Tobacco smoke exposure is associated with multiple diseases including, respiratory diseases like asthma and chronic obstructive pulmonary disease. Tobacco smoke is a potent inflammatory trigger and is immunosuppressive, contributing to increased susceptibility to pulmonary infections in smokers, ex-smokers, and vulnerable populations exposed to secondhand smoke. Tobacco smoke exposure also reduces vaccine efficacy. Therefore, mitigating the immunosuppressive effects of chronic smoke exposure and improving the efficacy of vaccinations in individuals exposed to tobacco smoke, is a critical unmet clinical problem. We hypothesized that specialized proresolving mediators (SPMs), a class of immune regulators promoting resolution of inflammation, without being immunosuppressive, and enhancing B cell Ab responses, could reverse the immunosuppressive effects resulting from tobacco smoke exposure. We exposed mice to secondhand smoke for 8 wk, followed by a period of smoke exposure cessation, and the mice were immunized with the P6 lipoprotein from nontypeable Haemophilus influenzae, using 17-HDHA and aspirin-triggered–resolvin D1 (AT-RvD1) as adjuvants. 17-HDHA and AT-RvD1 used as adjuvants resulted in elevated serum and bronchoalveolar lavage levels of anti-P6–specific IgG and IgA that were protective, with immunized mice exhibiting more rapid bacterial clearance upon challenge, reduced pulmonary immune cell infiltrates, reduced production of proinflammatory cytokines, and less lung-epithelial cell damage. Furthermore, the treatment of mice with AT-RvD1 during a period of smoke-cessation further enhanced the efficacy of SPM-adjuvanted P6 vaccination. Overall, SPMs show promise as novel vaccine adjuvants with the ability to overcome the tobacco smoke-induced immunosuppressive effects.
Tobacco smoke in the form of mainstream tobacco smoke (MTS) or secondhand smoke (SHS) is proinflammatory, immunosuppressive, and associated with diseases like cancer, cardiovascular disorders, and pulmonary disorders like chronic bronchitis and chronic obstructive pulmonary disease (COPD) (1, 2). Around 8 million people die each year because of tobacco smoke exposure (3). Chronic respiratory diseases are the third-most common cause of death in the United States and fifth worldwide, putting a huge socioeconomic burden on society (2–6). Tobacco smoke triggers proinflammatory physiological changes in the airway epithelium, inducing pulmonary damage and immunosuppression that collectively augment susceptibility to respiratory infections and exacerbate infection-induced pulmonary inflammation (1, 7–9). Whereas acute inflammatory responses are self-limiting processes (10), chronic inflammation due to smoking persists long after smoking cessation because of the production of excess proinflammatory cytokines and reactive oxygen and nitrogen species by the infiltrating activated immune cells themselves (11, 12). It is also established that smokers and ex-smokers, with and without COPD, children, and others involuntarily exposed to SHS in the home or workplace are at increased risk of respiratory infections (1, 13–15). Respiratory infections among COPD patients account for 75% of acute exacerbation events, which are responsible for the majority of morbidity, mortality, and medical costs in this population (16–20). SHS exposure in young children correlates with acute otitis media episodes, whereas in patients with COPD, increased incidence of infections with nontypeable Haemophilus influenzae (NTHI) are common and lead to disease exacerbations (1, 13–15). We have previously shown that chronic exposure to MTS or SHS suppresses Ag-specific adaptive immunity to NTHI and leads to increased bacterial burden in the lungs of mice following infection (8, 9). Vaccination is of limited effectiveness, possibly due in part to the immunosuppressive effects of tobacco smoke exposure (1, 8, 9). Thus, effective solutions are urgently needed to treat chronic inflammation and enhance immune responses in these populations.
Acute inflammatory responses, crucial to fight pathogens and critical for host tissue repair and homeostasis, are self-limiting processes (10, 21). A newly identified class of lipid mediators called specialized proresolving mediators (SPMs) actively regulate the resolution phase of inflammation (22–24). SPMs have proresolving as well as anti-inflammatory properties without inducing immunosuppression (24–26). SPMs are endogenous mediators derived from the dietary ω-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid and are found in a variety of tissues and organs, including bone marrow, spleen, and blood (27–29). SPMs exert multiple functions in promoting resolution of inflammation, including decreasing transmigration of neutrophils, enhancing nonphlogistic monocyte recruitment, and augmenting macrophage engulfment of apoptotic neutrophils (29–33). SPMs inhibit the production of proinflammatory mediators such as TNF-α, IL-6, CXCL1, and IL-12 while enhancing anti-inflammatory cytokines like IL-10 (26, 34–36).
