Our experiments are the first, to our knowledge, to describe an antimicrobial effect of human ELF obtained via BAL against S. pneumoniae
, the most common pathogen in CAP, particularly among those with AUDs. Further, this study identified novel causes for the increased susceptibility to pneumococcal pneumonia in individuals with AUDs. Differences in antimicrobial protein composition, and activity of lysozyme and lactoferrin, probably contribute to the alterations in innate observed in those with AUDs. Importantly, the present study identifies potentially addressable factors that may decrease the incidence of pneumococcal pneumonia among those with AUDs. A decrease in the incidence of pneumonia among these individuals is an important public health issue, as an estimated 50% of patients with pneumonia have an AUD (Goss et al., 2003
). Moreover, these patients have more severe symptoms, longer and more expensive hospitalizations and higher mortality (Fernandez-Sola et al., 1995
; Perlino and Rimland, 1985
; Saitz et al., 1997
). As a result, there are more AUD-associated deaths from pneumonia when compared with the number of deaths due to alcohol-related liver disease (Kung et al., 2008
), traffic accidents (Anonymous, 1993
) or alcohol-related pancreatitis (Steinberg and Tenner, 1994
Lysozyme and lactoferrin are manufactured by serous epithelial cells and myeloid lineage cells, including neutrophils and alveolar macrophages. Alcohol consumption has been reported to affect innate immune functions of alveolar macrophages, including their production of cytokines important in host defense (Standiford and Danforth, 1997
), and decreasing the expression of the granulocyte–monocyte colony stimulating factor receptor (Joshi et al., 2005
). AUDs are also known to result in excessive oxidation in ELF that may have potential ramifications on transcription and translation of antimicrobial proteins by serous epithelial cells or resident alveolar macrophages (Yeh et al., 2007
). Additionally, breakdown products of ethanol could affect antimicrobial proteins post-translationally. For example, metabolism of alcohol produces acetaldehyde that in vitro
has been shown to decrease the activity of lysozyme by 62% (Brecher et al., 1995
). It is unclear which of these alterations in anti-pneumococcal defense elicited by alcohol is most important in increasing the predisposition for pneumococcal disease among those with AUDs, but each has a potential role.
Our group focused on examining differences in lysozyme and lactoferrin in acellular BAL fluid as these are quantitatively the most abundant antimicrobial proteins in lung (Travis et al., 2001
) and therefore potentially have the most potent effect on eliminating the pneumococcus in the airways. Lysozyme functions in antimicrobial defense through its ability to cleave N
-acetylmuramic acid, the cell wall material that helps bacteria maintain their shape. It is found ubiquitously throughout the airway, and has been associated with better killing and improved survival in infection with group B Streptococci and mucoid Pseudomonas aeruginosa
in animal models (Akinbi et al., 2000
). Lactoferrin has not only antibacterial but anti-inflammatory properties. The synergism that has been reported between various antimicrobial proteins may explain the presence of multiple antimicrobial proteins in ELF, including those present in only minute quantities. More recently, antimicrobial proteins and peptides have gained additional recognition as immune regulators, possessing activity in the neutralization of LPS, chemotactic activity, wound healing and activity in adaptive immunity (Diamond et al., 2009
It is certainly possible that alcohol consumption may have affected not only lysozyme and lactoferrin, but also additional proteins important in the immune response against pneumococci that ultimately contributed to our killing assay observations. In order to specifically identify antimicrobial proteins whose concentration or activity are most prominently affected by AUDs, investigations with BAL fluid utilizing more sophisticated techniques (i.e. proteomics, crystallography) would be helpful (Baker and Baker, 2004
; Merkel et al., 2005
) to clarify the type and quantity of antimicrobials present in BAL fluid. One important family of proteins to assess in future investigations consists of the surfactant proteins. In ovine models, alcohol exposure of pregnant ewes in late gestation has been determined to result in decreased expression of mRNA for surfactant proteins A, B and D (Lazic et al., 2007
; Sozo et al., 2009
). Although the effect of AUDs on human surfactant proteins is not known at present, decreased expression of these proteins could be an additional contributor to the susceptibility for pulmonary infections, and might also be a relevant factor in these individuals’ susceptibility to ALI (Moss et al., 1996
). Future investigations can also help establish if modifications resulting from alcohol or its metabolites are operative that might affect protein function. For example, our lactoferrin assay did not allow us to differentiate between apolactoferrin (iron-desaturated) and hololactoferrin (iron-saturated) forms of this protein in BAL fluid, although the presence of iron binding has been reported to result in conformational and functional changes to this protein (Baker and Baker, 2004
; Norrby, 2004
). By clarifying the distribution and function of these pulmonary antimicrobial proteins in the context of AUDs, mechanisms underlying the predisposition to pulmonary infections (and, potentially, their severity) in this population may be established.
