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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Alcohol. 2018 Sep 5;80:119–130. doi: 10.1016/j.alcohol.2018.08.016

The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

Jeanette Gaydos 1, Alicia McNally 1, Ellen L Burnham 1
PMCID: PMC6401331  NIHMSID: NIHMS1011587  PMID: 30195043

Abstract

Community-acquired pneumonia due to Streptococcus pneumoniae occurs commonly in alcohol use disorders (AUDs). Pneumonia in the AUD patient is associated with poorer outcomes, and specific therapies to mitigate disease severity in these patients do not exist. Numerous investigations have attributed increased severity of pneumonia in AUDs to aberrant function of the alveolar macrophage (AM), a lung immune cell critical in host defense initiation. No studies have examined the response of human AMs to S. pneumoniae in AUDs. We hypothesized that the inflammatory mediators released by AMs after S. pneumoniae stimulation would differ quantitatively in individuals with AUDs compared to non-AUD participants. We further postulated that AM inflammatory mediators would be diminished after exposure to the antioxidant, N-acetylcysteine (NAC). For comparison, responses of peripheral blood mononuclear cells (PBMCs) to pneumococcal protein were also examined. Otherwise healthy participants with AUDs and smoking-matched controls underwent bronchoalveolar lavage and peripheral blood sampling to obtain AMs and PBMCs, respectively. Freshly collected cells were cultured with increasing doses of heat-killed S. pneumoniae protein, with and without exposure to N-acetylcysteine. Cell culture supernatants were collected, and inflammatory mediators were measured, including interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. IFN-γ and IL-6 were significantly higher in unstimulated AM cell culture supernatants from subjects with AUDs. After stimulation with pneumococcal protein, a dose-response and time-dependent increase in pro-inflammatory cytokine production by both AMs and PBMCs was also observed; differences were not observed between AUD and non-AUD subjects. Addition of NAC to pneumococcal-stimulated AMs and PBMCs was generally associated with diminished cytokine production, with the exception of IL-1β that was elevated in AM culture supernatants from subjects with AUDs. Our observations suggest that AUDs contribute to basal alterations in AM pro-inflammatory cytokine elaboration, but did not support consistent differences in pneumococcal-stimulated AM or PBMC inflammatory mediator secretion that were referable to AUDs.

Keywords: alcoholism, alveolar macrophage, monocytes, bronchoalveolar lavage, lung, translational research

Introduction and Background

It has been appreciated for decades that the risk for development of community-acquired pneumonia is higher among individuals with alcohol use disorders (AUDs), particularly those with the heaviest alcohol consumption (Torres, Peetermans, Viegi, & Blasi, 2013; Samokhvalov, Irving, & Rehm, 2010). Worldwide, individuals with AUDs are disproportionately affected by Streptococcus pneumoniae, the most common etiologic agent in bacterial pneumonia (van der Poll & Opal, 2009; DeRoux A. et al., 2006; Centers for Disease Control and Prevention., 2015). Moreover, infections with S. pneumoniae among patients with AUDs are marked by increased severity (Beatty, Majumdar, Tyrrell, Marrie, & Eurich, 2016), including extrapulmonary spread of disease (Falguera et al., 2011), need for ICU admission with mechanical ventilation (O’Brien, Jr. et al., 2007; Clark et al., 2013), and development of the acute respiratory distress syndrome (ARDS) (Moss et al., 2003). It follows that health care costs associated with S. pneumoniae infection among people with AUDs are disproportionately higher than infections in non-alcohol abusers (Molina, Gardner, Souza-Smith, & Whitaker, 2014; Zaridze et al., 2009). Unfortunately, efforts to minimize disease incidence and severity in AUDs by pneumococcal vaccination have been limited by poorer response to available vaccines (Benin et al., 2003), as well as difficulties in reaching target populations for vaccination efforts, such as those with unhealthy alcohol consumption, (Grau et al., 2014; Merrick et al., 2008).

Chronic alcohol exposure has been implicated in an excessive inflammatory response in tissues including the brain (Umhau et al., 2014; Abdul-Muneer et al., 2017), liver (Heymann, Trautwein, & Tacke, 2009), and lung.(O’Halloran et al., 2016) In the lung, tissue-specific alveolar macrophages (AMs) are critical in initiating and sustaining a response to pathogens (Aggarwal, King, & D’Alessio, 2014). AMs represent the majority (approximately 90%) of cells that are obtained during the course of bronchoalveolar lavage (BAL) in healthy individuals. During infection, AMs recognize pathogens, such as S. pneumoniae, via Toll-like receptors, whereupon they are ingested (Koppe, Suttorp, & Opitz, 2012; Quinton & Mizgerd, 2015). After encountering pathogens, AMs produce the pro-inflammatory mediators interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and type I interferons (IFNs) that alert neighboring cells to pathogen invaders. After infection has been addressed, AMs are re-directed towards curbing inflammation and promoting tissue repair by ingesting and eliminating immature leukocytes, preventing release of danger signals by necrotic cells, and suppressing further inflammation. Therefore, dysfunction of AMs related to harmful alcohol consumption could be one driver of poorer clinical outcomes among individuals with AUDs.

Prior studies suggest that alcohol exposure may alter human pulmonary immune activity in the absence of overt pulmonary illness. For example, enhanced interleukin-6 and −8 have been measured in proximal airway washings from humans with AUDs,(Bailey et al., 2015) and in human AM cell lysates, AUDs were associated with a pro-inflammatory profile of cytokines, chemokines and growth factors (O’Halloran et al., 2016), although measured differences in these proteins within cell-free BAL fluid from distal lung appear to be more modest.(Burnham, Kovacs, & Davis, 2013) Further, alcohol exposure contributes to formation of pulmonary immune protein adducts ingested by AMs that are associated with differences in pro-inflammatory cytokines and immunoglobulins (McCaskill et al., 2011; Wyatt et al., 2012; Sapkota et al., 2017). It has been postulated that these alterations may be attributed to alcohol’s effects on pulmonary oxidative stress (Moss et al., 2000; Yeh, Burnham, Moss, & Brown, 2007; Burnham, Brown, Halls, & Moss, 2003). Therefore, investigators have conducted translational projects to delineate mechanisms underlying AUD-associated oxidative stress on pulmonary innate immunity involving the AM. Chronic alcohol exposure has been reported to alter normal activities of AM NADPH oxidases (Yeligar, Harris, Hart, & Brown, 2012) and AM xanthine oxidoreductase (XOR) (Fini, Gaydos, McNally, Karoor, & Burnham, 2017). These alcohol-elicited AM abnormalities may potentially influence pathogen recognition and clearance in lung, predisposing individuals with AUDs to pneumonia. Importantly, clinically available therapeutics exist that may modulate signaling pathways altered by alcohol-induced oxidative stress, including the glutathione precursor N-acetylcysteine (NAC), thiazolidinediones such as rosiglitazone, and xanthine oxidoreductase inhibitors. Nevertheless, translational investigations that explore the response of AMs to pathogens, such as S. pneumoniae, and mechanisms to normalize aberrant cellular pathways in the AUD setting are lacking.

