Alcohol induced mitochondrial oxidative stress and alveolar macrophage dysfunction

Yan Liang, Frank L. Harris and Lou Ann S. Brown

BioMed Research International. (Annual 2014):

DOI: http://dx.doi.org/10.1155/2014/371593

Copyright: COPYRIGHT 2014 Hindawi Publishing Corp.

http://www.hindawi.com/journals/bmri/

Abstract:

An alcohol use disorder increases the risk of invasive and antimicrobial resistant community-acquired pneumonia and tuberculosis. Since the alveolar macrophage (AM) orchestrates the immune response in the alveolar space, understanding the underlying mechanisms by which alcohol suppresses AM phagocytosis is critical to improving clinical outcomes. In the alveolar space, chronic alcohol ingestion causes severe oxidative stress and depletes antioxidants which are critical for AM function. The mitochondrion is important in maintaining cellular redox balance and providing the ATP critical for phagocytosis. The focus of this study was to understand how alcohol triggers mitochondrial reactive oxygen species (ROS), stimulates cellular oxidative stress, and induces AM dysfunction. The current study also investigated the capacity of the mitochondrial targeted antioxidant, mitoTEMPOL (mitoT), in modulating mitochondrial oxidative stress, and AM dysfunction. Using in vitro ethanol exposure and AMs from ethanol-fed mice, ethanol promoted mitochondrial dysfunction including increased mitochondrial ROS, decreased mitochondrial membrane potential, and decreased ATP. Treatment with mitoT reversed these effects. Ethanol-induced decreases in phagocytosis and cell viability were also attenuated with mitoT. Therefore, antioxidants targeted to the mitochondria have the potential to ameliorate ethanol-induced mitochondrial oxidative stress and subsequent decreases in AM phagocytosis and cell viability.

1. Introduction

Both acute and chronic alcohol consumption have well-documented effects on the immune system leading to increased susceptibility to community acquired pneumonia and tuberculosis [1]. When subjects with an alcohol use disorder get pneumonia, they are more likely to be infected with serious Gram-negative bacteria [2] and these increased risks occur even in those who do not meet the diagnostic criteria for an alcohol use disorder [3]. This results in a higher rate of intensive care use, longer inpatient stays, higher healthcare costs, and a 2-4 times greater mortality rate [4]. There is also an increased risk of ventilator-associated pneumonia which worsens the morbidity and mortality rates [5]. Alcohol abuse is also associated with a 2-3-fold increased risk of the acute respiratory syndrome (ARDS), representing ~50% of all ARDS cases with an average age of 30-35 3]. For subjects without a history of alcohol abuse, pneumonia will lead to sepsis in ~35% of the cases and ~30% will progress to ARDS. In contrast, pneumonia will lead to sepsis in ~60% of the cases if the subject has a history of alcohol abuse and 70% will progress to ARDS [3].

A seminal feature is that chronic alcohol abuse causes severe oxidative stress in the fluid lining the alveolar space, which includes the depletion of the critical antioxidant glutathione (GSH) and oxidation of the GSH/GSSG redox state by ~40 mV in subjects with an alcohol use disorder [6,7]. GSH depletion and oxidation within the alveolar space are particularly critical for alveolar macrophages (AM) since they are constantly bathed by this fluid and depend on this GSH pool for cellular uptake and protection against the oxidative stress generated during immune responses. Residing at the inner epithelial surfaces of airway and alveoli, AMs are the only lung phagocytes exposed directly to the environment. Therefore, AMs represent the first line of cellular defense in the lower respiratory tract [8]. However, oxidative stress can impair AM phagocytosis [9, 10]. In addition to impaired clearance of microbes, impaired phagocytosis can cause insufficient clearance of dying or dead cells and lead to pathological inflammation. Therefore, alcohol-induced oxidative stress can be a critical contributor to pulmonary pathophysiology, risk of infection, and contribute to the increased risk of tissue injury associated with ARDS.

There are multiple cellular sources of reactive oxygen species (ROS) including the mitochondria, the cytochrome P450 family, xanthine oxidoreductase, peroxisomes, cyclooxygenases, lipoxygenases, and the family of NADPH oxidases [11]. The consequences of the ROS depend on the type of the ROS generated, the amount of ROS, and where it is generated. Under resting conditions, the majority of the cellular ROS generated is derived from the mitochondria where ~90% of the oxygen used by a cell is consumed during energy metabolism [12]. In this mitochondrial process, nicotinamide adenine dinucleotide (NADH) is oxidized to support electrochemical coupling of oxidative phosphorylation and ATP synthesis [13-16]. However, respiration also generates ROS such as superoxide anions ([O.sub.2.sup.*-]), hydrogen peroxide ([H.sub.2][O.sub.2]), and hydroxyl radicals ([sup.*]OH). To protect against the ROS generated during respiration, mitochondria also maintain redox balance through numerous ROS defense systems including mitochondrial manganese superoxide dismutase (MnSOD), GSH, thioredoxin 2 (Trx2), and catalase [17]. Neutralization of mitochondrial ROS is critical for mitochondrial function and, ultimately, cellular functions but low-level concentrations of ROS are also required for signal transduction [18]. During respiration, the NADH is oxidized to [NAD.sup.+] and the [NAD.sup.+]/NADH ratio has been recognized as a key regulator in energy metabolism, aging, and immunological functions [19]. For example, decreases in [NAD.sup.+] or in the [NAD.sup.+]/NADH are associated with increased production of superoxide by the mitochondria and subsequent alteration of the mitochondrial redox system [20-22].

Alcohol metabolism can interrupt this complex integrated redox system within the mitochondria. Whether it is metabolized by alcohol dehydrogenase or cytochrome P450, the primary metabolite produced during alcohol metabolism is acetaldehyde. Within the mitochondria, acetaldehyde is metabolized by mitochondrial aldehyde dehydrogenase (ALDH2) [23] which uses [NAD.sup.+] as a cofactor. Thus, acetaldehyde metabolism decreases mitochondrial [NAD.sup.+] pools and increases NADH. The resulting decreases in the [NAD.sup.+]/NADH ratio and subsequent increases in mitochondrial ROS can change the mitochondrial redox balance leading to cellular oxidative stress and damage. Our research team has previously demonstrated that chronic alcohol ingestion resulted in impaired phagocytosis by AMs and chronic oxidative stress was central to the impaired immune functions [9, 10], while alcohol-induced upregulation of ROS through NADPH oxidases is linked to impaired phagocytosis; we speculated that there was also a role for alcohol-induced mitochondrial ROS generation in impaired AM phagocytosis. The results presented in this paper demonstrate that chronic alcohol ingestion in a mouse model induced mitochondrial ROS generation and mitochondrial dysfunction which contribute to impaired AM phagocytosis. Treatment with mitochondrial specific antioxidants reversed mitochondrial dysfunction and restored phagocytosis.