While AUDs are related to a myriad of effects on the pulmonary host response, concomitant tobacco might also be expected to influence this response. For our current investigations, we examined the effects of AUDs in cohort of predominantly smokers, believing this to be a clinically relevant approach in that the majority of individuals with AUDs also smoke. Tobacco smoke exposure has been associated with an increased risk for pulmonary infections, including invasive pneumococcal disease (Nuorti et al., 2000
). Many alterations in host immunity elicited by tobacco smoke have been reported, including depressed mucociliary clearance (Foster et al., 1985
), increased bacterial adhesion (Raman et al., 1983
), decreased pulmonary surfactant (Honda et al., 1996
) and impaired function of host innate immune cells (e.g. alveolar macrophages) (Hodge et al., 2007
). As discussed previously, several of these same immune mediators are similarly altered by AUDs, suggesting the possibility of an additive or even synergistic effect between AUDs and smoking. However, the previously reported effects of tobacco smoking on lysozyme and lactoferrin concentrations in lung contrast with our current observations in those with AUDs. For example, alveolar macrophages from smokers have been reported to secrete significantly more lysozyme than alveolar macrophages from non-smokers (Hinman et al., 1980
). Moreover, concentrations of airway fluid lysozyme and lactoferrin among asymptomatic smokers were determined previously to be ~2-fold higher than in non-smokers (Thompson et al., 1990
), and more recent data utilizing proteomic techniques have confirmed this 2-fold increase in lysozyme (Merkel et al., 2005
). Our investigations accounted for the potential effect of smoking on lysozyme activity and lactoferrin concentrations in acellular BAL fluid by sampling a similar percentage of smokers in the AUD and control groups; therefore, differences in these outcome variables independent of smoking history are more likely to be detected. We ultimately enrolled very few non-smokers with AUDs due to the high prevalence of tobacco use in our population. As such, we cannot make assumptions about the effect of AUDs in the absence of smoking on our outcome variables.
Although this work provides impetus for additional investigations of antimicrobial proteins in those with AUDs, it is not without certain limitations. Although we attempted to perform all described assays on samples from every subject and control, we were limited by the amount of BAL fluid obtained from each individual, and that a modest number of individuals were enrolled. However, subjects with AUDs and controls were balanced in terms of smoking history and had no co-morbidities. This served to diminish between-subject variability and strengthens the validity of our comparisons. The killing assay that we utilized to measure the antimicrobial effect of acellular BAL fluid proteins does not precisely replicate the in vivo environment. For example, antimicrobial effects of innate immune cells, such as alveolar macrophages, were not measured with this assay, and protein concentrations utilized in these experiments could differ from those in native ELF. However, use of this assay enabled us to experiment specifically with antimicrobial proteins from human subjects rather than a commercially available product. Although some lysozyme activity was found to be lost during vacuum concentration, we were able to perceive an effect on pneumococcal killing proportionate to the amount of protein present, and the effect was similar between AUD subjects and controls. The assay could be modified to explore the specific interactions between antimicrobial proteins and cells within collected BAL to further address these issues. We acknowledge that we examined the antimicrobial effects of one strain of S. pneumoniae, and our results might differ with other strains or pathogens. We elected to utilize a strain of a pathogen that had been utilized successfully in killing assay experiments in our laboratory, and is an important pulmonary pathogen in humans, particularly those with AUDs. Modifying this assay with the use of other strains of S. pneumoniae or other pathogens important in CAPs in those with AUDs should be examined in the future.
Pulmonary antimicrobial proteins are an important component of innate immunity. The effect of AUDs on these proteins has not been examined; however, AUDs are known to deleteriously affect other innate immune functions, so it seems plausible that antimicrobial proteins could be affected as well. Acellular BAL fluid from subjects with AUDs, who were otherwise healthy save for their excessive use of alcohol, tended to have a less potent anti-bacterial effect on the S. pneumoniae than did acellular BAL from healthy individuals, suggesting that antimicrobial proteins may be affected by the consumption of alcohol. These effects may be mediated by differences in the antimicrobial proteins lysozyme and lactoferrin, as well as the activity of other proteins and peptides. Clarifying the composition and activity of ELF proteins in AUDs can help establish their contribution to the prevention of pulmonary infections in this setting, and may provide avenues to develop novel prophylactic therapies against the development of pneumococcal pneumonia for individuals with AUDs.