The overarching goal of these experiments was to extend prior observations regarding the association of AUDs and inflammatory mediator secretion by pulmonary and systemic immune cells after pathogen stimulation. We also wished to examine the superimposed effect of antioxidant exposure on these relationships. More specifically, we wished to measure inflammatory mediators produced by AMs and peripheral blood mononuclear cells (PBMCs) in response to varying doses of protein derived from S. pneumoniae using samples from human subjects, with and without AUDs. We hypothesized that AMs and PBMCs from subjects with AUDs would exhibit a pro-inflammatory response to stimulation with heat-killed S. pneumoniae, secreting increased quantities of IFNγ, IL-1β, IL-6, and TNFα, compared to non-AUD participants. Further, we postulated that addition of the antioxidant N-acetylcysteine (NAC) after a period of pneumococcal protein stimulation would attenuate pro-inflammatory cytokine secretion by AMs and PBMCs compared to no treatment, providing indirect evidence that oxidative stress may be in part responsible for regulation of the cells’ inflammatory responses to S. pneumoniae.

Materials and Methods

Management of Participants in Research

Subject screening, recruitment, and enrollment

Participants were recruited and enrolled as previously described (Gaydos et al., 2016). Subjects with AUDs were recruited from an inpatient detoxification facility in Denver, CO, while non-AUD subjects were recruited via approved flyers and web advertisements in the Denver metropolitan area. The Colorado Multiple Institutional Review Board approved this study. All participants provided written informed consent prior to participation.

Participant Inclusion and Exclusion Criteria.

AUD participants were eligible if they met all of the following criteria: 1) an Alcohol Use Disorders Identification Test (AUDIT) score (Reinert & Allen, 2002) of ≥8 for men or ≥5 for women, 2) alcohol use within the 7 days before enrollment, and 3) age of ≥21. To meet eligibility as a non-AUD participant, AUDIT values were required to be <8 for men or <5 for women. Exclusion criteria were chosen in an effort to minimize potential confounding related to medical comorbidities, and such as serious medical conditions requiring specialty care or prescription medication, including the need for antibiotics in the past month, as previously described (Gaydos et al., 2016). Further, an abnormal chest radiograph or spirometry resulted in exclusion, along with concurrent illicit drug use. Those subjects >55 years of age were also excluded to minimize the presence of concomitant but asymptomatic comorbidities. Whether or not participants had received pneumococcal vaccines was not assessed specifically. The ultimate sample size was chosen based on feasibility to recruit comparable numbers of AUD subjects and controls who were similar in terms of cigarette smoking, based on our group’s prior investigations (Burnham et al., 2012; Fini et al., 2017; Gaydos et al., 2016).

Clinical Protocol in Participants.

All eligible participants were admitted to the University of Colorado Hospital’s Clinical and Translational Research Center (CTRC) for blood sampling and bronchoscopy. All bronchoscopy procedures were performed utilizing telemetry monitoring and standard conscious sedation protocols as previously described (Hunninghake, Gadek, Kawanami, Ferrans, & Crystal, 1979). AUD subjects undergoing bronchoscopy had been sober for an average of 72 hours prior to the procedure. The bronchoscope was wedged into a subsegment of either the right middle lobe or the lingula. Three to four 50-ml aliquots of sterile, room temperature 0.9% saline were sequentially instilled and recovered with gentle aspiration. All aliquots were combined and used in experiments as representative of the distal airspaces. BAL samples were transported to the laboratory in sterile 50-ml conical tubes. Whole blood samples were also collected prior to bronchoscopy in a subset of participants to isolate peripheral blood mononuclear cells (PBMCs).

Experimental assays with collected research materials.

Preliminary processing of BAL and blood samples.

All participants underwent identical procedures for recruitment and enrollment, and standardized protocols for sample collection. Due to limitations in sample quantity, not all experiments described were performed in samples from every individual enrolled as noted in the results.

The total amount of saline aspirated during the BAL procedure and the total amount of saline instilled into the lung was utilized to calculate the percent yield ([quantity aspirated/quantity instilled]*100) for each procedure. BAL specimens were immediately centrifuged (900xg, 10 min) after collection to separate cells from BAL fluid. Remaining BAL cells were then re-suspended in RPMI media (Corning, Inc., Corning, NY) with S. pneumoniae and other reagents as described below. PBMCs were isolated from 10mL whole blood using a BD Vacutainer CPT Cell Preparation Tube (BD, Franklin Lakes, NJ), that promotes purification of peripheral WBCs that contain, on average, 98% mononuclear cells with 2% neutrophil contamination after centrifugation (Anonymous, 2015). CPT tubes were centrifuged immediately at 1800xg for 30 min; supernatant was decanted, and then the cells washed twice in 1xPBS (450xg, 5 min), and finally re-suspended in RPMI (Corning, Inc., Corning, NY) with additives as indicated below.

Cellular viability was determined via trypan blue exclusion; cells from all subjects were estimated to exceed 95% viability prior to culture. Cell counts were performed with both BAL and PBMCs to determine total cell number. Cytospins of BAL cells were examined after staining to determine differential cell types from a minimum of 200 cells by an observer blinded to the subject history. On average, 90±5% of BAL cells were alveolar macrophages.

In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points. S. pneumoniae was grown into log-phase, snap frozen in liquid nitrogen, and then heat-killed by autoclaving. After autoclaving, 500 μg of protein was plated and cultured (37°C, 24 hours) to ensure no colony-forming units would emerge.