More recent evidence shows a role for SPMs in Ab responses by augmenting differentiation of B cells into Ab-secreting plasma cells to enhance human B cell Ab production in vitro (29). 17-Hydroxydocosahexaenoic acid (17-HDHA), which has both direct SPM activity and is also a precursor for resolvin D1 (RvD1), can act as a vaccine adjuvant by augmenting Ab responses to recombinant influenza hemagglutinin protein (30). 17-HDHA, RvD1, and protectin D1 were all found in the spleen (29), suggesting that endogenous SPMs play a role in regulating Ab responses and that treatment with SPMs might be a novel therapeutic option to increase immunity against infections.
In this study, we hypothesized that the SPMs 17-HDHA and aspirin-triggered–RvD1 (AT-RvD1) could overcome SHS exposure–induced suppression of pathogen-specific Ab responses following vaccination and infection. We report that SHS-exposed mice immunized with P6 plus 17-HDHA or with P6 plus AT-RvD1 developed increased systemic as well as mucosal Ag-specific Ab titers that translated into rapid bacterial clearance in the lungs of vaccinated mice following acute NTHI infection with reduced epithelial damage. Treatment of SHS-exposed mice with AT-RvD1 prior to immunization, further reduced lung inflammation and enhanced the efficiency of the immunization. Taken together, our results suggest that proresolving mediators will be an effective approach to reduce chronic lung inflammation and improve the effectiveness of immunization against lung pathogens, leading to reduced morbidity and mortality among people with lung disease resulting from chronic exposure to MTS or SHS.
Materials and Methods
Female C57BL/6J mice (8 wk old) were purchased from Jackson Laboratory (Bar Harbor, ME) and used in all experiments. Animals were housed under specific pathogen-free conditions with free access to food and water and a light/dark cycle of 12:12 h. Number of animals used per group in each experiment was 10, unless mentioned otherwise in the figure legends. All animal procedures performed were approved by the Animal Care and Use Committees of both institutions (Roswell Park Comprehensive Cancer Center, Buffalo, NY and the University of Rochester, Rochester, NY), and complied with all state, federal, and National Institutes of Health regulations.
Mice housed in the Inhalation Core Facility at the University of Rochester were exposed to SHS 5 h/d, 5 d/wk for 8 wk as described previously (9). Briefly, research cigarettes 3R4F were combusted in an automated smoking machine (TE-10; Teague Enterprises, Woodland, CA) and then 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 89%:11% ratio (37). Total particulate matter concentration of 99 ± 3 mg/m3 was achieved for these chronic exposure experiments by adjusting the number of cigarettes loaded. Control group of animals were exposed to filtered air in an identical manner. At the end of the final exposure cycle, animals were immediately transported to Roswell Park Comprehensive Cancer Center for infection and vaccination experiments.
AT-RvD1 treatment regimen
AT-RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13-Z,15E,19Z-docosahexaenoic acid) is an epimer of RvD1 that is produced when cyclooxygenase 2 (Cox-2) is acetylated by aspirin; it has similar in vivo and in vitro effects to RvD1 and is resistant to endogenous degradation (38). AT-RvD1 (Cayman Chemical) was dissolved in ethanol at 200 μg/ml, aliquoted, and stored frozen. Immediately prior to use, this was diluted with seven volumes of normal saline solution, resulting in a solution containing 25 μg/ml AT-RvD1 in 12.5% ethanol. A volume of 40 μl (1 μg of AT-RvD1) was administered to SHS-exposed mice by oropharyngeal aspiration (39), twice a week for 8 consecutive wk. Control animals received 12.5% ethanol in saline. In experiements pertaining to Fig. 1E–H, AT-RvD1 treatment on mice was done on weeks 5–12 before chronic infection or P6 vaccination, during which weeks 5–8 represent the second half of smoke exposure period and weeks 9–12 represent a period of cessation to smoke exposure.
