AT-RvD1 Mitigates Secondhand Smoke–Exacerbated Pulmonary Inflammation and Restores Secondhand Smoke–Suppressed Antibacterial Immunity

Cigarette smoke (CS) in the form of mainstream tobacco smoke (MTS) or secondhand smoke (SHS) globally kills around 8 million people annually (1). Smoking is a major risk factor in the development and progression of cancer, cardiovascular disorders, and respiratory diseases such as chronic obstructive pulmonary disease (COPD) (2–4). Chronic respiratory diseases are among the leading causes of death worldwide (2–6). CS as a proinflammatory trigger is immunosuppressive, enhancing the risk of infections associated with respiratory diseases such as COPD (4, 7–15). In the lung, many cells, including macrophages and fibroblasts, respond to CS by producing multiple proinflammatory mediators, resulting in an inflammatory microenvironment and inducing tissue damage (4, 9–14). Importantly, long-term smokers with and without COPD and people chronically exposed to SHS are at increased risk from acute exacerbations due to infections that are responsible for the majority of the morbidity, mortality, and costs of smoking-related lung diseases, which can persist even long after smoking cessation (7, 8, 14–19). Nontypeable Haemophilus influenzae (NTHI) is an opportunistic Gram-negative bacterium commonly found in the upper respiratory tract. It causes otitis media in children, bronchitis in adults, and the majority of invasive H. influenzae disease in all age groups (20). It is a major cause of acute exacerbations and worsening of symptoms in individuals with COPD (21). Vaccination trials against NTHI in individuals with recurrent exacerbations of chronic bronchitis or COPD have not been successful (22). Moreover, NTHI infections are typically persistent in COPD patients, with some strains acquiring drug resistance and the potential to avoid phagocytosis, thus leading to airway bacterial colonization (7, 23–25). NTHI infections induce a potent and prolonged inflammatory response in COPD patients, thus repeated infections could further worsen the inflammatory microenvironment and lead to extensive lung tissue destruction (7, 8, 13, 26). We have demonstrated that MTS and SHS worsen NTHI-induced pulmonary inflammation and suppress antibacterial immunity (9, 10). Importantly, SHS exposure significantly impaired the protective Ab response to immunization with NTHI P6 protein in Freund adjuvant. We have also reported that SHS exposure has long-term consequences on the lung microenvironment (10), and we thus reasoned that SHS may also induce defects in the resolution of inflammation.

Timely resolution of inflammation is critical to tissue remodeling and wound healing to minimize tissue damage (27–29). Resolution of inflammation, previously thought to occur passively, is an active process mediated by a family of endogenous lipid-derived mediators, termed specialized proresolving mediators (SPMs) (30–35). SPMs offer both anti-inflammatory and proresolving actions without inducing immunosuppression (34, 36–40). There is growing interest in evaluating the efficacy of SPMs in resolving CS- and infection-associated pulmonary inflammation to allow the design of strategies to manage lung diseases having an inflammatory component as a causal factor. SPMs play two distinct roles that are of special interest when considering the problem of respiratory infections in smokers and people exposed to SHS. First, SPMs can mitigate acute and chronic inflammation. There is evidence that smokers with COPD have lower levels of resolvin D1 (RvD1) in their serum and bronchoalveolar lavage (BAL) fluid (41) and higher levels of eicosanoid oxidoreductase, an enzyme that degrades SPMs, in their lung tissue (42). Aspirin-triggered RvD1 (AT-RvD1) is a stereoisomer of RvD1 that is formed when the enzyme cyclooxygenase-2 is acetylated by aspirin. AT-RvD1 exhibits increased biological activity and a longer in vivo half life than RvD1, in part because of its resistance to degrading enzymes (43, 44), and is generally preferred for in vivo experiments for this reason.

Second, SPMs have important properties in modulating adaptive immune responses. SPMs inhibit maturation of dendritic cells, inhibit Th17 responses, and promote regulatory T cells, all of which tend to promote a return to homeostasis (reviewed in Ref. 45). SPMs can either up- or downregulate Ab production, depending on the specific SPM and the cellular context. We recently reported that AT-RvD1 or its precursor 17-hydroxydocosahexaenoic acid (17-HDHA) as vaccine adjuvants could restore Ab responses to NTHI P6 Ag in mice exposed to SHS, resulting in improved clearance of NTHI following an acute infectious challenge (46). This raises the exciting possibility that SPMs could be used as a novel class of adjuvants to enhance vaccine responses to common respiratory pathogens and mitigate the effects of chronic SHS exposure.