Cell culture experiments utilizing BAL cells.

Experiments performed to measure cytokines and chemokines used prospectively collected, fresh BAL cells. After BAL centrifugation, 5 × 105 BAL cells/well were plated in 2 ml of RPMI medium with antibiotics (Corning) into three 12-well plates, one plate for each time point. In an effort to more closely replicate serum-free conditions in lung, serum was not added to BAL culture medium. At the time of plating, between 0 μg/mL to 10μg/mL of pneumococcal protein was added to the wells. Plates were cultured for 2 hours, 18 hours, or 24 hours (37°C, 10% CO2); then cell culture supernatant and cells were collected separately and stored at −80°C.

An additional set of experiments to examine the effect of delayed addition of N-acetyl cysteine (NAC) on AM secretion of cytokines was also performed. At the time of plating, 5 × 105 BAL cells/well were plated in 2 ml of RPMI medium with antibiotics into two separate 12-well plates. Pneumococcal protein was added to two wells on each plate at a concentration of 10μg/mL. One plate was cultured for 18 hours (37°C, 10% CO2); then cell culture supernatant and cells were collected separately and stored at −80°C. The second plate was cultured for 18 hours (37°C, 10% CO2), then NAC was added to 1 of the pneumococcal protein-treated wells at a final concentration of 2.5mM. The second plate was cultured for an additional 24 hours (37°C, 10% CO2) for a total of 42 hours, before the cell culture supernatant and cells were collected separately and stored at −80°C.

Cell culture experiments utilizing peripheral blood mononuclear cells.

After PBMC isolation, 5 × 105 cells were plated in 2 ml of RPMI medium containing 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and antibiotics in 12-well plates. Like the BAL cells, at the time of plating, between 0μg/mL to 10μg/mL of pneumococcal protein was added to the wells, and plates were cultured for 2 hours, 18 hours, or 24 hours (37°C, 10% CO2). Cell culture supernatant and cells were then collected separately and stored at −80°C.

An additional set of experiments was performed to compare the effect adding NAC in a delayed fashion, after pneumococcal protein addition, on PBMC cytokine secretion. 5 × 105 PBMCs/well were plated in 2 ml of RPMI medium with 10% fetal bovine serum and antibiotics into two 12-well plates. On both plates, pneumococcal protein was added to 2 wells at a final concentration of 10μg/mL. The first plate was cultured for 18 hours (37°C, 10% CO2); then cell culture supernatant and cells were collected separately and stored at −80°C. The second plate was cultured for 18 hours (37°C, 10% CO2), then NAC was added to 1 of the pneumococcal protein-treated wells at a final concentration of 2.5mM. The second plate was then cultured for an additional 24 hours (37°C, 10% CO2), for 42 hours total, before the cell culture supernatant and cells were collected separately and stored at −80°C.

Measurement of cytokine/chemokine secretion by BAL cells and PBMCs in culture supernatants.

IFN-γ, IL-1β, IL-6, and TNF-α were assayed in culture supernatants (Pro-inflammatory One 4-plex for tissue culture (K15052D), MesoScale Discovery, Rockville, MD) according to the manufacturer’s directions. Specific analytes were chosen based on prior work by our laboratory demonstrating differential expression of cytokines associated with the presence of AUDs. Further, these cytokines are relevant in the innate immune response to S. pneumoniae. (Gaydos et al., 2016; Jones, Simms, Lupa, Kogan, & Mizgerd, 2005; O’Halloran et al., 2016; Rabes, Suttorp, & Opitz, 2016) For all condition types, experimental replicates were performed.

Statistical analyses

Demographic data between AUD subjects and controls were compared using a t test for continuous data and Fisher’s exact test for categorical data.

The major outcome variables examined between the non-AUD and AUD subject groups were IFNγ, IL-1β, IL-6, and TNFα quantities measured in cell culture supernatants from cultured AMs and PBMCs ex vivo. Culture supernatants were assessed: (1) over a time course, without pneumococcal protein stimulation; (2) over a time course, with pneumococcal protein stimulation; (3) with varying amounts of stimulation using pneumococcal protein at a single time point; and (4) over a time course, with and without pneumococcal protein stimulation, and delayed addition of the antioxidant NAC. To compare data regarding changes in outcome variables over time, or changes in outcome variables with increasing doses of pneumococcal protein, repeated measures ANOVA statistical tests were utilized. Non-parametric Wilcoxon/Kruskal-Wallis testing was also used to examine the differences in outcome variables between non-AUD and AUD subject groups across single conditions. When comparing two conditions only (e.g. values at a single time point, with and without NAC), paired non-parametric t tests were also used.

All statistical analyses were performed using JMP 14 (SAS Institute, Inc., Cary, NC). A significance level of 0.05 was assumed throughout the study.

Results

Demographics of the study population undergoing BAL for collection of AMs.

Twenty-nine non-AUD subjects and 32 AUD subjects were enrolled between 2013 and 2015 (Table 1). The two groups were similar in terms of age, sex, and the percentage of current and former cigarette smokers. AUDIT scores were substantially higher among the AUD group. AUD participants began consuming alcohol at an average age of 16±5 years. Over 95% of AUD subjects reported drinking alcohol more than twice a week, and 85% reported consuming more than 5 drinks on a typical day. Almost 50% of AUD subjects endorsed daily binge drinking of 6 or more drinks on a single occasion. Pack-year smoking was higher in the non-AUD group. BAL leukocyte count did not differ between non-AUD and AUD groups. As previously noted (Burnham et al., 2013), smoking subjects had more elevated BAL leukocyte counts (p=0.02). However, the BAL leukocyte counts in non-smoking subjects (regardless of AUD habits) did not differ, nor did BAL leukocyte counts in smoking subjects (regardless of AUD habits). The yield of the BAL procedure did not differ between non-AUD and AUD subjects. Laboratory values, including liver function testing, did not substantially differ between the two groups.

Table 1.

Demographics of Alcohol Use Disorder Subjects and non-Alcohol Use Disorder Controls.