Chronic and acute pulmonary infection with NTHI
For all the experiments, NTHI bacterial strain 1479 (clinical isolate from a COPD exacerbation) from a single frozen glycerol stock was streaked onto chocolate agar plates to grow bacterial cultures using a standard operating procedure to ensure that the NTHI doses were comparable in all chronic and acute pulmonary infection studies as described previously (8, 9). Briefly, for chronic NTHI-mediated pulmonary inflammation, mice received 1 × 106 live bacteria twice per week for 8 consecutive wk via intratracheal route. For acute bacterial infection, mice were given a single intratracheal challenge with 1 × 106 live bacteria and euthanized 4 and 24 h later. After preparing lung homogenates, multiple serial dilutions were plated onto chocolate agar plates and the resulting colonies are quantitated after overnight incubation.
Mice were immunized i.m. with 40 μg of purified P6 Ag + 1 μg of SPM (17-HDHA or AT-RvD1) in a final ethanol concentration of 4%. Sham-immunized mice received 4% diluent in PBS. For the experiments in Fig. 1, air and SHS-exposed mice were immunized i.p. with 40 μg of purified native P6 lipoprotein emulsified in CFA and boosted 1 wk later with P6 in IFA and 2 wk later with Ag alone in PBS (40). Sham-immunized mice received PBS only. To quantify Ag-specific Abs in serum and BAL, mice were bled on a weekly basis to collect sera, and the bronchoalveolar lavage (BAL) was harvested at the time of euthanasia.
Upon completion of experimental protocol, mice were euthanized by injecting i.p. 1 ml of warmed 2.5% avertin solution (2,2,2-tribromethanol). Thoracic cavity was exposed to cannulate trachea and lungs were gently lavaged twice by injecting 750 μl of ice-cold 1% BSA solution in PBS as described previously (9). The cell-free BAL fluid was stored at −80°C and used to measure the levels of cytokines, albumin, and Ag-specific Ab titers.
Isolation of lung lymphocytes, splenocytes, and bone marrow cells
Lung lymphocytes were isolated as described previously (9). Briefly, after euthanasia, lungs were excised and minced into small pieces in a 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 incubated for 1 h on a rotator at 37°C. Single-cell suspension was passed through a 40-μm filter to remove debris and undigested tissue, and then centrifuged for 5 min and resuspended in complete RPMI-1640 culture media containing 10% FBS. The cell suspension was gently overlaid on top of Ficoll-Paque and centrifuged at 700 × g for 30 min with the brake-off. Immune cells at the interface were collected and washed twice with PBS to remove residual Ficoll-Paque, and the cell count was determined.
Immune cell numbers in the lungs of mice was determined by flow cytometry as described previously (9). Briefly, 0.5 million lung lymphocytes from each sample were stained with 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 Cytofix (BD Biosciences). To stain intracellular markers, cells were first treated with permeabilizing solution (BD Biosciences) and then stained with cell-type specific fluorophore-tagged Abs. Samples were acquired on LSRII-A flow cytometer and FlowJo software was employed to analyze the data. Gating strategy that was used is shown in Supplemental Fig. 1.
ELISA assays were performed as described previously (8, 9) to determine cytokine and albumin levels in the BAL and Ag-specific Ab titers in the serum and BAL. Briefly, the levels of cytokines in the BAL were measured using the eBioscience ELISA kits following the manufacturer’s protocol: IL-17 (catalog no. 88-7371-77), IL-6 (catalog no. 88-7064-77), TNF-α (catalog no. 88-7324-77), and IL-1β (catalog no. 88-7013-77). Mouse albumin levels in BAL fluid (as a surrogate marker of lung-epithelial damage) were quantified using a mouse-specific albumin-quantifying ELISA Kit from Bethyl Laboratories (Montgomery, TX), with no cross-reactivity to BSA. Ag-specific Ab titers in the serum and BAL were determined using purified P6–coated ELISA plates as described previously (8, 9). Dilutions of serum and end point BAL samples 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 (Southern Biotech, Birmingham, AL). Plates were developed with eBioscience 3,3′,5,5′-tetramethylbenzidine (TMB; for HRP) solution, and absorbance was read at 450 nm on an ELISA plate reader (Synergy HTX Multi-Mode Reader, Winooski, VT).