However, improving vaccine efficacy does not address the underlying cause of the increased susceptibility to respiratory infection, which is chronic inflammation resulting from SHS exposure. Therefore, we set out to determine whether proresolving AT-RvD1 given during smoke exposure or during cessation could mitigate lung inflammation due to chronic infection and improve the adaptive immune response to the classically adjuvanted Ag vaccination. We report, in this study, that AT-RvD1 given during SHS exposure or during cessation reduced lung inflammation and inflammatory cytokines and blocked the pathological Th17 response, whereas it enhanced the Ab response to NTHI. Treatment with AT-RvD1 during smoke exposure or after cessation also improved Ab responses to vaccination using conventional adjuvants that began after the final treatment with AT-RvD1. Collectively, these results demonstrate that AT-RvD1 mitigates lung inflammation and improves immune responses to NTHI, suggesting that AT-RvD1 has a high translational potential in people formerly exposed to SHS.

Eight-week-old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Animals were maintained under specific pathogen–free conditions. Number of animals per group in each experiment was n = 10, unless mentioned otherwise in the figure legends. All procedures performed on animals were approved by the Animal Care and Use Committees of both institutions (Roswell Park Comprehensive Cancer Center [Buffalo, NY] and University of Rochester [Rochester, NY]) and complied with all state, federal, and National Institutes of Health regulations.

Animals were housed in the Inhalation Exposure Facility at the University of Rochester and exposed to SHS 5 h/d, 5 d/wk for 8 wk, as described previously (10). Briefly, 3R4F research cigarettes were combusted using an automated smoking machine (TE-10; Teague Enterprises, Woodland, CA). MTS was generated in a puff volume of 35 ml in 2-s duration once per minute (the Federal Trade Commission protocol). SHS was generated by collecting and mixing sidestream smoke with the MTS at a ratio of 89%:11% (47). Total particulate matter concentration of 99 ± 3 mg/m³ was achieved for these experiments by adjusting the number of cigarettes loaded and the flow rate of the dilution air. Control mice were exposed to filtered air using the same exposure protocol.

AT-RvD1 (Cayman Chemical) was dissolved in ethanol at 200 μg/ml, aliquoted, and stored frozen at −20°C. Immediately prior to administration, the AT-RvD1 was diluted with seven volumes of saline, and 40 μl per mouse was administered by oropharyngeal aspiration under isoflurane anesthesia (48). Thus, each mouse received 1 μg of AT-RvD1 in 40 μl of 12.5% ethanol in saline. Control animals received 40 μl of 12.5% ethanol in saline. Mice were treated twice per week (Monday and Thursday or Tuesday and Friday), 1 h prior to the start of the smoke exposure. For one experiment, mice were treated in weeks 5–8 of an 8-wk smoke exposure (Fig. 1). For a second experiment, mice were treated in weeks 5–12, for which weeks 5–8 were the second half of smoke exposure and weeks 9–12 were a 4-wk smoke-free cessation period (Supplemental Fig. 1).

Chronic and acute pulmonary infection experiments with NTHI were done similar to those described previously (9, 10). A frozen glycerol stock of NTHI bacterial strain 1479 (clinical isolate from a COPD exacerbation) was streaked onto chocolate agar plates and incubated overnight at 35°C under 5% CO₂ conditions. A loop full of NTHI colonies was grown in a liquid culture of brain–heart infusion media supplemented with 10 mg/ml hemin and 10 mg/ml β-NAD (Sigma-Aldrich, St. Louis, MO) for 3–4 h in a 35°C shaking incubator. OD_600nm was measured to dilute the required number of CFUs in PBS to 2 × 10⁷ CFUs/ml. Bacteria were pelleted by centrifugation at 13,000 × g for 5 min and washed twice in PBS. When air or SHS exposures and AT-RvD1 treatments were completed, NTHI was administered by intratracheal route, as described previously (10). To achieve NTHI-induced chronic pulmonary inflammation, mice were given 1 × 10⁶ live bacteria twice per week for eight consecutive weeks. For acute challenge, mice were given a single intratracheal instillation with 1 × 10⁶ live NTHI bacteria and euthanized 4 and 24 h later to collect the BAL and whole lungs for evaluating the clearance of bacteria from the lungs of mice and quantifying albumin leak as a marker of lung epithelial cell damage.

When air or SHS exposures and AT-RvD1 treatments were completed, mice were vaccinated with 40 μg of purified native P6 emulsified in CFA and boosted 1 wk later with P6 in IFA and 2 wk later with P6 alone, as described previously (9, 10, 49). To quantify the titers and levels of Ag-specific Abs by ELISA, mice were bled retro-orbitally on a weekly basis to collect immune sera.

