This study provides evidence that lipids are secreted to mucosal surfaces and contribute to the inherent antimicrobial activity of mucosal secretions. To examine the potential role of lipids in innate mucosal host defense, we used nasal mucosal secretions. Nasal mucosa is a primary microbial exposure site; nasal mucosa is not exposed to alimentary lipids, its secretions are easily accessible and its antibacterial activity has been previously established in respect to antimicrobial polypeptides (27
We found all major lipid classes in nasal fluid collected from healthy adults and, to our knowledge, this study is the first quantification of lipids in human nasal fluid. Glycerophospholipids and cholesterol, as well as to a lesser extent triglycerides and free fatty acids, have already been described in bronchioalveolar and nasal lavages, whereby the lipids were mainly thought to originate from lung surfactant reaching the upper airways through mucociliary propulsion (35
). However, the presence of cholesteryl esters in native nasal fluid and their identification in the apical secretions of human bronchial epithelial cells suggest that the epithelial cells of the upper airways contribute to the lipids found in nasal fluid. This is also supported by the recent discovery of surfactant lamellar bodies in normal sinus mucosa (38
). Nonetheless, we can not exclude transudation of plasma lipids through endothelial cells of the upper respiratory tract (39
In aqueous environments, at submicellelar concentrations, lipids require carrier molecules such as albumin or lipoproteins. Our results demonstrate the presence of lipoproteins in nasal fluid. Specifically, by employing immunoblotting, apolipoprotein A-I (apoA-I) was identified, which has also been detected by Ghafouri et al. (41
) in nasal fluid lavage. We found apoA-I in individual samples in multiple forms possibly reflecting proteolysis of apoA-I by enzymes in nasal fluid (42
). ApoA-I is a mainly found in HDL and one of the major functions of apoA-I is cholesterol binding and reverse cholesterol transport from tissue to bile (43
). This is consistent with our findings showing the presence of both cholesterol and apoA-I. However, we were not able to detect distinct lipoprotein bands co-migrating with HDL in the lipoprotein gels, but, in contrast, observed diffuse staining. Such diffuse mobility in agarose gels has been also shown for protease-modified HDL particles (42
). Furthermore, our results could also reflect the presence of other lipid binding proteins. For example, lipocalin, which is produced by nasal mucosa (44
) has been previously detected in nasal fluid (27
). Alternatively, nasal fluid may contain unique lipoprotein particles synthesized by epithelial cells in the upper airways. The specific lipoproteins in nasal fluid and their origins remain to be defined.
To achieve reasonably selective lipid depletion from nasal fluid while minimally altering its natural, highly complex composition, we developed an SPE procedure. Though there are several widely accepted SPE protocols for the purification of lipids from biological fluids (reviewed in (47
)), these do not aim to preserve the depleted fractions for further testing. Initial pilot studies suggested an association between inherent killing capacity of nasal fluid with cholesteryl ester contents and we focused on removing primarily non-polar lipids while allowing the presence of phospholipids. We found that the inherent antibacterial activity of nasal fluid was significantly diminished when non-polar lipid concentrations were reduced. Even though the SPE procedure may have altered other constituents of the nasal fluid, such as electrolytes and mucins, the observation that the re-addition of lipids restored in part the antibacterial activity, and the direct antibacterial activity of lipid extracts and commercial cholesteryl esters strongly suggest that host-derived antimicrobial lipids contribute to the inherent antimicrobial activity of nasal fluid, which was previously speculated by Widdicombe (48
). Antimicrobial functions of cholesteryl esters are supported by a study by Georgel et al. (49
), in which mice with a mutation in the stearoyl coenzyme A desaturase 1 gene develop spontaneous chronic dermatitis and also exhibit a decrease in cholesteryl ester content in the affected skin.
