Background Airway secretions contain endogenous antimicrobial factors (AMFs) which contribute to the innate host defense of the respiratory tract. Antibacterial peptides as well as host-derived lipids including cholesteryl esters have been detected in maxillary lavage fluid. Sterol O-acyltransferase 1 (SOAT1) is a key enzyme in cholesteryl ester production. The purpose of this study is to determine if such intrinsic microbicidal molecules are acutely expressed within sinus tissue and to compare levels of expression between patients with and without chronic rhinosinusitis (CRS). Methods Sinus tissue was obtained from subjects with (24) and without (9) a history of CRS. Six CRS patients had nasal polyposis (CRSwNP). Immunofluorescence staining for human neutrophil peptide (HNP) was done as a marker for inflammation. RT-PCR following RNA extraction was used to quantify the expression of SOAT-1, the epithelial beta-defensins (HBD2,3), and the cathelicidin LL37 with ribosomal protein RPLP0 as the housekeeping gene. Results Immunofluorescence showed significant increase in HNP staining in CRS patients without nasal polyposis (CRSsNP) versus non-CRS specimens (p=0.010), in agreement with clinical inflammation status. SOAT1 mRNA expression was also upregulated in CRSsNP compared to non-CRS (p=0.041) and CRSwNP (p=0.005) patients; while increases for HBD2 and HBD3 were less prominent. LL37 was either absent or expressed at very low levels in all samples. Conclusions Increased biosynthesis of SOAT1, a key enzyme for antimicrobial cholesteryl ester production, was observed in the sinus tissue of CRSsNP but not in CRSwNP patients. This further supports the novel concept of lipid-mediated innate mucosal defense and delineates CRS with and without nasal polyposis as distinct subtypes.
INTRODUCTION: Consistent, moderate-to-vigorous intensity exercise has been associated with a lower risk of upper respiratory tract infection (URI). However, the molecular basis for this apparent protection has not yet been fully resolved. Host-derived lipids such as cholesteryl esters (CEs) have emerged as important effector molecules of innate defense against infections. Here, we compared antimicrobial CEs in nasal fluid before and after moderate-to-vigorous exercise between active and inactive subjects. METHODS: Nasal fluid was collected from fourteen healthy, recreationally-active subjects (32 ± 11 yr, 7 males, 7 females) and 14 healthy, inactive subjects (25 ± 3 yr, 7 males, 7 females) before and after treadmill exercise at 70% heart rate reserve. Nasal fluid was analyzed for lysozyme, cholesteryl linoleate (CL), cholesteryl arachidonate (CA), and albumin (Alb) concentrations. RESULTS: Baseline concentrations (means ± SEM, inactive vs. active) of lysozyme (117.7±31.1 μg/mL vs. 122.9±15.5 μg/mL), CL+CA (15.3±1.8 μg/mL vs.26.2±10.05 μg/mL), and albumin (156.6± 54.5μg/mL vs.126.9±32.8 μg/mL) were similar to previously reported levels and did not differ significantly between study groups. However, post-exercise, CL+CA concentration was significantly lower in inactive compared to active subjects (7.8 ± 1.5 μg/mL vs. 20.1 ± 4.8 μg/mL, p = 0.036) dropping below the antimicrobial effective range. Once adjusted to albumin concentrations the changes were no longer significant, suggesting that plasma transudation accounted for the increased CA+CL concentration post-exercise in the active group relative to the inactive group. CONCLUSION: Moderate-to-vigorous aerobic exercise acutely decreases the antimicrobial CE response in inactive subjects, but does not modify baseline levels of CEs between active and inactive subjects. This suggests that compared to active individuals, inactive individuals may be at greater risk for URI immediately post-exercise.
Airway epithelial cells contribute to the first line of mucosal defense by secretion of antimicrobial peptides and antimicrobial lipids, including the cholesteryl ester (CE) cholesteryl arachidonate. Sterol O-acyltransferase 1 (SOAT1) is a key enzyme for CE biosynthesis. However, little is known about the regulation of the lipid-mediated arm of innate host defense. Polarized human bronchial epithelial cells (HBE) were incubated for 6 h with 100 ng/mL LPS, 10 μg/mL peptidoglycan (PG), 25 μg/mL PamCys3, and solvent control. SOAT1 mRNA was quantified by qRT-PCR with duplexing and RPLP0α as housekeeping gene. Lipid content of apical HBE secretions was analyzed by rpHPLC. SOAT1 expression was blocked with siRNA/lipofection. Antibacterial activity of HBE secretions was assessed by a 3 h colony forming unit assay with Pseudomonas aeruginosa (PA). SOAT1 gene expression was significantly increased by LPS (14.6 ±11.4, means ± SD, n=4, p=0.037). Stimulation with PG and PamCys3 increased SOAT1 expression by ~ 4- and 2-fold, respectively. Secretions from LPS-stimulated HBE showed an increase of total lipids including CA by ~30-50%. Selective inhibition of SOAT1 mRNA expression by 80% reduced the antibacterial activity of HBE whereby PA showed enhanced bacterial growth reaching >170% of the controls. These data suggest that SOAT1 is regulated by conserved innate immunity pathways and further support the role of antimicrobial lipids as effector molecules in mucosal airway defense.
Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1.
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