At the end of the chronic pulmonary infection, animals were euthanized, and lungs were harvested. Lungs were fixed overnight in 10% neutral buffered formalin (Merck, Gibbstown, NJ), then transferred to ethanol, embedded in paraffin, sectioned, and stained with H&E by the Mouse Tumor Model Shared Resource at Roswell Park Comprehensive Cancer Center. Images were taken on an Olympus light microscope equipped with a charge-coupled device camera and SPOT image analysis software (v25.4; Diagnostics Instruments, Sterling Heights, MI). Lung pathology was evaluated by a pathologist (P.N. Bogner) who was blinded to treatment groups, as described previously (10).

To determine total leukocyte counts, numbers of specific immune cells, the levels of various cytokines, albumin, and Ag-specific Ab titers in the lung lavage, we harvested the BAL from the lungs of mice, as described previously (9, 10). Briefly, after treatments were completed, mice were euthanized by injecting i.p. 1 ml of warmed 2.5% avertin solution (2,2,2-tribromethanol). After no pinch reflex was observed in the animals, the thoracic cavity was opened, and the trachea was cannulated. Lungs were gently lavaged twice by injecting 750 μl of ice-cold 1% BSA solution in PBS, as described previously (10). Total leukocyte counts in BAL samples were calculated by a hemocytometer before staining with cell type-specific Abs for FACS analysis. Cell-free BAL fluid was stored at −80°C until it was used to quantitate the levels of cytokines and albumin and also to measure the titers of Ag-specific Abs by ELISA.

After mice were euthanized, lungs were excised and minced into small pieces in a 6-cm petri dish on ice. The resulting lung slurry was mixed with 1 mg/ml type IA-S collagenase solution containing 50 U/ml DNase I (Sigma-Aldrich) and placed for 1 h on a rotator at 37°C. A single-cell suspension was passed through a 40-μm filter to remove debris and undigested tissue, then centrifuged for 5 min and resuspended in complete RPMI 1640 culture media containing 10% FBS. The cell suspension was then gently overlaid on top of Ficoll-Paque and centrifuged at 700 × g for 30 min with brake off. Immune cells at the interface were collected, washed twice in cell culture media to remove residual Ficoll-Paque, and counted. Single-cell suspensions of splenocytes were isolated in complete cell culture media. Bone marrow was collected by flushing femur and tibia with sterile ice-cold PBS. Splenocytes and bone marrow cells were centrifuged for 5 min at 500 × g at room temperature, resuspended in complete cell culture media, and counted using a hemocytometer.

Immune cell populations in the BAL and lungs of mice were determined by flow cytometry, as described previously (10). Briefly, 0.5 million BAL cells and lung lymphocytes from each sample were stained with specific fluorophore-tagged Abs in 100 μl of FACS staining buffer (1% BSA in PBS) for 30 min at 4°C and then washed in FACS buffer before fixing with BD Cytofix. To stain intracellular markers, cells were first treated with BD FACS Permeabilizing Solution (BD Biosciences) and then stained with cell type-specific fluorophore-tagged Abs. All the samples were acquired on an LSR II-A flow cytometer, and data were analyzed by FlowJo software.

To quantitate the cytokine levels, albumin leak into the BAL, and Ag-specific Ab titers, ELISA assays were performed as described previously (9, 10). Briefly, the levels of cytokines in the BAL fluid of mice were quantified by ELISA using the following eBioscience kits (San Diego, CA) following the manufacturer’s instructions: IL-1β (catalog no. 88-7013-77), IL-6 (catalog no. 88-7064-77), IL-17 (catalog no. 88-7371-77), TNF-α (catalog no. 88-7324-77), IFN-γ (catalog no. 88-7314-77). Albumin levels in BAL fluid (as a surrogate marker of lung epithelial damage) were quantified by a mouse albumin-specific kit from Bethyl Laboratories (catalog no. E90–134; Montgomery, TX). Titers of Ag-specific Abs in mouse serum and BAL fluid were measured using ELISA plates coated with 3 μg/ml purified P6 protein, as described previously (9, 10). Specific dilutions of weekly mouse serum and end point BAL fluid were incubated with P6-coated, BSA-blocked ELISA plates at room temperature, and bound anti-P6 Igs were detected with HRP-conjugated goat anti-mouse Ig(H+L), IgA, IgG1, and IgG2b Abs (SouthernBiotech, Birmingham, AL). All the ELISA plates were developed with eBioscience 3,3′,5,5′-tetramethylbenzidine (for HRP) solution, and absorbance was read at 450 nm on an ELISA plate reader (Synergy HTX Multi-Mode Reader; BioTek Instruments, Winooski, VT).