2. Materials and Methods

2.1. Mouse Model of Chronic Ethanol Ingestion.

All animal studies were performed in accordance with the National Institutes of Health guideline outlined in the Guide for the Care and Use of Laboratory Animals. All described protocols were reviewed and approved by the Emory University Institutional Animal Care and Use Committee. Mice (C57BL/6; age 6-8 weeks) were fed standard laboratory chow ad libitum with incremental increases of ethanol in the drinking water over 3 weeks (5%/week) to a final concentration of 20%. Mice were maintained at 20% ethanol (EtOH) in the drinking water for 10-12 weeks (n = 5/group) [24, 25]. The controls were pair-fed in order to control for the calories due to EtOH as well as any differences in food intake. The weight of the chow consumed by the mice with ethanol in the drinking water is routinely determined and this historical data was then used to establish a pair-feeding model for the controls. This regimen produced clinically relevant elevations in blood alcohol concentrations of 0.12% [+ or -] 0.03, as published by our group [26] and others [27, 28]. After euthanasia, tracheas were cannulated and a bronchoalveolar lavage (BAL; three 1 mL of saline) performed. Mouse AMs (mAMs) were then isolated from the fluid by centrifugation at 1000 xg for 10 min. After differential staining with Diff-Quik (Dade Behring, Newark, DE) and counting with a hemocytometer, the cell population was determined to be ~95% alveolar macrophages. The cell pellet was resuspended in RPMI 1640 medium containing 2% FBS and 1% penicillin/streptomycin and cells were incubated at 37 [degrees]C in 5% C[O.sub.2] atmosphere before the experiments outlined below were performed.

2.2. MH-S Cell Culture and EtOH Exposure.

The mouse AM cell line, MH-S (American Type Culture Collection, Manassas, VA), was used as a model system for studying the direct effects of EtOH exposure in vitro. Cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin and incubated at 37[degrees]C in a 5% C[O.sub.2] atmosphere. MH-S cells were treated with 0.2% EtOH for 5 consecutive days with the media changed daily. This EtOH concentration (0.2%) is representative of the blood alcohol content (BAC) when a 120 lb person consumes 5 drinks at a single sitting [28-30]. During the last 24hr of the 5d EtOH treatment, some cells were also treated with the mitochondria-targeted antioxidant mitoTEMPOL (mitoT, 100 [micro]M) [31].

2.3. Measurement of Intracellular ROS Generation.

After EtOH exposure, the cellular ROS sensitive probe CM-[H.sub.2]DCFDA (Invitrogen; Carlsbad, CA) and the mitochondrial superoxide probe mitoSOX (Invitrogen; Carlsbad, CA) were added to the medium (10 [micro]M, 30 min, 37 [degrees]C). Cells were then harvested, washed, and resuspended in phosphate buffered saline (PBS) for FACS analysis by BD Canto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). CM-[H.sub.2]DCFDA and MitoSOX were excited at 488 nm and detected at 530 [+ or -] 15 nm or 585 [+ or -] 42 nm, respectively. Data analysis was performed using Flowjo (http://www.flowjo.com/).

2.4. Measurement of Mitochondrial Membrane Potential.

After the different treatments, the mitochondrial membrane potential was determined by incubating the cells with tetra-methylrhodamine, ethyl ester (10 nM, 30 min, 37 degrees]C; TMRE; Sigma, St. Louis, MO). This cell-permeable, positively charged, red-orange fluorescent dye is readily sequestered by active mitochondria due to the relative negative charge of the fluorophore. However, depolarization of the mitochondrial membrane results in a failure to sequester TMRE. Cells were then harvested, washed, resuspended in PBS, and analyzed by FACS analysis. Data analysis was performed using Flowjo.

2.5. Measurement of Mitochondrial ATP Production.

ATP production was measured by a plate reader bioluminescence assay following the manufacturer's instructions (abcam, Boston, MA). In brief, MH-S cells were harvested after the appropriate exposures, stained with Diff-Quik (Dade Behring; Newark, DE), and counted using a hemocytometer. 10 [micro]L of a cell resuspension ([10.sup.3] - [10.sup.4] cells) was mixed with 100 [micro]L of the reaction mix for 5-10 min and then read in a luminometer. The ATP values were normalized to the cell count for each sample.

2.6. Colorimetric Assay for Measuring the Mitochondrial Ratio of NAD/NADH.

EnzyChrom [NAD.sup.+]/NADH assay kit (Bioassay Systems; Hayward, CA) was used to determine the mitochondrial ratio of [NAD.sup.+]/NADH. In brief, mitochondria were isolated from MH-S cells or mAMs using a Mitochondria Isolation Kit (Thermo Fisher Scientific, Rockford, IL). [NAD.sup.+] and NADH were then extracted with the extraction buffer provided in the assay kit, mixed with assay buffer, and absorbance-read at 565 nm.

2.7. Measurement of Phagocytosis and Cell Viability.

To determine the phagocytic capacity of macrophages, pHrodo Red S. aureus bioparticles conjugate (Invitrogen, Carlsbad, CA) was added to the culture media according to the manufacturer's recommendations with ~2 x [10.sup.6] cells per 2 mg vial of pHrodo-labeled bioparticles. Cells were incubated with the pHrodo labeled bioparticles for 2 hrs and then collected for FACS analysis and data analysis by Flowjo. This phagocytosis assay is based on the fact that there is a minimal fluorescence signal when the pHrodo Red S. aureus bioparticle conjugate is adherent to the outer surface of the phagocyte. Once the S. aureus is internalized and incorporated into the acidic environment of the phagosome, the bioparticle conjugates emit a strong red fluorescence. Internalization was verified by live cell confocal imaging (Olympus FluoView FV1000, Center Valley, PA). To assess changes in viability due to ethanol, MH-S cells were stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and Propidium Iodide (PI) (Invitrogen, Carlsbad, CA) before analysis by flow cytometry.

2.8. Fluorescence Microscopy and Image Analysis.

mAMs isolated from EtOH-fed and control mice were cultured overnight in 8-well cover glass bottom chambers (Lab-Tek; Scotts Valley, CA) with RPMI 1640 medium containing 2% FBS and 1% penicillin/streptomycin. Some mAMs were also treated with 500 [micro]M mitoT for 24hrs. ROS probes CM-[H.sub.2]DCFDA (10 [micro]M) or mitoSOX (10 [micro]M) was added to the media and images were taken after a 30 min incubation. Images were acquired with Olympus FluoView FV1000 Confocal Microscope using a 63 x oil objective. Images were viewed and analyzed by FV10-ASW 2.0 (Olympus, Center Valley, PA). For mitochondrial morphology analysis, acquired images were subjected to particle analysis using ImageJ Particle Analyzer (National Institutes of Health (http://rsbweb.nih.gov/ij/)). After thresholding, individual particles (mitochondria) were analyzed for area, perimeter, circularity (4[pi] x Area/([perimeter.sup.2])), and the lengths of major and minor axes of fit ellipse. From these values, form factor (FF; the reciprocal of circularity value) and aspect ratio (AR; major/minor) were calculated. Both FF and AR have a minimal value of 1 when a particle is a perfect circle and the values increase as the noncircle features of the particle increase. Specifically, AR is a measure of mitochondrial length and the increase of FF represents the increase of mitochondrial length and branching. This procedure is similar to mitochondrial morphology analysis as previously described [30, 32].