Non-AUD Controls, n=29 AUD Subjects, n=32 p value
Age 41±8 41±8 0.90
Sex, % men 79% 78% 1.0
AUDIT* Score, average 2±1 27±1 <0.0001
Current Smoker, % 66% 59% 0.79
 Pack-years 19±11 8±9 0.002
Fomer Smoker, % 20% 27% 1.0
 Pack-years 17±19 1±1 0.20
BAL** white blood cells (106) 12.1±7.6 11.0±5.3 0.49
 Non-current smokers 8.6±4.1 9.5±3.1 0.53
 Current smokers 13.9±8.5 11.9±6.2 0.41
Percent yield of BAL procedure 45±14% 46±14% 0.86
Peripheral white blood cells (x103), per cc3 blood 7.0±2.0 7.0±1.8 0.96
Serum total bilirubin, mg/dL 0.71±0.41 0.83±0.41 0.28
Serum albumin, g/dL 4.1±0.3 4.1±0.4 0.64
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*AUDIT=alcohol use disorders identification test;

BAL**=bronchoalveolar lavage. Percent yield of BAL procedure= (volume of fluid aspirated/volume of fluid instilled into lung)*100

AM secretion of cytokines and chemokines over time, with and without exposure to pneumococcal protein ex vivo.

For experiments, AMs from 14 non-AUD subjects and 17 AUD subjects were utilized that were similar in terms of age (p=0.19) and sex (p=1.0). The percent of current smokers between the non-AUD and AUD groups did not differ significantly (57% in non-AUD versus 53% in the AUD group, p=1.0), and pack-year history among smokers was similar (18±13 in non-AUD versus 12±11 in AUD subjects, p=0.26). Clinical laboratory values between the two groups did not differ.

In these participants, the relationship of alcohol use on ex vivo AM cytokine secretion was examined without pneumococcal exposure. Participants’ AMs were cultured as described, and supernatants were collected after two, 18, and 24 hours had elapsed (Figure 1). In the absence of any pneumococcal protein exposure, IFNγ and TNFα secretion by AUD subjects’ AMs were significantly higher compared to non-AUD subjects over the three time points. Univariable tests revealed small but statistically higher values of IL-1β at 2 hours, and IL-6 at 18 hours among subjects with AUDs. Values of IL-1β, IL-6, and TNFα increased significantly over time for both subject groups, but values of IFNγ did not change appreciably over time. In general, the most robust changes in analyte secretion for both subject types were observed to occur between the two and 18 hour time points.

Figure 1. Secretion of cytokines/chemokines over time by unstimulated alveolar macrophages.

Figure 1.

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Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured ex vivo without stimulation. Cell culture supernatant was collected after two, 18, and 24 hours had elapsed. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (1A) AUD subjects exhibited increased secretion of IFNγ compared with non-AUD subjects (p=0.007). Values did not appreciably change with time for either group. (1B) No significant differences in secretion of IL-1β were observed between AUD and non-AUD subjects in repeated measures analysis (p=0.07); however, in univariable analysis, IL-1β was relatively higher at 2 hours in AUD subjects (p=0.05, asterisk). Secretion of IL-1β increased over time (p=0.0004) in both subject types. (1C) Secretion of IL-6 tended to be higher in AUD than in non-AUD subjects (p=0.06), and in univariable analysis, IL-6 secretion was higher in AUD subjects at 18 hours (p=0.05, asterisk). Secretion of IL-6 increased over time for both subject types (p=0.008). (1D) Secretion of TNFα was relatively elevated in AUD subjects compared to non-AUD subjects (p=0.04), and TNFα increased in each group of subjects over time (p<0.0001).

Subsequently, the effect of a single dose of pneumococcal protein on ex vivo cytokine secretion by AMs over time was also assessed in participants. With 5μg pneumococcal protein stimulation, similar to what was observed in media-only conditions, AM secretion of all four analytes increased significantly between two and 24 hours (p≤0.0003 over time for each of the four analytes), regardless of subject type, Figure 2. As in the media-only conditions, the most robust changes in secretion were observed between the two hour and 18 hour time points. No significant between-groups differences in analyte secretion were observed between non-AUD and AUD groups.

Figure 2. Secretion of cytokines/chemokines over time by alveolar macrophages stimulated with single dose S. pneumoniae protein.

Figure 2.

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Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured ex vivo with 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected after two, 18, and 24 hours had elapsed. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (2A) AUD subjects exhibited higher secretion of IFNγ, but this did not achieve statistical significance (p=0.10). IFNγ quantity increased significantly over time for both subject types (p<0.0001). (2B) No significant differences in secretion of IL-1β were observed between different subject types, however, secretion of IL-1β increased over time (p=0.0003). (2C) Secretion of IL-6 by AMs from subjects with AUDs tended to be higher (p=0.12). Secretion of IL-6 increased significantly over time in each subject group (p<0.0001). (2D) Secretion of TNFα did not differ between groups (p=0.26), but did increase in each group of subjects over time (p<0.0001).

Given that cytokine secretion values had increased significantly the 18 hour time point, this time point was chosen to evaluate the impact of differing doses of pneumococcal protein on AM cytokine and chemokine secretion. Freshly collected AMs from the same non-AUD (n=14) and AUD (n=17) participants were cultured with pneumococcal protein doses ranging from 0 μg to 10 μg. At the 18 hour time point, AM secretion of IFNγ, IL-1β, IL-6, and TNFα were noted to rise significantly with increasing pneumococcal exposure in both subject types (Figure 3). Absolute values of IFNγ secretion were higher in cell culture supernatants from AUD subjects (p=0.008, 3A). At the 10μg dose, AUD subjects’ IFNγ secretion was approximately four-fold higher than in non-AUD subjects (p=0.04). For IL-1β, IL-6, and TNFα measured in cell culture supernatants, although absolute values tended to be higher with increasing pneumococcal protein exposure in samples from AUD subjects, they did not significantly differ compared to non-AUD subjects.

Figure 3. Secretion of cytokines/chemokines by alveolar macrophages stimulated with increasing doses of S. pneumoniae protein.

Figure 3.

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Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured in the presence of heat-killed S. pneumoniae protein, at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (3A) IFNγ secretion by AMs rose with exposure to increasing doses of pneumococcal protein (p=0.0002). IFNγ secretion was more elevated in the AUD group (p=0.008). (3B) IL-1β secretion by AMs rose with increasing doses of pneumococcal protein (p=0.02), but was not different between non-AUD and AUD groups. (3C) IL-6 secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups. (3D) TNFα secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups.