ELISPOT assays were performed to quantitate the frequency of Ag-specific, cytokine-secreting T and Ag-specific, Ab-secreting B cells, as described previously (10). Briefly, for T cell ELISPOTS, multiscreen HA plates (Millipore, Carrigtwohill, Ireland) were coated with 3 μg/ml anti–IL-17A, anti–IL-4, or anti–IFN-γ Abs in ELISPOT Coating Buffer overnight at 4°C. On the next day, lung or splenic lymphocytes were cocultured with 1 μM P6_41–55 peptide-pulsed APCs (49). After a 48-h coculture, ELISPOT plates were washed, and cytokines were detected with cytokine-specific biotinylated Abs (anti–IL-17A, anti–IL-4, or anti–IFN-γ) followed by addition of streptavidin-HRP (all reagents from Thermo Fisher Scientific, Suwanee, GA). To quantify the frequency of Ag-specific, Ab-secreting B cells in bone marrow and spleen, ELISPOT plates were coated with 3 μg/ml native P6 protein overnight at 4°C. On the next day, B cells were incubated in P6-coated, BSA-blocked ELISPOT plates at 37°C and 5% CO₂ for 48 h. After plates were gently washed, bound anti-P6 Abs were detected with HRP-conjugated secondary Abs to mouse IgG1 and IgG2a (SouthernBiotech). After washing the plates, spots were developed with 3,3′,5,5′-tetramethylbenzidine substrate (Vector Labs, Burlingame, CA), air-dried, and enumerated. Spots were enumerated using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).

NTHI bacterial burden in the lungs of mice following acute infection was assessed as described previously (10). Briefly, the mice were euthanized 4 and 24 h after acute bacterial infection, and whole lungs were excised. Lungs were then gently homogenized in 1 ml of PBS on ice. Under sterile conditions, serial dilutions of lung homogenates were plated on chocolate agar plates and incubated for 16 h at 35°C at 5% CO₂ conditions. On the next day, NTHI colonies were counted, the bacterial burden in the lungs was determined, and data were expressed as the number of bacterial CFUs recovered compared with the 1 × 10⁶ CFUs instilled initially.

Results are expressed as mean ± SEM. Testing for differences between mean values was determined using a two-tailed unpaired Student t test or two-way ANOVA with a Tukey posttest comparison using GraphPad Prism 8 software. The difference between two groups was considered to be significant at p ≤ 0.05.

Because SPMs are considered to be potent anti-inflammatory and proresolving mediators, we evaluated the impact of AT-RvD1 on SHS-augmented infection-induced pulmonary inflammation. Air- or SHS-exposed mice treated with AT-RvD1 were chronically infected in the lungs with NTHI (Fig. 1A). Histopathological examination of lung sections revealed characteristic leukocytic accumulation surrounding airways and bronchovasculature in all the groups. We found that prior exposure to chronic SHS aggravated NTHI-induced pulmonary inflammation (Fig. 1B; panel 3 versus panel 1), which agreed with our previous findings (10). Notably we observed that when mice were treated with AT-RvD1 during the second half of the smoke exposure period (weeks 4–8), the extent of immune cell infiltration into the lungs markedly decreased compared with untreated mice (Fig. 1B; panel 4 versus panel 3). The histological data were additionally supported by quantitation of inflammatory cell infiltrates in the BAL and lung. Chronic SHS exposure increased total leukocyte infiltrate in the BAL and lungs after chronic infection, and AT-RvD1 treatment resulted in significant decreases (Fig. 1C). Of note, infiltration in the BAL of SHS-exposed, AT-RvD1–treated mice was reduced to the level comparable with that observed in mice exposed to air, demonstrating that SPM AT-RvD1 potently ameliorates pulmonary inflammation (Fig. 1C).

We also evaluated the impact of AT-RvD1 treatment in SHS-exposed mice on the phenotype of immune cell infiltrates in the BAL and lungs. The numbers of neutrophils and macrophages in the BAL and lungs of mice exposed to chronic SHS were significantly elevated compared with air exposure. AT-RvD1 treatment of SHS-exposed mice significantly reduced the numbers of these cells compared with SHS-exposed, vehicle-treated mice (Fig. 1D, 1E). Furthermore, chronic SHS augmented the accumulation of CD4⁺ T, CD8⁺ T, and CD19⁺ B cells in the BAL and lungs, and AT-RvD1 treatment of SHS-exposed mice significantly reduced this lymphocytic accumulation in the BAL (Fig. 1F) and also decreased the numbers of B cells in the lungs (Fig. 1G). Elevated numbers of proinflammatory CD4⁺RORγt⁺ and CD4⁺IL-17A⁺ Th17 T cells in the BAL and lungs were also significantly reduced by AT-RvD1 treatment in SHS-exposed mice (Fig. 1H). We also assessed the effect of smoking cessation by interposing a 4-wk smoke-free cessation period between the final smoke exposure and first bacterial infection (Supplemental Fig. 1A). We found that even following a 4-wk period of cessation of exposure, mice previously exposed to SHS had significantly elevated lung inflammation compared with air-exposed mice (Supplemental Fig. 1B, 1C). However, AT-RvD1 treatment lead to marked reduction in the total numbers of immune cells, numbers of neutrophils, macrophages, lymphocytes (CD4⁺ T and CD19⁺B cells) and proinflammatory IL-17A⁺ Th17 and RORγ t⁺ Th17 T cells infiltrating the lungs after chronic NTHI infection (Supplemental Fig. 1B–E). Our gating strategy is shown in Supplemental Fig. 2.