3. Results

3.1. Chronic EtOH Exposure Induced Mitochondrial ROS Generation.

CM-[H.sub.2]DCFDA is oxidized to DCF (dichlorofluorescein) by cellular ROS [33] and ethanol increased DCF fluorescence (images in Figure 1(a)). MitoSOX Red, which selectively targets mitochondria and is rapidly oxidized by superoxide, was used to monitor mitochondrial superoxide production (images in Figure 1(a)). In MH-S cells, five days of EtOH exposure increased both cellular ROS and mitochondrial superoxide production by ~ 100% and 50%, respectively (Figures 1(b) and 1(c)). However, EtOH-induced upregulation of cellular ROS and mitochondrial superoxide in MH-S cells were reversed by treatment with the mitochondrial targeted antioxidant, mitoT. To determine whether chronic EtOH ingestion induced ROS generation in vivo, mAMs were isolated from control or EtOH-fed mice and stained with MitoSOX and CM-[H.sub.2]DCFDA before flow cytometry or confocal imaging. Chronic ethanol ingestion upregulated cellular ROS in mAMs by ~2-fold and mitochondrial superoxide by ~3-fold (Figures 2(a) and 2(b)). Similar to that observed with MH-S cells, 24 h in vitro treatments of the mAMs with mitoT reversed EtOH-induced cellular and mitochondrial ROS production.

[FIGURE 1 OMITTED]

3.2. Chronic EtOH Exposure Resulted in Mitochondrial Dysfunction.

Mitochondrial membrane potential is a key indicator of mitochondrial function and integrity. It can be determined with TMRE, a cell permeable cationic dye that readily accumulates in active mitochondria because of the relative negative charge of the mitochondrial membrane potential. EtOH exposure resulted in two mitochondrial populations with TMRE staining. The population with higher TMRE intensity represents those with polarized mitochondria and greater capacity to transport the fluorophore. The population with lower TMRE intensity represents the depolarized mitochondria and decreased capacity to transport the fluorophore. In mAMs, chronic EtOH ingestion decreased the population of cells with higher TMRE staining by 10% (Figures 3(a) and 3(b)) suggesting loss of mitochondrial membrane potential. Similarly, EtOH exposure of MH-S cells incrementally decreased the percentage of cells with higher TMRE staining relative to the period of EtOH exposure (Figure 4(a)). In addition, in vitro and in vivo alcohol exposure also decreased the ratio of [NAD.sup.+]/NADH (Figures 4(c) and 3(c)). Since NADH is oxidized to [NAD.sup.+]+ in the process of transferring electrons in the mitochondrial electron transfer chain, decreases in the [NAD.sup.+]/NADH ratio indicate a mitochondrial redox imbalance and loss of mitochondrial function. Indeed, this EtOH-induced loss of mitochondrial membrane integrity and perturbations in the [NAD.sup.+]/NADH ratio were accompanied by a ~25% decrease in ATP production (Figure 4(b)). Ethanol-induced decreases in ATP and [NAD.sup.+]/NADH levels were both normalized through the addition of mitoT (Figures 4(b) and 4(c)).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

3.3. Chronic EtOH Exposure Induced Mitochondria Condensation and Perinuclear Clustering.

Mitochondria are dynamic organelles which constantly change their size and shape by fusion and fission and their morphological dynamics are linked to the regulation of normal cell physiology and disease. We next examined whether the EtOH-induced mitochondrial ROS generation and mitochondrial depolarization were linked to changes in mitochondrial morphology. In control MH-S, the mitochondrial network was spread throughout the cell (Figure 5(a)). With 5 consecutive days of EtOH exposure (0.2%), the majority of MH-S cells had mitochondria that were condensed and located in the perinuclear region (Figure 5(a)). To quantitatively address mitochondrial morphology changes, we analyzed mitochondrial morphology using a computer-assisted morphometric analysis, which calculates form factor (FF) and aspect ratio (AR) as discussed above. With a minimal value of 1 representing a perfect circle (major axis = minor axis), the mitochondria within control MH-S cells had AR values distributed above 4 suggesting that the mitochondria were elongated. With EtOH exposure, the majority of the AR values were below 4, suggesting a transition from an elongated shape to a more spherical shape. In addition, mitochondrial areas exceeded 15 [micro][m.sup.2] in the EtOH treatment group suggesting mitochondrial clustering. We next investigated the mitochondrial morphology after chronic EtOH ingestion. Figure 5(b) is comprised of representative confocal microscopic images of AMs isolated from control and EtOH-fed mice. Because primary mAM cells were taken from their original environment in mouse lungs, they are more fragile, and their morphology was not well retained like that for the MH-S cell line. However, the mitochondria in the AMs from the EtOH-fed mice were more fragmented and clustered at the perinuclear area when compared to the AMs from the control mice.

3.4. EtOH-Induced Impairment of Macrophage Phagocytosis Was Reversed by mitoT.

As demonstrated previously [9, 10], EtOH exposure decreased the phagocytic capacity of mAMs and MH-S cells. Since mitoT attenuated cellular and mitochondrial ROS, we next examined whether mitoT would reverse the effects of ethanol on phagocytosis. As demonstrated in our previous studies, EtOH exposure suppressed phagocytosis of the S. aureus bioparticle conjugates by 30% (Figure 6). Treatment with mitoT during the last 24 h of EtOH exposure restored the phagocytic ability of MH-S cells suggesting that mitochondrial-derived oxidative stress was central to EtOH-induced disruptions in phagocytosis. We also examined whether mitoT could restore phagocytosis to mAMs from ethanol-fed mice. Similar to that observed with MH-S cells, in vitro treatments with mitoT restored phagocytosis to the mAMs suppressed by chronic ethanol ingestion (Figure 7). These results further confirmed the association between EtOH-induced mitochondrial oxidative stress and impaired mAM phagocytosis.

[FIGURE 5 OMITTED]

3.5. EtOH Induced Early Apoptosis but Was Prevented by mitoT Treatment.

As demonstrated previously [9, 10], chronic ethanol ingestion increases AM apoptosis 3-fold with ~30% of the cells expressing markers of apoptosis. In MH-S cells with 5 days of ethanol exposure, the percentage of cells with an early marker of apoptosis, Annexin V positive staining, increased 5-fold (Figures 8(a)-8(c)) when compared to the control group. For cells positive for Annexin V plus loss of cytoplasm, ethanol increased the percentage of cells positive for late apoptosis but statistical significance was not achieved. There also was no statistically significant increase in the percentage of cells positive for a marker of necrosis, propidium iodide staining of DNA (Figure 8(c)). When MH-S cells were pretreated with mitoT, ethanol-induced early apoptosis, Annexin V positive staining, was attenuated (Figure 8(d)).

[FIGURE 6 OMITTED]

4. Discussion

Chronic alcohol abuse is associated with an increased risk of respiratory infections, pneumonia, and tuberculosis, even in those without a clinical diagnosis of an alcohol use disorder [1, 3]. However, the underlying mechanisms by which alcohol abuse increases the risk of respiratory infections are unclear. One central effect of chronic alcohol ingestion is severe oxidative stress and depletion of critical antioxidants [7]. Within the alveolar space, GSH levels in the alcoholic subjects were significantly decreased when compared with those of nonalcoholic subjects. In the alveolar epithelial lining fluid, alcohol abuse caused an -40 mV change in the glutathione and glutathione disulfide (GSH/GSSG) redox potential (Eh) [6, 7, 34]. Changes in the extracellular GSH/GSSG redox status were echoed in the intracellular GSH/GSSG redox balance of alveolar type II cells. Indeed, chronic alcohol ingestion caused a 60% decrease in GSH and induced GSH/GSSG oxidation by 40 mV in alveolar type II cells from ethanol-fed adult male rats [35]. In the mitochondria of type II cells, chronic alcohol ingestion also induced a 60 mV oxidation of the GSH/GSSG redox potential when compared to the cells from control rats [35]. For AMs, the GSH/GSSG redox state was oxidized by -30 mV after chronic ethanol ingestion [36]. Across intracellular and extracellular GSH pools in alveolar cells, the GSH/GSSG redox state was consistently oxidized by 30-60mV. AMs are the only intra-alveolar phagocyte that responds to inflammation [37] and their function is dependent on the oxidation/reduction balance in the alveolar lining fluid. Indeed, ex vivo GSH antioxidant supplementation can reverse the EtOH-induced suppression of phagocytosis in rodent models of chronic alcohol abuse [36].