Differences in AM secretion of cytokines and chemokines over time with exposure to pneumococcal protein, in the presence and absence of the antioxidant N-acetylcysteine (NAC).

Samples from 30 (non-AUD, n=15; AUD, n=15) of the total participants in the cohort were subjected to experiments where NAC was added in a delayed fashion after 18 hours of exposure to 10 μg pneumococcal protein. These experiments were performed to determine if addition of NAC after AM exposure to of S. pneumoniae protein for several hours would alter cytokine secretion; this would more closely approximate the clinical condition. We also wished to verify the observations already described. Non-AUD and AUD participants for these experiments did not differ in terms of age (p=0.29) and sex (p=1.0). The percentage of current smokers was similar between the two subject types (73% in non-AUD versus 67% in AUD subjects, p=1.0); non-AUD subjects had a more robust pack-year consumption than did AUD subjects (20±9 pack-years in non-AUD versus 6±7 in AUD subjects, p=0.001). White blood cell count, serum total bilirubin, and albumin did not differ between the two groups.

After AMs had been cultured for 18 hours without pneumococcal protein, cell culture supernatants from AUD subjects contained significantly greater IFNγ (p=0.02), as well as IL-6 (p=0.03), confirming prior observations. Values of IL-1β and TNFα did not differ between the two groups in unstimulated conditions (Figure 4). With exposure to pneumococcal protein over the 18 to 42 hour time point, in the absence of NAC, quantities of IFNγ, IL-1β, IL-6, and TNFα measured in both non-AUD and AUD subjects’ cell culture supernatants increased (p≤0.002 for each comparison), and were higher than unstimulated conditions. Examining pneumococcal-stimulated AM cell culture supernatants at the 42 hour time point, where NAC was added at the 18 hour time point compared to those where NAC was not added, quantities of IFNγ, IL-6, and TNFα were diminished (p<0.0001 for all comparisons). This was true regardless of participants’ AUD history. However, addition of NAC at 18 hours did not appreciably impact the quantity of IL-1β at 42 hours among non-AUD subjects (p=0.06), and was associated with a relative increase in IL-1β by AUD subjects’ AMs at the 42 hour time point (p=0.009).

Figure 4. Differences in AM secretion of cytokines/chemokines over time and exposure to pneumococcal protein, in the presence and absence of the antioxidant N-acetylcysteine (NAC).

Figure 4.

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Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=15) and AUD subjects (n=15) were cultured up to 42 hours, with and without the addition of heat-killed S. pneumoniae (Sp, 10μg). In a subset of wells, N-acetylcysteine (NAC) was added after 18 hours of exposure to S. pneumoniae. Cell culture supernatants were collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (4A) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IFNγ (p=0.02) (denoted with asterisk). With pneumococcal protein stimulation, IFNγ secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.002). The addition of NAC at 18 hours during the 42-hour time course was associated with less IFNγ in cell culture supernatants compared to non-NAC treated AMs at 42 hours (p<0.0001 for both non-AUD and AUD subjects). (4B) After pneumococcal protein stimulation, IL-1β secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.0004). Compared to cell culture supernatants at 42 hours without NAC, the addition of NAC at 18 hours during the 42-hour time course was associated with a non-significant rise in supernatant IL-1β among non-AUD subjects (p=0.06), and significantly higher IL-1β in AUD subjects (p=0.009). Cell culture supernatants from pneumococcal protein-stimulated, NAC-treated AMs at 42 hours compared supernatants from pneumococcal protein-stimulated AMs at 18 hours contained IL-1β values that were higher both in non-AUD (p=0.003) and AUD subjects (p=0.01). (4C) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IL-6 (p=0.03) (denoted with asterisk). With pneumococcal protein stimulation, IL-6 secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.009). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 secretion than that by non-NAC treated AMs at 42 hours (p=0.0001 for both non-AUD and AUD subjects). Supernatant IL-6 quantities at the 18 hour time point were significantly higher than those in supernatants from pneumococcal protein-stimulated, NAC-treated AMs in both subject types (p=0.05 for non-AUD, p=0.0001 for AUD). (4D) With pneumococcal protein stimulation, TNFα secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.004). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in supernatants than what was measured in non-NAC treated AM culture supernatants at 42 hours (p=0.0001 for both non-AUD and AUD subjects), with values that were significantly less than AM culture supernatants at the 18 hour time point (p=0.02 for non-AUD, and p=0.0006 for AUD). * indicates p≤0.03 between non-AUD and AUD subjects, without pneumococcal stimulation, at 18 hours in culture. # indicates p<0.0001 between NAC and non-NAC treated conditions at 42 hours. & indicates p≤0.05 between non-NAC-treated 18-hour condition and NAC-treated, 42-hour condition.

IFNγ secretion by pneumococcal-stimulated, NAC-treated AMs at 42 hours was not quantitatively different from IFNγ secretion by pneumococcal-stimulated cells the 18 hour time (p=ns). However, IL-1β secretion by pneumococcal-stimulated, NAC-treated AMs at 42 hours was more elevated compared to pneumococcal-stimulated AMs at the 18 hour time point, both in non-AUD (p=0.003) and AUD (p=0.01) subjects. For both IL-6 and TNFα measured in cell culture supernatants from pneumococcal-stimulated, NAC-treated AMs at 42 hours, diminished cytokine secretion was evident compared to pneumococcal-exposed cells the 18 hour time point. This was true for both non-AUD subjects (p≤0.05) and in AUD subjects (p≤0.0006).

To assess the possibility that addition of NAC at the 18 hour time point decreased cellular viability, secretion of cytokines by AMs from (1) cells exposed to media only for 42 hours, and (2) cells exposed to NAC at 18 hours, followed by additional culture to 42 hours, were compared. For all analytes, cytokine secretion was low, and not significantly different from the media-only (non-pneumococcal stimulated) conditions (data not shown).

Demographics of the study population with PBMCs examined experimentally.