We have previously shown that SHS exposure induces augmented levels of proinflammatory cytokines in response to pulmonary infection (10). Because AT-RvD1 reduced chronic SHS-induced pulmonary inflammation and suppressed accumulation of proinflammatory immune cells in the lungs, we next evaluated its influence on the levels of proinflammatory cytokines (Fig. 2A). AT-RvD1 cotreatment markedly reduced levels of the proinflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α in the BAL of mice, although they were still above the levels seen in air-exposed mice (Fig. 2B–E). Notably, the decrease in IFN-γ levels seen in the BAL of SHS-exposed mice was reversed when these mice were treated with AT-RvD1 (Fig. 2F). The efficacy of AT-RvD1 was enhanced by giving the mice a 4-wk smoke-free period prior to the first bacterial infection, as proinflammatory cytokine levels were restored to the baseline levels seen in vehicle-treated, air-exposed mice (Supplemental Fig. 3, Supplemental Table I). Additionally, we observed that IFN-γ levels in the BAL of SHS-exposed mice after a period of cessation were augmented compared with or without cessation, irrespective of RvD1 treatment (Supplemental Table I). Albumin leak, a measure of epithelial barrier integrity, was significantly reduced in SHS-exposed mice cotreated with AT-RvD1 (Fig. 2G), and the efficacy of AT-RvD1 at reducing alveolar leakage was also enhanced by a 4-wk smoking cessation period (Supplemental Fig. 3G, Supplemental Table I).

We have previously reported that chronic exposure to SHS diminishes the development of adaptive immunity to chronic respiratory infection (10). We therefore examined whether AT-RvD1 treatment (schema in Fig. 3A) could augment Ag-specific Ab responses in the serum and BAL of mice chronically infected with NTHI. We measured Abs against the immunodominant P6 Ag of NTHI in the serum and BAL. Although total anti-P6 Ab levels were considerably reduced in the serum and BAL of SHS-exposed mice compared with air-exposed mice, AT-RvD1 treatment modestly increased anti-P6 Ab levels in the serum and markedly restored anti-P6 Abs in the BAL Fig. 3B). Furthermore, whereas the levels of Ab subclasses IgG1 and IgG2b in serum and IgA in the BAL were significantly reduced in SHS-exposed mice, treatment with AT-RvD1 markedly elevated these levels (Fig. 3C). When AT-RvD1 treatment was continued during a 4-wk smoke-free cessation period, it was even more effective at improving anti-P6 Ab levels (SHS plus RvD1 versus SHS plus vehicle in Fig. 3D–F), which were now similar to the responses observed in air-exposed controls (SHS plus RvD1 versus air plus vehicle in Fig. 3D–F). This clearly indicates that, although the effect of SHS persists despite cessation (SHS plus vehicle versus air plus vehicle in Fig. 3D–F), continued SPM treatment significantly rescues and boosts the Ab responses (SHS plus RvD1 versus air plus vehicle in Fig. 3D–F).

T cell responses are essential for antibacterial immunity and critical to the generation of a robust Ab response against the pathogen. Because AT-RvD1 alleviated the effects of SHS on the Ab response to NTHI, we therefore assessed the impact of AT-RvD1 on T cell responses using the ELISPOT assay. We observed that SHS exposure caused a marked increase in the frequencies of P6-specific Th17 cells. AT-RvD1 treatment in SHS-exposed mice decreased the frequencies of IL-17A–secreting inflammatory Th17 T cells both in the lung and spleen (Fig. 4A). Furthermore, treatment with AT-RvD1 increased the frequency of IL-4– and IFN-γ–producing T cells, both in the lungs and spleen of mice exposed to chronic SHS (Fig. 4B, 4C). Continued treatment with AT-RvD1 for another 4 wk following cessation resulted in a small additional beneficial effect, with further reduction in the frequency of IL-17A–secreting Th17 T cells and an increase in the frequency of IL-4–producing T cells in both organs (Fig. 4D, 4E). A period of cessation plus treatment with AT-RvD1 did not provide additional enhancement in the numbers of IFN-γ–producing T cells in the lungs and spleen when compared with treatment with AT-RvD1 (Fig. 4F versus Fig. 4C).