Previous studies in our laboratory demonstrated that EtOH promotes oxidative stress in AMs through increased ROS production by NADPH oxidases (Nox) [10]. In that mouse model, chronic EtOH ingestion increased the level of mRNA and protein expression of Nox1, Nox2, and Nox4. Since mitochondria prodduce 90% of cellular ROS compared to a 10% cytosolic ROS contribution under baseline conditions [38], we examined the potential contribution of mitochondria to the ROS. Like the alveolar type II cells [35], the current study demonstrated that EtOH exposure (in vitro or in vivo) also upregulated mitochondrial ROS generation. Under baseline conditions, increased mitochondrial superoxide production can be enzymatically dismutated to hydrogen peroxide which is subsequently removed by catalase or glutathione peroxidase. However, chronic increases in mitochondrial ROS generation can overwhelm normal scavenging mechanisms, promote the uncoupling of the respiratory chain, and result in more ROS generation, loss of mitochondrial membrane potential, and decreased ATP. In the current study, a role for ethanol-induced mitochondrial ROS was further demonstrated by treatment of AM cells with mitoTEMPOL (mitoT) which reversed EtOH-induced mitochondrial ROS. MitoT contains a lipophilic triphenylphosphonium cation added to the TEMPOL antioxidant moiety which promotes its accumulation in the mitochondria. The TEMPOL moiety is a piperidine nitroxide, which has been widely used as a mitochondrial specific antioxidant for in vivo and in vitro studies. The TEMPOL moiety has super-oxide dismutase activity that promotes the detoxification of ferrous iron and prevents toxic hydroxyl radicals formation in the reaction of [H.sub.2][O.sub.2] with ferrous iron [39, 40]. Although our analysis of ROS by redox sensitive fluorophores has its limitations, treatment with mitoT blocked or reversed EtOH-induced ROS generation in the mitochondria further supporting that EtOH promoted mitochondrial ROS. Whether EtOH-induced mitochondrial ROS is due to interference with mitochondrial redox balances [41] or other mechanisms remains to be determined. Furthermore, mitoT also blocked the ethanol-induced increases in cytosolic ROS suggesting that mitochondrial ROS contributes to the generation of cytosolic ROS. Additional studies are needed to determine whether ROS generation through NADPH oxidases, CYP2E1, or other ROS generators are dependent on mitochondrial ROS.

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

EtOH-induced mitochondrial ROS was also associated with mitochondrial dysfunction. Mitochondrial integrity and function were interrupted by EtOH as evidenced by decreased mitochondrial membrane potential in MH-S cells. More importantly, mAMs from EtOH-fed mice displayed decreased mitochondrial function as evidenced by decreased mitochondrial membrane potential and ATP. The ratio of [NAD.sup.+]/NADH was also decreased in both the in vitro and in vivo models of ethanol exposure. Although the isolation protocol may have increased [NAD.sup.+] and NADH leak from the mitochondria, it is unlikely that the leak of one component would be preferential over the other. Since the actual leak may differ between mitochondrial preparations, we decided that expressing the concentrations of the two components as a ratio would be more accurate. In these studies, NAD+ was 0.33 [+ or -] 0.03 a.u. (absolute unit) and NADH was 0.20 [+ or -] 0.01 a.u. for the control group. For the ethanol group, NAD+ was 0.25 [+ or -] 0.03 a.u. and NADH was 0.76 [+ or -] 0.20 a.u. Therefore, the decrease in [NAD.sup.+] and increase in NADH resulted in a decrease in the mitochondrial [NAD.sup.+]/NADH ratio suggesting its oxidation in the mitochondria.

Mitochondria are organelles which supply energy for normal cellular functions making it a key regulator of cell function. For the AM, the energy intensive cellular process such as phagocytosis is particularly dependent on the capacity of the mitochondria to generate ATP. In the current studies, EtOH exposure promoted significant mitochondrial morphological changes, a central indicator of the organelle's integrity and function. In the control group, there was a network of mitochondria with an elongated shape. With EtOH exposure, the mitochondria became more spherical in shape and were present in condensed perinuclear clusters. These EtOH-induced mitochondrial morphological changes are generally associated with cellular oxidative stress and are associated with cell death [35]. As with ethanol-induced mitochondrial ROS, treatment with mitoT normalized mitochondrial [NAD.sup.+]/NADH as well as the ATP pool after ethanol exposure. This further supports a causative role for ethanol-induced mitochondrial ROS in the corresponding mitochondrial dysfunction. As observed in previous studies with ethanol-fed animals [36], there was impaired phagocytosis, a key immune function of AMs. However, in vitro mitoT treatments reversed the injurious effects of EtOH on the mitochondria and restored the phagocytic capacity even in mAMs from mice fed ethanol for 12 weeks. In addition to impaired bacterial clearance, ethanol decreased cell viability as evidenced by increased early markers of apoptosis. The capacity of mitoT treatment to block apoptosis suggested a central role for ethanol-induced mitochondrial dysfunction in the apoptotic process.

Since mitoT decreased ethanol-induced mitochondrial-and cytosolic-derived ROS, one potential mechanism for the beneficial effects of mitoT could be through its positive cytosolic effects. In previous studies, we demonstrated that chronic EtOH ingestion increases the production of TGF-[beta] and IL-13 in AMs which subsequently promotes alternative activation (M2 activation) [7]. In that study, TGF-[beta] and IL-13 activated an autocrine loop that was central to AM alternative activation. EtOH-induced upregulation of TGF-[beta] expression also promoted another self-activating autocrine loop with the constitutively active NOX 4 resulting in chronic cytosolic ROS generation [10]. Additional studies are needed to determine whether the ethanol-induced activation of TGF-[beta]/IL-13 and the TGF[beta]/NOX 4 autocrine loops are causative or secondary to ethanol-induced mitochondrial dysfunction. Alternatively, the primary driver could be through ethanol-induced mitochondrial ROS that overwhelm the large antioxidant capacity of the mitochondria, leak into the cytosol, and activate various mechanisms for cytosolic ROS such as the TGF-[beta]/NOX4 autocrine loop. In the current study, mitoT attenuated ethanol-induced mitochondrial dysfunction such as ROS, decreased ATP levels, and decreased NAD+/NADH. The mechanisms by which mitoT maintains these events that are critical for the highly energy-dependent processes of phagocytosis and maintenance of cell viability are unclear but may be through maintenance of mitochondrial GSH/GSSG, a critical event for type II cells [35].