A subset of non-AUD (n=8) and AUD (n=8) subjects from the cohort where AMs had been obtained for experiments with and without NAC, described above, also had PBMCs isolated from whole blood that were examined concurrently. The non-AUD and AUD groups in this subset were similar in terms of age (p=0.20), sex (p=1.0), and current smoking (p=1.0). Peripheral WBC counts were also similar between the two groups (p=0.48). Collected PBMCs were cultured as described, and culture supernatants were collected after two, 18, and 24 hours had elapsed.

Differences in PBMC secretion of cytokines and chemokines with increasing time exposure, and increasing pneumococcal protein exposure.

Similar to what was observed in AM experiments using these participants’ specimens, PBMC secretion of all four analytes in unstimulated conditions was relatively low, but increased over time (data not shown). Among AUD subjects, values for IL-1β (p=0.03) in supernatants was noted to be greatest at the 18 hour time point, as was IL-6 (p=0.06). Values of IFNγ and TNFα did not appreciably vary over time in unstimulated conditions (data not shown).

All four analytes secreted by PBMCs from either non-AUD or AUD subjects significantly increased over time after exposure to 5μg pneumococcal protein (p≤0.002 for each of the four analytes), Figure 5. The most robust increase occurred between the two and 18 hour time points, as was observed previously in experiments with AMs. No significant differences in the quantities of the four analytes in PBMC culture supernatants were found to be present between the non-AUD and AUD groups at any time point. The magnitude of cytokine elaboration after exposure to pneumococcal protein at the 5μg dose was similar between PBMCs and AMs.

Figure 5. Secretion of cytokines/chemokines over time by peripheral blood mononuclear cells stimulated with single dose S. pneumoniae protein.

Figure 5.

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Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected at two, 18, and 24 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (5A) For IFNγ, AUD subjects exhibited non-significantly higher secretion (p=0.10). Values increased significantly over time (p<0.0001). (5B) For IL-1β, no significant between-groups differences in secretion were observed (p=0.97), however, secretion increased over time (p=0.002). (5C) For IL-6, secretion values from AUD PBMCs did not differ between groups (p=0.35), but secretion increased over time (p=0.003). (5D) For TNFα, values did not differ between groups (p=0.54), and but did increase in subjects over time (p=0.007).

The 18 hour time point was again chosen to evaluate the impact of escalating doses of pneumococcal protein exposure (0 μg to 10 μg) on cytokine/chemokine secretion by PBMCs. Secretion of IFNγ, IL-1β, IL-6, and TNFα by PBMCs measured in cell culture supernatants significantly increased with increasing pneumococcal protein exposure in both non-AUD and AUD subjects (p≤0.04 for each analyte), Figure 6. For all of these four analytes, values between non-AUD and AUD subjects did not differ. In univariate analyses, cell culture supernatant from unstimulated PBMCs obtained from AUD subjects contained more IL-6 (exposure to 0 μg pneumococcal protein).

Figure 6. Secretion of cytokines/chemokines by peripheral blood mononuclear cells stimulated with increasing doses of S. pneumoniae protein.

Figure 6.

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Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of heat-killed S. pneumoniae (Sp), at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours, and interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured. (6A) IFNγ secretion over the dose range increased with increasing doses of pneumococcal protein (p=0.04), but values between groups did not differ. (6B) IL-1β secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups. (6C). IL-6 secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups, except at the 0μg (media only) condition (p=0.04, asterisk). (6D) TNFα secretion increased with increasing pneumococcal protein doses (p=0.001), but were not different between non-AUD and AUD groups.

Differences in PBMC secretion of cytokines and chemokines with increasing time exposure to pneumococcal protein, with and without the antioxidant N-acetylcysteine (NAC).

The impact of adding NAC on cytokine elaboration by PBMCs was then addressed (Figure 7). After 10 μg pneumococcal exposure, cytokine or chemokine quantities in supernatants did not vary by AUD history at either the 18 hour or 42 hour time points. In paired analyses, examining cytokines and chemokines present in pneumococcal protein-stimulated PBMC cell culture supernatants collected at the 42 hour time point, cytokine values were determined to be relatively diminished when PBMCs were treated with NAC at 18 hours. This was true regardless if the PBMCs originated from either non-AUD or AUD subjects. In general, cytokine and chemokine values measured in cell culture supernatants at 42 hours from the previously NAC-exposed PBMCs were on par with PBMC cell culture supernatants collected from pneumococcal protein-stimulated supernatants at 18 hours, though values were statistically lower at the 42 hour time point for IFNγ and TNFα among non-AUD participants.

Figure 7. Differences in peripheral blood mononuclear cell secretion of cytokines/chemokines over time and exposure to pneumococcal protein, in the presence and absence of the antioxidant N-acetylcysteine (NAC).

Figure 7.

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Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=10) and AUD subjects (n=10) were cultured up to 42 hours, with and without the addition of 10 μg heat-killed S. pneumoniae (Sp). In some wells, N-acetylcysteine (NAC) was added after 18 hours in culture. Culture media was collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (7A) With pneumococcal protein stimulation, IFNγ secretion by PBMCs rose significantly between the 18 and 42 hour time points (p=0.04). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IFNγ secretion than that by non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects), with values that were significantly different than PBMCs at the 18 hour time point in non-AUD subjects only (p=0.02). (7B) With pneumococcal protein stimulation, IL-1β secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.92). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-1β secretion than that by non-NAC treated PBMCs at the 42 hour time point (p=0.002 for non-AUD subjects; p=0.02 for AUD subjects). Values among NAC-treated cells at the 42 hour time point approximated those in untreated cells at the 18 hour time point (p=ns). (7C) With pneumococcal protein stimulation, IL-6 secretion by PBMCs tended to rise between the 18 hour and 42 hour time points, but not significantly (p=0.09). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 in cell culture supernatants than that found in supernatants from non-NAC treated PBMCs at the 42 hour time point (p=0.002 for both non-AUD and AUD subjects); values were not significantly different than PBMCs at the 18 hour time point for both subject types. (7D) With pneumococcal protein stimulation, TNFα secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.44). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in cell culture supernatants than in non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects); TNFα quantity in cell culture supernatant from pneumococcal protein -stimulated, NAC treated cells at 42 hours were significantly less than pneumococcal protein-stimulated PBMCs at the 18 hour time point among non-AUD subjects only (p=0.002). # indicates p≤0.02 between NAC and non-NAC treated conditions. & indicates p≤0.02 between 18-hour condition and NAC-treated, 42-hour condition.