We also evaluated the impact of At-RvD1 in modulating the frequency of Ag-specific B cell responses following chronic infection. Although SHS exposure suppressed NTHI-specific IgG1- and IgG2a-secreting B cell frequency in bone marrow and spleen, AT-RvD1 treatment enhanced their frequencies (Fig. 4G, 4H). Furthermore, we observed that SHS exposure followed by a period of cessation and continuation of treatment with AT-RvD1 during this interval was at least equally as effective as cessation alone in augmenting the NTHI-specific IgG1-secreting B cell frequency (Fig. 4I); however, this treatment strategy was beneficial in further augmenting the NTHI-specific IgG2a-secreting B cell frequency in both bone marrow and spleen (Fig. 4J).

Having established that AT-RvD1 treatment reduced inflammation and augmented immunity to infection, we evaluated whether AT-RvD1 treatment in SHS-exposed mice could enhance the efficacy of P6 vaccination (schema in Fig. 5A, 5C). Our results, in agreement with our previous findings (10), showed that prior chronic SHS exposure suppresses the generation of Ab responses to P6 vaccination in mice compared with air-exposed mice (Fig. 5B, 5D). AT-RvD1 treatment that started either immediately after the end of the SHS exposure (schema in Fig. 5A) or midway into the SHS exposure and continued for 8 wk (schema in Fig. 5C) significantly enhanced the production of both systemic (serum Ig) as well as mucosal (BAL total Ig and IgA) Ag-specific Abs following vaccination compared with vehicle-treated, SHS-exposed mice (Fig. 5B, 5D).

Sham-immunized air or sham-immunized SHS-exposed mice served as controls to evaluate the effectiveness of vaccination in decreasing the bacterial burden measured 4 and 24 h after acute NTHI infection (Fig. 6A). Although the bacterial clearance from the lungs of sham-immunized, SHS-exposed mice was nonsignificantly reduced at the 4-h time point. It was significantly inhibited compared with sham-immunized, air-exposed mice at 24 h after acute NTHI infection, although this challenge infection was given 16 wk after the last smoke exposure (third bar versus first bar in both panels in Fig. 6B). P6 vaccination enhanced NTHI clearance in both air- and SHS-exposed mice compared with their respective sham-immunized controls but was not as effective at the 4-h time point in SHS-exposed mice relative to air-exposed mice (Fig. 6B). However, clearance was markedly enhanced when mice were treated with AT-RvD1 prior to vaccination (Fig. 6B). With a physiological milieu of rapid bacterial clearance in the lungs of mice, we predicted less lung epithelial damage. Albumin leak into BAL of mice served as a surrogate marker of lung epithelial integrity. SHS-exposed, P6-vaccinated mice that were treated with SPM AT-RvD1 had significantly lower levels of albumin in the BAL at 4 and 24 h following acute NTHI infection (Fig. 6C).

We also evaluated the model in which mice were treated with AT-RvD1 during the second half of the smoke exposure period, and treatment continued during a 4-wk period of cessation to smoke exposure (Fig. 6D). We again observed that AT-RvD1 treatment prior to vaccination resulted in enhanced bacterial clearance compared with SHS-exposed, vehicle-treated, P6-vaccinated mice (Fig. 6E). These results also indicate that AT-RvD1 treatment started either during SHS exposure or at the end of the exposure has similar beneficial effects in enhancing bacterial clearance.

CS is a powerful inducer of inflammatory mediators in the lung that play a critical role in pulmonary inflammation and damage (4, 9, 10). Persistence of inflammatory mediators induces lung damage, impaired lung function, and increased susceptibility to infections (4, 9, 10, 50, 51). In fact, there is evidence that the pathology of COPD is driven by chronic unresolved inflammation, even in the absence of ongoing smoke exposure, as activated inflammatory cells produce reactive oxygen and nitrogen species, cytokines and chemokines, and enzymes that cause progressive lung tissue destruction, airway remodeling, and impaired lung function (4, 7, 8). Additionally, CS impairs the function of innate immune cells, such as neutrophils and macrophages, by prolonging inflammatory signaling, thus accelerating tissue damage (4, 13, 52–54). Smoking-related disorders resolve poorly, with the inflammatory damage to the lung persisting long after smoking cessation (10, 55, 56). Individuals, such as flight attendants, hospitality workers, and children exposed to chronic SHS, display an increased risk of smoking-related complications, including respiratory infections (57, 58). Indeed, we have recently demonstrated that chronic SHS exposure exacerbates pulmonary inflammation and diminishes immunity to pulmonary bacterial infections (10).