Although each piece of data is not singularly definitive, the collective data from diverse measures indicated that ethanol increased mitochondrial ROS: (1) increased cellular ROS as indicated by CM-[H.sub.2]DCFDA oxidation which was blocked by the mitochondrial specific antioxidant mitoT; (2) increased mitochondrial ROS as indicated by MitoSOX fluorescence which was also blocked by mitoT; (3) increased cellular and mitochondrial ROS with in vitro and in vivo EtOH exposure; (4) the ability of mitoT to attenuate cellular and mitochondrial ROS in the AMs even after chronic EtOH ingestion; and (5) increased oxidation of the mitochondrial [NAD.sup.+]/NADH ratio. This mitochondrial oxidation was also associated with mitochondrial dysfunction as evidenced by loss of mitochondrial morphology, depolarization of the mitochondrial membrane potential, and decreased ATP generation. The mitochondria-targeted antioxidant mitoT not only reversed EtOH-induced mitochondrial and cytosolic ROS generation, it also reversed EtOH-induced mitochondrial dysfunction, restored AM phagocytosis, and maintained cell viability. Phagocytosis and cell viability are both complex cellular processes that are controlled at multiple points; additional studies are needed to determine the actual roles of mitochondrial ROS in EtOH-induced disruption of these cellular events. Chronically, alcohol can dampen the inflammatory responses of alveolar macrophages and the chronic suppression of phagocytosis decreases the capacity of alveolar macrophages to clear microbes. Therefore, EtOH-induced mitochondrial ROS and dysfunction in AMs may be pivotal in the increased risk of respiratory infections and ARDS in subjects with an alcohol use disorder.

http://dx.doi.org/10.1155/2014/371593

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by a NIAAA T32 Training Grant (5T32AA013528-08), the Emory Alcohol and Lung Biology Center (1P50AA135757), and NIAAA (R01 AA12197). The authors acknowledge the facilities and the scientific and technical assistance of the Flow Cytometry Core Facility and Imaging Core Facility at Emory + Children's Pediatrics Research Center.

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[31] K. Nakah

Using social media, online social networks, and internet search as platforms for public health interventions: a pilot study

Marco D. Huesch, Aram Galstyan, Michael K. Ong and Jason N. Doctor

Health Services Research. 51.3 (June 2016): p1273. From InfoTrac Humanities Collection 2017.

Abstract:

Objective. To pilot public health interventions at women potentially interested in maternity care via campaigns on social media (Twitter), social networks (Facebook), and online search engines (Google Search).

Data Sources/Study Setting. Primary data from Twitter, Facebook, and Google Search on users of these platforms in Los Angeles between March and July 2014.

Study Design. Observational study measuring the responses of targeted users of Twitter, Facebook, and Google Search exposed to our sponsored messages soliciting them to start an engagement process by clicking through to a study website containing information on maternity care quality information for the Los Angeles market.

Principal Findings. Campaigns reached a little more than 140,000 consumers each day across the three platforms, with a little more than 400 engagements each day. Facebook and Google search had broader reach, better engagement rates, and lower costs than Twitter. Costs to reach 1,000 targeted users were approximately in the same range as less well-targeted radio and TV advertisements, while initial engagements--a user clicking through an advertisement--cost less than $1 each.

Conclusions. Our results suggest that commercially available online advertising platforms in wide use by other industries could play a role in targeted public health interventions.

Key Words. Social media, social networks, maternity care quality

Full Text:

Widespread attempts have been made to publicize clinical performance measures aimed at improving delivered quality (Agency for Healthcare Research and Quality, 2011). Such "report cards" exist for a number of reasons: to educate consumers and referrers, to illustrate and to enhance potential choices of provider, to allow greater autonomy, and to improve efficiency in decision making.

Today, the National Quality Forum currently endorses 743 standards (National Quality Forum, 2011). Public report cards administered by health insurance plans are similarly available nationwide, with, for example, the National Committee for Quality Assurance listing 136 national plans reporting some measure of physician or hospital-specific performance (National Committee for Quality Assurance, 2011), culminating in the launch of the Hospital Compare public reporting website by CMS in 2005.

Given this generation of potentially accessible valuable information, it is clear that stakeholders need to and wish to inform the public of these resources. Traditional marketing campaigns may be less potent in an increasingly digital, technology-enabled society with home broadband and high bandwidth mobile Internet on ubiquitous smartphones that allow real-time searches for online information and live interactions on social media and social networks. Consumers wish to search for and create content online and interact with like-minded others. The Pew Internet and American Life Project finds that more than 50 percent of online adults between the ages of 18 and 55 years use social networking sites, while one in four Americans report that the Internet has helped them deal with at least one major health-related life decision (Pew Research Internet Project, 2014).

However, public health messaging that does attempt to use these virtual channels is typically highly passive. Merely tweeting broadly about the importance of seasonal flu vaccination is not likely to be sufficient. Using static websites to offer comparative hospital quality information-based messages will not work unless consumers become aware of and have confidence in the information (Huesch, Currid-Halkett, and Doctor 2014).

It is reasonable to ask whether we have fully exploited technology-based solutions to empower and better inform patients. In other industries, digital advertising through social media, online social networks, and Internet search engines is premised on the valuable data that such online platforms build up on their users.

Objective

In this article, we describe a pilot study of an intervention to provide information on maternity care quality to Los Angeles consumers plausibly interested in such information using three commercial campaigns on social media, an online social network, and an Internet search engine. Our objective is to understand whether we can quickly and cheaply reach out to prospective consumers of public health information using new digital technology. This pilot explicitly did not seek to actually change the health-related behavior of consumers nor did it ascertain whether consumers understood and acted on such public health information.

Empirical Setting

We chose to focus on the maternity care setting for a number of important reasons, aside from the obvious aspect that maternity care is a highly "shoppable" condition in which consumers have substantial time to acquire information and make decisions on health care utilization.

Maternity care is the second most common reason for hospitalization, the fourth most common reason for seeking ambulatory care (Sakala and Corry 2008) includes the top three procedures billed to Medicaid or private payors, and accounts for more than fourth of all Medicaid-billed hospital charges, and nearly a sixth of all private insured-billed hospital charges (Agency for Healthcare Research and Quality, 2008).

Yet on key objective evidence-based metrics such as the appropriate use of Cesarean sections (Main et al. 2011), the proportion of women who received antenatal care within the first trimester, low birth weight infant deliveries, infant mortality, and maternal death rates, progress has either been away from or incompletely toward federal targets (U.S. Department of Health and Human Services, 2006).

Reflecting these quality imperative, a number of easily accessible state government, federal government, and commercial entities' websites provide substantial data on local, regional, and national maternity care quality by named hospitals. Our study then provided links to these online report cards on the study website; the first author will provide by request detailed additional information on the quality metrics listed on each of these online report cards.

CONCEPTUAL FRAMEWORK

We have employed three complementary frameworks in this study, each of which represents a different perspective on what we seek to do. At the highest level, we see this pilot study as being a classical public health intervention. The theoretical underpinnings of this intervention are in social cognitive theory, while the practical bases lie in standard commercial marketing management and sales management.

Public Health Intervention Perspective

Using a classic public health model (Keller et al. 1998), our study is a population-based, individual-focused, primary prevention, public health intervention which combines elements of Outreach, Social Marketing, and Health Teaching.

As an Outreach intervention, we seek to locate individuals at risk of receiving less than optimal maternity care and ensure their access to information that can improve their maternity and delivery care by choosing a hospital of higher quality as reported by a public hospital reporting website. As a Social Marketing intervention, we seek to utilize commercial marketing tools and techniques to influence these individuals and their beliefs and decisions. As a Health Teaching intervention, we intend to communicate facts and ideas to change the beliefs and behaviors of those individuals.

Social Cognitive Theoretical Perspective

The theoretical grounding of our intervention is in social cognitive theory. The principles and processes underlying a target's susceptibility to outside influences are grounded in light of three goals fundamental to rewarding human functioning (Cialdini and Goldstein 2004). Specifically, targets are motivated to form accurate perceptions of reality and react accordingly, to develop and preserve meaningful social relationships, and to maintain a favorable self-concept. Of particular importance are social norms, behavior expectations within a particular group that can influence behavior of group members due to a desire to conform with actual behavior (the descriptive norm) or sanctioned behavior (the injunctive norm).