The contributions of apoA-I and possibly other lipoproteins to the observed antimicrobial activity of lipids is not clear yet. ApoA-I was partially co-depleted during SPE, in contrast to other proteins, and lipid supplement alone did not fully restore the antibacterial activity of LD. Hence, apoA-I may exert antimicrobial activity alone (50
) or by enabling host-derived lipids to function similarly to the reported antibacterial activity of lipoproteins in conjunction with antimicrobial peptides (52
Host-derived antimicrobial lipids may exert their activity in conjunction with antimicrobial (poly) peptides, which have a well documented membrane-perturbing activity (53
). It is conceivable that antimicrobial lipids disrupt microbial membranes by embedding their hydrophobic acyl chains or side chains, and this activity could be facilitated by lesions initially created by antimicrobial (poly) peptides. Here, we have shown that lipids extracted from nasal fluid act synergistically with the antimicrobial peptide HNP-2. Tollin et al. (25
) reported in vitro
synergistic activity between selected fatty acids and the antimicrobial peptide LL37, and we are currently addressing in our laboratory the mode of synergistic action between fatty acids and lysozyme (manuscript submitted). Similarly, lipopeptaibols, naturally occurring lipopeptides with antimicrobial activity (54
) and in vitro
modified antimicrobial peptides that contain acyl chains and exhibit enhanced antimicrobial activity have been described (55
). The antimicrobial properties of host-derived lipids may also extend to intracellular targets (56
) and direct or indirect toxicity due to oxidation of lipids. Polyunsaturated fatty acids, such as linoleic acid and arachidonic acid, which have been found in our study either as free fatty acid or esterified in cholesteryl linoleate and cholesteryl arachidonate, are prone to peroxidation in the presence of oxygen and can form cytotoxic by-products (57
Cole et al. (27
) reported previously that the cationic protein extract of nasal fluid retained most of the bactericidal activity of nasal fluid. However, the lipid contents has not been analyzed in this study and it cannot be ruled out that some antibacterial lipids associated with highly hydrophobic antimicrobial proteins such as lysozyme may have been co-extracted. Even though lysozyme exerted rapid bactericidal activity and was able to fully restore the bactericidal activity of cationic protein- depleted nasal fluid when tested at high concentrations in vitro
suggesting a predominant role in mucosal defense, its relative contribution to the inherent antibacterial activity of nasal fluid (and other bodily fluids) may be more complex in vivo.
Cholesteryl esters exhibited prolonged activity in a cation adjusted, nutrient rich medium, unlike most antimicrobial peptides (60
), and it is conceivable that antimicrobial peptides and antimicrobial lipids cover different aspects of mucosal defense, rapid initial mass destruction and retardation of the proliferation of survivors, respectively. Similarly, considering the varying spectrum of activity among both, antimicrobial (poly)peptides and cholesteryl esters, mucosal defense may relay on both components to cover the entire spectrum of potential invaders. A careful analysis of antimicrobial (poly)peptides and antimicrobial lipids in mucosal secretions and their in vitro
activities will reveal their respective relative contributions to mucosal defense.
Lipid secretion may contribute to shaping the resident microbiota on mucosal surfaces like antimicrobial peptides (61
). We did not observe a significant activity of nasal fluid lipids against S. aureus
, which often colonizes the nostrils (62
), but a pronounced activity against PA which is not routinely present in normal subjects. Furthermore, secretion of antimicrobial lipids may be also important for keeping the total number of the resident microbiota low. Mucosal surfaces populated by squamous epithelial cells with less secretory functions such as in the oral and vaginal cavity are typically heavily colonized. Hence, antimicrobial lipid secretion may be a feature of columnar epithelial cells in the airways and possibly other body sites with little colonization.
In conclusion, we have shown that lipids are present in mucosal secretions of the upper airways in significant quantities, and have provided evidence that lipids contribute to the inherent antibacterial activity of nasal fluid alone and in synergism with antimicrobial peptides. This suggests a role of host-derived lipids as direct antimicrobial effector molecules in innate mucosal immunity. The concept of antimicrobial lipids may unveil new mechanisms of host resistance to infections and microbial pathogenesis, as well as new avenues for prophylactic and therapeutic strategies in infectious diseases.