We recently demonstrated the ability of SPMs to function as vaccine adjuvants (46). We now show that treatment with the proresolving mediator AT-RvD1 during smoke exposure or immediately after initiation of smoking cessation decreases SHS-exacerbated, infection-induced lung inflammation, with decreased recruitment of inflammatory cells and cytokine production. Furthermore, AT-RvD1 treatment alleviated smoke-induced impairment of T cell and B cell function and generation of anti-NTHI Abs. Importantly, although the efficacy of AT-RvD1 was improved by smoking cessation, it was also effective when given concurrently with smoke exposure. This suggests that proresolving mediators could be developed as new clinical therapies to reduce the burden of infectious diseases in smokers, ex-smokers, children, and other vulnerable populations exposed unwillingly to SHS.

Anti-inflammatory drugs, cytokine receptor antagonists, antioxidants, and oxidative stress response modifiers have been shown to reduce CS-induced inflammation (59–61). However, anti-inflammatory agents that do not simultaneously induce immunosuppression are highly desired. SPMs not only diminish production of proinflammatory factors, but also induce a cellular phenotypic shift to promote anti-inflammatory effects, thus resetting the balance between pro- and anti-inflammatory processes to promote resolution (45, 62).

It is known that CS-induced macrophage and neutrophil dysfunction impairs bacterial killing and increases apoptotic cell-accumulation, thus impairing resolution of inflammation (4, 63, 64). RvD1 has been reported to inhibit neutrophilic inflammation and promote efferocytosis in experimental colitis and allergic airway models (65–67). Macrophages assist inflammation resolution via a tissue healing process and are recruited to ward off exacerbated lung inflammation (68). However, CS is shown to alter the phenotype of alveolar macrophages and inhibit bacterial phagocytosis that might be implicated in SHS-induced suppression of Ag-specific immunity and resolution of inflammation (19, 63, 69, 70). RvD1 is reported to enhance macrophage phagocytic activity, thus promoting inflammation resolution (71–74). Collectively, these observations suggest that AT-RvD1 might reduce SHS-exacerbated pulmonary inflammation by diminishing neutrophilic influx to the lungs and modulating macrophage function, thus promoting inflammation resolution and augmenting immunity.

Based on epidemiological findings, inflammatory diseases in smokers are associated with a Th17 phenotype. Chronic CS promotes Th17-polarized immunity that is associated with lung damage during emphysema and is characterized by increased ROR-γt, IL-17, and IL-6 expression, with a concomitant decline in Foxp3 and IL-10 expression (75–79). Such an induction of proinflammatory IL-17 signaling and differentiation to a Th17 cellular profile is attributed to the presence of multiple aryl hydrocarbon receptor (AhR) ligands in CS (80, 81). Our studies show that SHS causes an imbalance in Th1, Th2, and Th17 responses against NTHI infection, resulting in an augmentation of the IL-17 response. Furthermore, SHS-induced augmentation of IL-17A cytokines in the BAL, CD4⁺IL17A⁺, and CD4⁺RORγt⁺ T cell subsets in the lung and supports the involvement of the IL-17 axis in the sustenance of SHS-exacerbated, infection-induced chronic inflammation. Our findings are supported by an earlier report that IL-17A is required for CS-augmented, NTHI-induced pulmonary neutrophilia in acute exacerbation of COPD (82). Reports also show that RvD1 suppresses LPS -induced lung injury (83) and that D-series resolvins abrogate IL-17–induced inflammatory effects (84). From these perspectives, we propose that AT-RvD1–mediated amelioration of SHS-exacerbated, infection-induced pulmonary inflammation might be in part due to suppression of IL-17–dependent mechanisms.

Although CS-induced oxidative stress is reported to increase immune cell recruitment to cause pulmonary inflammation and damage associated with emphysema (85–87), augmented immune cell infiltration in the lungs itself is one of the critical mechanisms involved in oxidative stress induction in airway diseases (88). Although AT- RvD1 is not an antioxidant at the concentration used, it could contribute to the overall antioxidant activity via an indirect effect of decreased pulmonary recruitment of activated neutrophils and macrophages, immune cells that are a major source of oxidative stress during inflammation (50, 88, 89). Additionally, although CS-induced oxidative stress suppresses dendritic cell function (90), putting smokers at a higher risk of mucosal infections and lung damage, resolution of inflammation could reduce the detrimental impact. Indeed, RvDs decrease CS-generated, ROS-induced levels of carbonylated proteins (41) and thus could help reverse CS-mediated, oxidative stress-induced immune dysfunction and improve antipathogen host responses.