Discussion

In these investigations, we sought to define the impact of S. pneumoniae protein exposure on the inflammatory response by the lung’s most important cellular innate immune effector, the alveolar macrophage, as well as by systemic immune cells in the bloodstream. These data are unique in that they examine live human immune cells subjected to long-term, harmful alcohol exposure in vivo after exposure to proteins from a key pulmonary pathogen, S. pneumoniae, during the first moments of the immune response. Without stimulation, AMs in the AUD setting exhibited enhanced secretion of pro-inflammatory cytokines, particularly IFNγ and TNFα, compared to controls; in a separate cohort of AUD participants and controls, augmented basal secretion of IFNγ by AMs was again observed, along with elevations in IL-6 secretion. Addition of heat-killed pneumococcal protein was associated with robust secretion of all four cytokines in a dose-response fashion, regardless of AUD history. Adding the antioxidant NAC to cultured AMs hours after pneumococcal stimulation was associated with suppressed secretion of IFNγ, IL-6, and TNFα in supernatants at 42 hours both in AUD and non-AUD subjects. In contrast, NAC exposure did not alter IL-1β secretion by non-AUD AMs, and was associated with higher IL-1β at 42 hours in supernatants from AUD subjects. In parallel experiments with PBMCs, no basal differences in analytes present in cell culture supernatants were observed. As with AMs, most analytes in PBMC cell culture supernatants increased with increasing doses of pneumococcal protein. Addition of NAC to PBMCs after 18 hours’ pneumococcal stimulation was associated with diminished quantities of all analytes (including IL-1β) at 42 hours compared to no NAC treatment. Our results suggest a potential effect of antioxidants in altering immune cell inflammatory responses to pneumococcus. Although our findings do not support a substantial relationship between AUDs and immune cell inflammatory mediator production after pneumococcal stimulation, methods we have established may be useful to query alternative mediators and mechanisms in human cells to advance knowledge regarding the morbidity of pneumococcal disease among people both with and without AUDs.

Our prior investigations using cells from human BAL have consistently demonstrated a pro-inflammatory relationship between AUDs and inflammation. Initial investigations using BAL cells containing more than 90% AMs indicated a correlation between AUDs and elevated IFNγ and TNFα in cell protein, along with IL-6 mRNA expression.(O’Halloran et al., 2016) In subsequent studies, employing a fresh cell culture strategy analogous to the current work, increased expression of IFNγ and IL-1β was observed in LPS-stimulated AMs from AUD subjects cultured ex vivo compared to controls. (Gaydos et al., 2016) A similar pattern of heightened cytokine expression by AMs was also observed in AUD non-smokers after stimulation with lipoteichoic acid (LTA), found on the surface of gram-positive organisms. In this prior work, the magnitude of cytokine release by AMs was similar after LPS or LTA stimulation. In contrast, PBMCs from these same subjects exhibited a much more robust response with LPS. Interestingly, the magnitude of AM cytokine expression after stimulation with an identical amount of LTA in our prior study compared to cytokine expression after pneumococcal protein stimulation we report in this work was quantitatively greater. Collectively, these data suggest that the inflammatory response to a given stimulus can vary according to cell type and the stimulus itself, and less likely represents a non-specific response.

Elevations in serum cytokine levels have been reported in the setting of chronic alcoholic liver disease and alcoholic hepatitis that are believed to be derived from hepatocytes and Kupffer cells, the resident phagocyte of the liver (Fukui, 2005; McClain, Song, Barve, Hill, & Deaciuc, 2004). Moreover, elevations of serum TNFα (Gonzalez-Reimers et al., 2007) and IFNγ have been reported in alcohol-dependent subjects with and without cirrhosis (Gonzalez-Reimers et al., 2012), suggesting the potential for increased Th-1 lymphocyte activity as a driver. Collectively, these data implicate that enhanced inflammation elicited by excessive alcohol use promotes tissue damage, possibly from excessive activation of the immune system. However, any alterations in immune cell inflammatory response related to excessive alcohol consumption are certain to be complex, and involve factors including the pathogen involved, cell-cell interactions, and organ type, among others. Additionally, the impact of AUDs on inflammatory mediator production is only one dimension of their activity. Evaluation of additional AM functions, such as phagocytosis, apoptosis, or interactions with cells involved in adaptive immunity, are critical. (Boe et al., 2010; Yeligar, Harris, Hart, & Brown, 2014) Future investigations with human samples that employ single-cell techniques can better define cell type(s) responsible for elaborating pro-inflammatory mediators and the impact of AUDs on cellular phenotype. However, additional ex vivo manipulation of freshly collected samples to isolate single cells may contribute to functional alterations that could confound the cytokine expression or cell phenotype that occurs in vivo, and would eliminate immune cell-cell interactions that exist in vivo. Although it remains possible that lymphocytes contributed to cytokine expression, particularly IFNγ, AMs have been reported by others to secrete IFNγ after stimulation (Darwich et al., 2009; Robinson & Nau, 2008). Data presented here complement our earlier observations that employed freshly collected pulmonary and systemic immune cells in the context of AUDs. Further, use of a clinically relevant pathogen, S. pneumoniae, highlights the possibility of using this system to conduct additional translational investigations, including additional functional studies that will enable a more complete understanding of the nature of immune responses in the setting of chronic alcohol exposure. Complementary investigations examining the impact of freezing cells prior to performing functional assays may also streamline subsequent investigations.