Previous research has demonstrated that social norms of appropriate behavior can exert a stronger effect on behavior than modest economic incentives or self-interest (Heyman and Ariely 2004; Griskevicius, Cialdini, and Goldstein 2008; Nolan et al. 2008). We are especially likely to follow the lead of others whom we perceive to be similar to ourselves (Hornstein, Fisch, and Holmes 1968; Murray et al. 1984; White, Hogg, and Terry 2002).

From an economic perspective, social norms may convey information concerning appropriate behavior or social consequences of failing to conform; however, behavioral studies find that these effects persist even when behavior is unobservable (e.g., littering when nobody is around) and social information is not particularly informative to one's own preferences (e.g., towel recycling).

We expect that engaging with consumers to provide accurate perceptions of the reality of differences in hospital quality, and providing information regarding the actions of geographically and psychosocially similar consumers in choosing high-quality hospitals for maternity care (a descriptive social norm), as well as providing information that there exist nationally recommended guidelines for avoiding unnecessary Cesarean sections (an injunctive norm) will lead consumers to choose to visit the recommended website (i.e., the study website) and click-through to existing sources of publicly reported data on hospital quality.

Marketing Perspective

Well-known terms of sales management can be applied to electronic patient education and information provision. We recognize that patient education is an actual sales process in which information is being sold even when it's being given away. In line with this sales process, potential customers are initially prospects, then become qualified prospects, then are converted to actual customers (see Figure 1).

We compete for some share of the customers' mind in an environment where a patient's fixed attention span is increasingly divided among many screens (i.e., TV, PC, and mobile) and by many attention-seekers. Online consumers in the United States are already estimated (eMarketer, 2012) to be exposed to nearly $22 billion worth of small advertisements that appear on paid search engine results (e.g., Google, Bing, Yahoo), and another $21 billion in display advertisements (e.g., on most any websites and on online social media and social networks).

Although we may offer our information for free, the potential customer or prospect faces noncash costs in acquiring this information. The prospect must invest time and effort in accessing and understanding this information. Conversely, there are most often opportunity costs for that time and effort. Accordingly, we must sell the patient on the worth of these investments. Partly this is through attractive and eye-catching advertisements, partly through the intrinsic value of the information that we seek to make available.

Qualified prospects are those who have expressed an interest in the product or service offered. We estimated that our total qualified prospects would be approximately 500,000-750,000 women and close friends, relatives, and partners at any point in time in Los Angeles county. We based this estimate on the 300,000 annual births in the county and the simplifying assumption of persisting interest throughout a typical 9-month pregnancy. This estimate was in line with recent findings of more than 1 million searches per month on Google in Los Angeles regarding pregnancy, more than 50,000 monthly searches for maternity care providers, and around 20,000 monthly searches for hospital quality information (Huesch, Currid Halkett, and Doctor 2014).

In our setting, the process of qualification is predominantly owned by the advertising platforms we access, who serve us prospects that they have deemed to be interested in our information on maternity care quality. Each of these platforms has different strengths and weaknesses. For example, a young lady in Los Angeles who uses Google to privately search the web regarding early pregnancy care options may have left a digital footprint with Google which could include every search she has ever made (if she has signed up for gmail or if she uses Google's social network, Google+, or if she uses the same home computer to perform all her searches). If she mentions pregnancy concerns in private to her Facebook friends, publicly follows a maternity care account on Twitter, or privately clicks-through Tweets to maternity care websites, then Facebook and Twitter can similarly infer relevant interests.

Finally, qualified prospects are incented to undertake some behavior that closes the deal. By navigating to a website, liking or following a Facebook page, following a Twitter account, or retweeting a Tweet, our qualified prospects have been converted to actual customers in this conceptual framework. Clearly, the ultimate objective of public health interventions that seek to educate patients on health care quality is in effecting actual change in decision making and health care utilization. However, in this pilot study, we sought merely to achieve a proof of concept of being able to reach and provide such information to a large number of consumers.

METHODS

We used three online platforms, each a leading example of a type of value-added Internet service. We contracted with Facebook, a platform that allows users to form social networks online, with Twitter, a platform that allows users to post brief 140-character messages online, and with Google Search, a platform that allows users to search the Internet.

Our study team has produced detailed guides (see Appendix S2 and S3), including step-by-step platform website recording, as to how these commercial arrangements are set up, maintained and adjusted, and ultimately wound down.

Our terms of trade with all three platforms were generally similar, although the individual technical details and terms varied. We sought to purchase access to qualified prospects for information provision on maternity care quality. This qualification is important to the platform: too many poorly targeted advertisements can affect user loyalty. It was therefore in each platform's interest--as well as ours--to present our advertisements only to those users who were likely to be interested.

We provided overall requested demographics (women, Los Angeles city + 25 miles or Los Angeles county, aged 18-49 if available, including Spanish speaking if available and broken out separately) and customer interest information (e.g., keywords such as pregnant, dar a luz) to the platforms to facilitate their qualification. Platforms drew from their pool of users but did not make detailed lists of exposed users available to us for privacy reasons. We thus relied completely on the integrity of the platform's respective user databases. Especially with regard to Twitter, where users provide limited account information on location and demographics and interests, the pool of users may have been selected partially based on Twitter's inferences of user behavior and interests.

We wished to limit our financial risk and only pay per converted customer, where this study intends "conversion" to mean an initial engagement by the customer in clicking through an advertisement on Facebook or Google Search and arriving at our test website. For Twitter, conversion was measured in two ways. The first is analogous to the narrow definition of conversion of a qualified prospect on Facebook or Google Search. The second is a broader and looser measure of engagement tailored to Twitter's social media business model, which includes click-throughs but also comprises additionally the following actions: following our account, expanding an advertisement to read the full copy, favoriting an account, retweeting, or replying. Twitter insists on payment on the basis of this broader measure of engagement, so when we report comparable costs per click-through for Twitter users, these will include the costs paid for customers who did not click-through to our website, but otherwise engaged with our message in Twitter's definition.

For each platform, we negotiated prices per customer and/or set bids to reach such customers, and were able to coarsely tune such prices and/or bids throughout the campaign to test whether reach or conversion increased. However, it is important to understand that we are bidding in a "sealed bid" auction for position in search results and in Facebook feeds so that reach and conversion are jointly determined by the competitive actions of very many marketers, each seeking to reach similar sets of customers.

In general, bids were a function of our advertisement's popularity with users, our desired placement within a user's visibility, and our desired reach. We paid monthly on electronic invoicing, using credit cards as payment mechanism.

We provided each platform with advertising materials consisting of pictures and text copy embedding a URL (a hyperlink) to click-through (Figures 2A, B, and C). We chose standard commercial images of neutral images of a baby, or a mother and baby, and within the context of dramatically limited word or character counts used neutral, easy to understand language to "pitch" our messages. The lead author took overall responsibility for approving team pictures and text copy.

The platforms supplied us with detailed data on a daily frequency of how many users had been exposed to the advertisement (the number of impressions or the reach), and what behavior had resulted (whether the user had clicked through to our test website, as well as for Twitter only, the other measures of engagement listed above). With some restrictions and prior permission by the platforms, we were able to modify and fine-tune advertising copy to test different responses.

