existing antibiotics, thus providing an avenue for practical applications of the molecule. However, there are still major voids of information about cholesterol esters’ specific role and mechanism of action in innate immunity. Due to the incomplete nature of the situation, we cannot provide a definitive answer in their use as a substitute for current antibiotic treatments. However, all current research supports the idea to further investigate cholesterol esters as an alternative avenue in the fight against microbial infections and antimicrobial resistance, and we agree with this concept.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.