The association of NAC with diminished cytokine production hours after pneumococcal stimulation suggests the possibility that pneumococcal proteins promote cytokine release by immune cells through mechanisms involving oxidative stress. Although the effect of NAC to limit pro-inflammatory cytokine expression was not limited to immune cells from subjects with AUDs, chronic alcohol exposure has been specifically associated with pulmonary oxidative stress through depletion of glutathione (GSH), the lung’s most important antioxidant (Yeh et al., 2007; Burnham et al., 2003). NAC is a direct precursor for GSH synthesis, and has been used extensively for glutathione deficiency in a variety of settings (reviewed in (Elbini, I et al., 2016). Notably, additional investigators have similarly implicated GSH depletion in altering proper AM response to the pathogen K. pneumoniae (Yeligar et al., 2014). In an in vivo murine model, oral GSH normalized expression of NADPH oxidase synthase isoforms and reactive oxidant species production, leading to improved phagocytic function of AMs and clearance of K. pneumoniae from AMs and the airways in these animals. In prior investigations (Gaydos et al., 2016), adding NAC concurrently with either LPS or LTA in a culture system with AMs or PBMCs led to abrogation of pro-inflammatory cytokine secretion, particularly in individuals without AUDs. However, NAC’s ability to halt pro-inflammatory cytokine and chemokine secretion among individuals with AUDs was more limited. In the present investigations, the potential of NAC to halt cytokine expression after a several hour period of pathogen exposure more realistically examines NAC’s therapeutic potential in the clinical setting (i.e. as might be expected in pneumococcal pneumonia). Importantly, addition of NAC after pneumococcal exposure appeared to “arrest” cytokine production by both AMs and PBMCs, regardless of AUD history, with the exception of IL-1β. The lack of an appreciable effect of NAC on AM IL-1β secretion suggests a relative increase in oxidative stress in the setting of AUDs that was not attenuated by the 2.5mM dose of NAC. Importantly, NAC has other reported properties that might bolster interest in its therapeutic use, including the ability to reduce bacterial growth of S. pneumoniae in vitro (Volgers et al., 2017), synergistic effects with antibiotics commonly used to treat S. pneumoniae (Landini et al., 2016), and mucolytic capabilities (Hurst, Shaw, & LeMaistre, 1967). Investigations by our group and others have proposed remediation of alcohol-induced oxidative stress through administration of antioxidants; however, it should be recognized that chronic alcohol exposure may also influence the AM inflammatory profile via other mechanisms such as inflammasome activation (Hoyt et al., 2017) and enhancement of xanthine oxidoreductase activity (Fini et al., 2017).

The use of inhaled (nebulized) NAC therapy targeted to patients with pneumonia, particularly those with AUDs as a mechanism to improve cellular function may be a practical strategy to restore depleted GSH in clinical practice given NAC’s widespread availability and relatively low cost (reviewed in (Rushworth & Megson, 2014). Nebulized NAC has been used safely in humans at doses ranging from 176mg to 1000 mg in saline solutions at volumes from 2–10mL, from two to four times daily. Intravenous NAC has similarly been used for clinical indications at doses of 150–200mg/kg provided via bolus and infusion, and is similarly well-tolerated in humans. (Beximco Pharma, 2018; Klein-Schwartz & Doyon, 2011; Sun, Liu, & Zhao, 2016) In comparison, our cell culture experiments involved exposure of a small (0.5 million) number of cells to approximately 0.82 mg of NAC in media. Given the effect of NAC we observed on cytokine secretion by AMs and PBMCs, with manifold higher doses being commonly administered in clinical medicine, it appears possible that NAC administration using accepted strategies (either via nebulization or intravenously) would provide similar effects to those observed in our ex vivo experiments.

Our observations are not without limitations. First, the number of participants enrolled was relatively small, though in keeping with prior investigations. Moreover, we were able to replicate some observations across two independent cohorts of subjects with AUDs and controls that enhance their validity. Although AUD and control participants were similar in terms of current cigarette smoking, the smoking exposure (e.g. pack-years) was not specifically examined in these investigations, and may have confounded on our results. The additive impact of AUDs and smoking are important to consider in future studies. Notably, we did not specifically assess the vaccination status of participants; receipt of a prior pneumococcal vaccination may have influenced our results. However, it has been suggested that pneumococcal vaccination among adults with chronic illnesses affords less protection from pneumococcal infection (Moberley, Holden, Tatham, & Andrews, 2013), and it has also been reported that individuals with substance dependence, including alcohol addiction, are less likely to participate in strategies to manage their chronic illness, such as routine vaccines.(Kim et al., 2011) We acknowledge that our experiments employed ex vivo culture of fresh cells that may have contributed to unmeasured alterations in these cells’ phenotype and behavior. Nevertheless, the methods chosen enabled us to examine the phenotype of a relatively pure population of cells with a legitimate in vivo exposure to alcohol. An experimental design where human subjects with AUDs are challenged with live pneumococcus would be optimal, but not feasible, and would introduce additional complexities in interpretation of the data. Therefore, heat-killed, rather than live, bacteria were employed in these experiments to enable a focus on understanding the initial inflammatory response to pneumococcal proteins on immune cells. Subsequent investigations using live bacteria in animal models will be important to fully understand the impact of AUDs on both the innate and adaptive immune response that certainly entails involvement by numerous cell types. Additionally, it remains possible that NAC’s effect on inflammatory mediator expression occurred through impairing cellular viability. However, doses used were well within the range of those not associated with naïve macrophage viability (Volgers et al., 2017); moreover, NAC did not uniformly impact secretion of all analytes (e.g. IL-1β), further suggesting preservation of cellular viability. Finally, specific immune cell types responsible for cytokine expression are unclear. Additional experiments requiring ex vivo manipulation will need to be performed to determine cell(s) responsible for driving inflammation upon exposure to S. pneumoniae, including additional mechanistic studies.

In summary, basal secretion of specific cytokines involved in inflammation by AMs derived from subjects with AUDs were found to differ from AMs in a matching population of non-AUD participants. Exposure to protein derived from S. pneumoniae elicited a dose-dependent response by lung and circulating immune cells from AUD participants and controls without AUDs that was relatively similar. Elaboration of specific cytokines after pneumococcal protein exposure was attenuated by the addition of the antioxidant NAC. Future investigations to more completely understand the impact of AUDs on innate immune effectors using translational approaches will help understand reasons for severity of pneumonia among people with AUDs, and facilitate best practices to prevent or limit morbidity.

Acknowledgements

The authors wish to thank clients and staff from Denver CARES, as well as the staff from the University of Colorado Hospital’s Clinical and Translational Research Center, for their support of this investigation.

Funding Sources

This work was supported by the National Institutes of Health (R24AA019661 and UL1 TR002535).

Contributor Information

Jeanette Gaydos, Email: Jeanette.gaydos@ucdenver.edu.

Alicia McNally, Email: Alicia.mcnally@ucdenver.edu.

Ellen L. Burnham, Email: Ellen.burnham@ucdenver.edu.

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