Finally, we also used a fluent Latin American Spanish speaker to translate our promotional materials into Spanish for the large number of Hispanic prospects in our geographical area. We used ethnicity-appropriate images and culturally appropriate text, including Central and Southern American slang terms where appropriate. We ran this part of the intervention separately from the English-language campaigns.

We created a visually attractive study website that explained the study objective and that encouraged arriving users to consult the collated sources of local, state, or federal data on maternity care quality. For the Spanish-language campaign, we similarly translated this website into Spanish. We enabled Google Analytics on this website to analyze the origin of incoming web traffic to ensure that the platforms' reports of outgoing traffic for which we were billed matched our analysis of incoming traffic. This match proved to be almost exact, with some additional organic traffic (<1 percent) coming to our website from repeat visitors who could have bookmarked our site and subsequently returned to consult it.

We designed the campaign to use masters' student research assistants, one acting as "channel manager" for each of the three platforms and one to administer the study website. The lead author supervised and managed the channel managers and approved all commercial negotiations, text copy, bid prices, and changes in website design. All statistics reported are purely descriptive and no statistical comparisons or extrapolations to the population were attempted. This study was approved by the Institutional Review Board of the lead author's home institution.

RESULTS

The summary performance measures across the three channels are described in terms of daily metrics and overall study performance in Table 1.

The individual platform performance measures are further described below at a summary level, with detailed platform performance data, website designs, advertising copy, and other details available from the lead author on request.

[FIGURE 2 OMITTED]

Facebook

Our campaign on Facebook ran on consecutive days between March 20 and July 30, 2014. We spent a total of $13,689 to reach a nonunique total of 4,480,119 Facebook users. Of these, our total number of click-through engagements achieved was 19,923 nonunique clicks and 17,764 unique user clicks.

Our overall unique engagement rate was thus 0.4 percent of those reached. On average, we reached 33,685 unique users each day, soliciting 134 unique clicks each day and spending $0.77 per unique user to achieve that engagement. Overall, to reach 1,000 users, or the CPM metric, cost us $3.06.

In the subset of our results in which we ran our Spanish-language pilot, results were similar. Over 28 days we reached 1,496,818 nonunique Facebook users in Los Angeles and solicited 5,752 unique engagements at an average cost of $0.75 per unique user. The Spanish-language unique engagement rate of 0.38 percent was similar to the overall campaign results.

Google

Our campaign on Google search ran on the consecutive days between April 1 and July 28, 2014. We spent a total of $25,177 to reach a nonunique total of 10,959,961 Google search users. Of these, our total number of click-through engagements achieved was 27,676 without data on how many were unique individuals.

Our overall engagement rate was thus 0.25 percent of those reached. On average, we reached 92,100 nonunique searchers each day, soliciting 232 clicks each day and spending $0.91 per user to achieve that engagement. Overall, to reach 1,000 users cost us the least at $2.30.

In the subset of our results in which we ran our Spanish-language pilot, activity was different. Over 21 days we reached only 203,463 nonunique Google search users in Los Angeles and solicited 1,054 click-through engagements at an average cost of $4.65 per unique user. The Spanish-language engagement rate of 0.52 percent was higher than the overall campaign results.

Twitter

Our campaign on Twitter ran between March 26 and July 31,2014. We spent a total of $20,542 to reach a nonunique total of 2,223,493 Twitter users. Based on Twitter's broad measure of engagement, we achieved a total of 30,858 engagements for an engagement rate of 1.4 percent.

However, to properly compare our results on Twitter with Facebook and Google, we focus on the comparable and far narrower measure of nonunique click-through engagements. Here, our total was far less at 3,798 nonunique engagements. Our overall engagement rate was thus 0.17 percent of those reached. On average, we reached 17,507 nonunique users each day, soliciting 29.9 nonunique clicks each day and spending $5.41 per user to achieve that engagement. Overall, to reach 1,000 users cost us the most at $9.24.

In the subset of our results in which we ran our Spanish-language pilot, results were similar. Over 17 days, we reached 336,439 nonunique Twitter users in Los Angeles and solicited 4,030 broad engagements and 430 nonunique click-throughs at an average cost of $7.12 per unique user. The Spanish-language unique click-through rate of 0.13 percent, slightly lower than the overall campaign results.

DISCUSSION

This study used Twitter, Facebook, and Google Search to reach out to consumers with potential interest in maternity care quality information. Contracting on commercial terms, we spent a little more than $500 a day across these three platforms to obtain engagements from a little more than 400 consumers each day to our study website containing relevant information.

As an initial proof of concept, we believe that this pilot has shown that it is possible to drive consumer interest toward a static website for a little more than $1 per qualified consumer on average. This intervention was also relatively simple to design and launch, and was greatly facilitated by the professional counterparties at each of the three platforms.

It is important to put our results into perspective and understand other options for reaching consumers. These options depend on whether the patient is identified or not, and whether in-person or remote channels are used.

On one hand, if such patients are not yet identified, the total cost of in-person outreach to unidentified high-risk, low-income pregnant women to enroll them in high-risk antenatal care using case workers has previously been estimated in one study as $850 per enrollee. That study sought to enroll women at welfare offices, clinics, and in high-potential residential areas in an urban environment (Brooks-Gunn et al. 1989).

On the other hand, if such consumers are already identified by name, location, or phone number, then direct outreach to enroll them (i.e., to achieve a conversion) into a counseling or educational program is possible. Historically, the direct costs of in-person outreach to female low-income patients is around $50 per patient (typically 2-3 labor hours at $20/hour staff time) down to $3/patient for labor costs related to a phone call, and as low as $1/patient for labor and postage costs for a letter to a known patient (Wagner et al. 2007).

Beyond in-person outreach, cheaper health education and social marketing using traditional media channels has different cost structures. Here, almost all cost data are on reach to prospects or qualified prospects, not on actual conversions. The relevant cost metric is CPM or cost per thousand impressions, where one impression is synonymous with reaching one prospect. It costs more than $30 to reach a thousand prospects using newspaper advertisements, around $20 for magazines, about $7 for radio, and $5 for TV advertisements (Flannagan 2015). Other less well-targeted modalities are still lower such as billboard marketing with an estimated CPM of $3-$5 (Grunert 2015).

In our results, our CPM was comparable to these metrics, lying between $2 and $10, although the quality of our impressions is likely to have been better than the less targeted advertising media listed above due to the platforms' better information on users. Reassuringly, our social media and social network achieved CPM is also very similar to cited results for CPM achieved in these digital channels by other relatively unsophisticated small business marketers who tend to pay in the range of around $4-$20 on average (Grunert 2015).

Limitations

We readily acknowledge several well-known and important limitations of such campaigns. We do not know whether such information provision did or even can affect users' decision making and thus lead to a health benefit, and this was explicitly not a study objective or ascertainable in this design.

Future studies should track user behavior, identifying follow-on behavior (e.g., which websites were contacted subsequently), satisfaction (e.g., with depth and breadth of information obtained), and actions taken (e.g., whether choice of providers was changed). Relatively simple trackers added to the website can track online behavior; these include outbound link trackers such as, for example, www.[insert your site URL here]/linktracker.php?link=[insert the URL of a subsequent site you wish to track outward traffic to] or simply by exploiting Google Analytics (https://www.google.com/analytics/) functionality on your site, and these allow researchers to understand how effective a marketing campaign is at driving qualified consumers to destination sites.