Along with the improvement in immune responses to NTHI infection, AT-RvD1 also enhanced Ab responses after immunization with NTHI P6 Ag that lead to increased bacterial clearance following bacterial challenge. Vaccination to prevent acute exacerbations in people with smoking-related diseases has limited effectiveness, likely because of the immunosuppressive effects of CS exposure. Our results show that a short course of AT-RvD1 treatment given after SHS exposure but prior to immunization can augment the immune response (Fig. 5), leading to higher Ab titers and more rapid clearance of bacteria following acute infection. This is consistent with the earlier and recent results from our group, showing that RvD1 or its precursor 17-hydroxydocosahexaenoic acid are effective adjuvants when given concurrently with the Ag (39, 45, 46, 91). We found that AT-RvD1–augmented antibacterial Ab responses could be due to restoration of Ag-specific B cell responses that were suppressed in the bone marrow and spleen of SHS-exposed mice. This immune-enhancing effect could be a direct translation of AT-RvD1–induced increased frequency of Ag-specific T cells secreting IL-4 and IFN-γ, cytokines that are required for Ab class switching to IgG1 and IgG2a, respectively (92–94). This immune-enhancing impact of AT-RvD1 on Ab production was further improved with a period of SHS cessation.

AT-RvD1 thus appears to have multiple beneficial effects in the context of bacterial infection following CS exposure. AT-RvD1 diminishes SHS-exacerbated, infection-induced pulmonary inflammatory cells and cytokines, whereas it enhances antibacterial adaptive immunity and augments Ag-specific systemic and mucosal Ab responses after vaccination. This enhanced efficacy of vaccination translated into decreased pulmonary bacterial burden with the induction of less lung epithelial cell damage following infection. Additionally, AT-RvD1 was effective when given during an ongoing SHS exposure as well as after smoking cessation.

RvD1 is only one of a large family of SPMs, and it is possible that other SPMs may also have significant use in smoke-induced inflammation and respiratory illness. However, there are several lines of research supporting the hypothesis that endogenous SPMs in general and RvD1 in specific act to mitigate inflammation and regulate immunity and that excess inflammation is associated with deficiencies in SPMs (62). For example, in mice, RvD1 and AT-RvD1 signal via the G protein–coupled receptor ALX/FPR2. Using a self-limiting model of peritonitis, in which the role of endogenous SPMs is well described, ALX/FPR2–overexpressing mice were more responsive to the effect of exogenous RvD1 treatment in reducing inflammation, whereas ALX/FPR2 knockout mice exhibited excess inflammation that could not be controlled with exogenous RvD1 (44). Lung tissue from COPD patients had elevated levels of eicosanoid oxidoreductase, an enzyme that degrades RvD1 (42), and RvD1 was decreased in the serum and BAL fluid of COPD patients relative to non-COPD subjects (41). Interestingly, CS also downregulates the RvD1 target miR-208a (95). Other SPMs implicated in the resolution of CS-induced inflammation include protectin D1, lipoxin A4, and RvD2 (62). Future work to explore the cellular sources and role of endogenous RvD1 could be performed using inhibitors of the lipoxygenase synthesis enzymes, soluble epoxide hydrolase inhibitors that block degradation of RvD1, and using ALX/FPR2 knockout and knock-in mice (96, 97), which include the possibility of creating cell- or tissue-specific knockouts using the Cre-Lox system.

SPMs may represent an important new therapeutic tool for preventing CS-related diseases in vulnerable populations, including children, the elderly, and immunocompromised individuals who have been previously exposed to MTS or SHS. SPMs can both reduce the burden of chronic smoking-related lung inflammation and also increase the efficacy of Ab responses to pathogens or vaccines, thereby potentially leading to an overall decrease in morbidity and mortality. This may be especially important in the elderly because, although the rate of active smoking has decreased, there remains a large population of elderly individuals who are former smokers or were formerly exposed to SHS in the home or workplace, in whom smoke exposure has lifelong consequences. The elderly and immunocompromised individuals are also less proficient at mounting adaptive immune responses to infection and vaccination (98, 99). Thus, the elderly in general and specifically persons with smoking or SHS exposure history, may benefit from therapeutic use of SPMs to reduce lung inflammation and increase adaptive immune responses.

In conclusion, our current findings demonstrate that SPM AT-RvD1 helps resolve SHS-exacerbated, chronic infection-induced pulmonary inflammation and might represent one of the mechanisms through which AT-RvD1 enhances adaptive immunity to infection and efficacy to vaccination in SHS-exposed mice. Importantly, the proresolving and anti-inflammatory effects of AT-RvD1 contrast with classical anti-inflammatory therapies, such as nonsteroidal anti-inflammatory drugs and corticosteroids, and underscore its potential use as a therapeutic agent to manage or treat infectious comorbidities of disorders having chronic inflammation as a causal factor.

The authors have no financial conflicts of interest.