We also know that much reach is repetitive: identical users are repeatedly exposed to the same advertisement, so that overall reach must be interpreted as being nonunique. Except for Facebook, where unique user engagements are tracked by Facebook, we have to assume that engagements achieved from Twitter and Google include nonunique user behavior.

CONCLUSIONS

In our results, Google and Facebook appeared to be of greater potential for other researchers and public health stakeholders undertaking similar interventions. Overall reach was greater, click-throughs higher and cheaper to obtain, and CPM and click-through rates higher than for Twitter. We assume that the greater knowledge built up by Facebook and Google over its members and users could better be leveraged to serve up more qualified customers.

Similarly, the more private nature of information exchange of those two properties (i.e., completely privately in Google searches, and semiprivately among friends in Facebook) compared to the more public nature of Twitter probably led to more genuine interest. Our results are directionally consistent with the relative amounts of per user revenue earned each year by these three platforms. Google is estimated to earn $45, Facebook $7.24, and Twitter $3.55 in advertising revenues per user per year (Meeker 2014).

In interesting but only introductory results, it appeared that Spanish-language users of Google search were substantially less likely to be exposed to our campaign, compared to English-language users. Daily reach was only a tenth, although engagement rates were double. It is not clear whether this indicates that this platform is used less often by Spanish speakers, perhaps due to insufficient access to Internet or desktop computers, or whether Spanish speakers use English-language searches. It was, however, the case that in Twitter and Facebook campaigns, there were essentially no differences between the English- and Spanish-language campaign responses.

Public health interventions can reach qualified prospects through many possible communication channels. Yet overall, traditional channels remain overrepresented in marketing communications: 45 percent of American's media time last year was spent on the Internet and on mobiles, yet only 26 percent of media spend went to those new channels (Meeker 2014). Specifically in health care marketing communications, the entire health care industry accounts for just 2.5 percent of total online digital advertising (eMarketer, 2013), and much of that is direct to consumer advertising by the pharmaceutical industry.

This disproportionately small share of these increasingly important communication channels highlights an opportunity for better, more tailored, closer, and more cost-effective outreach. While such campaigns require often substantial budgets, there are cost less options too. For example, Google offers in-kind advertising space (through https://www.google. com/grants/) similar to the offering of free public service announcements on traditional media.

This study showed that it was possible to reach out to qualified consumers and at least initiate an engagement with these customers at marketing costs that were comparable to traditional media. Still lacking are data on whether such initial engagement or the overall impressions actually predisposes users to obtain, understand, and internalize such health-related information and act appropriately on it by choosing high-quality health care providers.

Some leading examples for similar outreach exist. Directly targeting smokers through carefully placed online advertising has proved to be an inexpensive way of attracting traffic to the California Department of Public Health's TobaccoFreeCA website (personal communication, Valerie Quinn, June 12, 2014). Similar campaigns could target vaccine skeptics or users of complementary and alternative medicine. But more broadly, we are not aware of large-scale uses of such platforms except in relatively passive modes by hospitals with, for example, a Facebook page or a Twitter feed, or an advertisement on Google Search for their care delivery business.

It is our hope that this study conveys a sense of the relative ease and simplicity, and the relatively low costs and circumscribed financial risk of such campaigns to market public report cards. For our health system to overcome the serious challenges that threaten our entire nation fiscally, we need to have better informed patients taking charge and participating in their own health care and wellness. Understanding the role that Internet and social media-based custom education approaches could play to inform patient decision making and possibly incent patient behavioral change appears to be an important next step for public health stakeholders.

DOI: 10.1111/1475-6773.12496

ACKNOWLEDGMENTS

Joint Acknowledgment/Disclosure Statement The authors gratefully acknowledge financial support by the Agency for Healthcare Research and Quality (AHRQ) under grant R21 HS21868, and helpful project advice and ongoing support by Brent Sandmeyer and Galen Gregor at AHRQ. The authors also acknowledge the excellent research assistance of the following graduate students at USC over the period 09/30/2012-12/31/2014: Brent Costa, Shakeh Missian, Natalie Abidjian, Don Marshall, and Shyamala Shastri.

Disclosures: None.

Disclaimers: None.

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SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article:

Appendix SA1: Author Matrix.

Appendix SA2: Building the Back-End--The Study Website and Its Analytics Function.

Appendix SA3: Building the Front-End--The Facebook Channel and Its Advertising Function.

Address correspondence to Marco D. Huesch, MBBS, Ph.D., USC Leonard D. Schaeffer Center for Health Policy and Economics, University of Southern California, Verna & Peter Dauterive Hall (VPD), 635 Downey Way, Los Angeles, CA 90089-3333; e-mail: huesch.marco@gmail.com. Aram Galstyan, Ph.D., is with the Information Sciences Institute, University of Southern California; Department of Computer Science, Viterbi School of Engineering, University of Southern California, Marina del Rey, CA. Michael K. Ong, M.D., Ph.D., is with the Division of General Internal Medicine and Health Services Research, David Geffen School of Medicine at the University of California; Veterans Administration Los Angeles, Los Angeles, CA. Jason N. Doctor, Ph.D., is with the USC Leonard D. Schaeffer Center for Health Policy and Economics, University of Southern California; School of Pharmacy, University of Southern California, Los Angeles, CA.

Table 1: Daily and Overall Campaign Performance Measures

                                            Platform

                           Google

                           Search      Facebook   Twitter    Total

Daily (all nonunique)

  Reach                    92,100      33,685     17,507     143,292

  Engagements              232         149        30         411

Campaign (all nonunique)

  Reach                    10,959,961  4,480,119  2,223,493  17,663,573

  Engagements              27,676      19,923     3,798      51,397

  Spending                 $25,177     $13,689    $20,542    $59,408

  Cost per 1,000 reached   $2.30       $3.06      $9.24

  Cost per click-through   $0.91       $0.69      $5.41

Note. Reach or impressions identifies number of users who saw or

potentially saw the advertisement because they were exposed to it.

Engagements identifies number of users who responded to the

advertisement by clicking through the website link contained in the

advertisement.

Figure 1: Conceptual Framework

Marketing                                ... Safes

                     Qualified

                     Prospects           Conversions

Prospects

                     * Has expressed

* Potentially a        the desire to

  consumer of          consume such      * Has started the

  maternal care        info or this        engagement process with the

  quality info         has been            info provider

                       inferred

* Has not                                * (May not yet have

  expressed a        * Ready to            consumed the information)

  desire to            start an

  consume this,        engagement        * (Has not yet changed

  or it has not        process             their behavior, or such

  been inferred                            behavior is not known by

                                           study)

* Anyone who         * Of all            * Of all qualified

  may potentially      prospects, only     prospects, only those users

  at some stage        those users of      who have responded to study

  be pregnant or       social media        ad on social media or

  may know             and social          social network by 'clicking

  someone who may      networks whose      through' to reach the study

  be pregnant          patterns of         website

                       use, shopping

                       and privately     * ('Closing the deal' by

                       disclosed           actually changing behavior

                       content             and choosing higher quality

                       suggests an         provider not ascertained in

                       interest in         this pilot study)

                       pregnancy

                     * Targeted by

                       study ads

* Correspond to Health Teaching          * Corresponds to an Outreach

  and Social Marker ting types             public health pilot

  of public health pilot                   intervention

  interventions

Huesch, Marco D.^Galstyan, Aram^Ong, Michael K.^Doctor, Jason N.