Assessment of NTHI clearance
NTHI bacterial clearance in the lungs of mice following acute infection was performed as described previously (9). Briefly, animals were given an acute intratracheal bacterial challenge with live 1 × 106 CFUs and euthanized 4 and 24 h later. Under aseptic conditions, whole lungs were excised and gently homogenized in 1 ml of PBS on ice. Serial dilutions of lung homogenates were plated onto chocolate agar plates and incubated at 35°C at 5% CO2 conditions for 16 h. NTHI colonies were counted the next day and bacterial burden in the lungs was determined and expressed as the total number of bacterial CFUs recovered from the lungs.
The statistical objective was to assess the differences in marker measurements across exposure groups and time. Central tendency of the markers was quantified by estimates for the geometric mean, with two-sided 95% confidence intervals (C.I.) indicating the precision of these estimates. For each marker in experiments with one observation per mouse, an ANOVA model was specified with fixed effects for exposure group, time, and the interaction terms. When repeated observations per mouse were available (Figs. 2B, 3B), linear-mixed models were specified with the same fixed effects, and a random subject effect with compound symmetric covariance structure. Throughout, marker measurements were assumed to be lognormally distributed. Compliance with distributional assumptions was confirmed using quantile plots of the model residuals. Each statistical model analyzed one marker within each experiment. For each model, Type 1 error was controlled at the α = 0.05 level using Tukey multiple-testing methods. All the data are expressed as geometric mean ± 95% C.I. For Fig. 1, difference between mean values was tested using a two-tailed unpaired Student t test with a Mann–Whitney posttest comparison using GraphPad Prism 8 software; the difference between two groups was considered significant at p ≤ 0.05.
SPMs 17-HDHA and AT-RvD1 overcome immune suppression induced by SHS exposure
First, we confirmed that the model was working as expected. As previously reported (9), chronic exposure of mice to SHS for 8 consecutive wk markedly suppressed the induction of systemic Ag-specific Ab titers after chronic NTHI infection (Fig. 1A, 1B) and following vaccination with NTHI P6 Ag (Fig. 1C, 1D). We next evaluated if treatment of SHS-exposed mice with AT-RvD1 could rescue the SHS-induced immunosuppressive effects following infection and vaccination (schema Fig. 1E, 1G). We observed that AT-RvD1 treatment in SHS-exposed mice significantly enhanced the Ag-specific Ab responses following both infection as well as vaccination with NTHI P6 Ag (Fig. 1F, 1H), thus clearly demonstrating in vivo immunoenhacning potential of SPM AT-RvD1. We next examined the potential of docosahexaenoic acid–derived SPMs, 17-HDHA, and AT-RvD1 to function as adjuvants to enhance the efficacy of vaccination in SHS-exposed mice. To address this question, mice exposed to SHS for 8 wk were vaccinated with purified P6 Ag plus 17-HDHA or P6 plus AT-RvD1 or vehicle control (Fig. 2A), and the efficacy of vaccination was evaluated by measuring the anti-P6 Ab titers in serum and the BAL of mice. Vaccination with P6 in the presence of 17-HDHA or AT-RvD1 accelerated the kinetics of Ab response as well as markedly elevated the absolute serum Ab titers compared with vaccination with P6 plus vehicle control (Fig. 2B). The efficacy of vaccination was greater with 17-HDHA than AT-RvD1. Furthermore, end point Ag-specific Ab titers in the BAL of SHS-exposed mice were significantly elevated after vaccination with P6 Ag either in the presence of 17-HDHA or AT-RvD1 compared with vaccination with P6 plus vehicle control (Fig. 2C). Additionally, the levels of P6-specific IgG1 and IgG2b Ab subclasses were significantly augmented in the serum and BAL of SHS-exposed mice vaccinated with P6 Ag plus 17-HDHA or P6 Ag plus AT-RvD1 compared with the levels elicited in mice vaccinated with P6 Ag plus vehicle (Fig. 2D–F). Importantly we report that vaccinating SHS-exposed mice with P6 Ag in the presence of SPM adjuvants 17-HDHA or AT-RvD1 significantly augmented mucosal P6-specific IgA Ab levels in the BAL (Fig. 2G).
AT-RvD1 treatment of SHS-exposed mice prior to vaccination further enhances SPM adjuvant–augmented vaccination efficacy
We next determined if pretreating SHS-exposed mice with AT-RvD1 prior to vaccination would further augment the efficacy of vaccination in the presence of SPM adjuvant 17-HDHA. To examine this, mice chronically exposed to SHS were treated with AT-RvD1 twice a week for 8 consecutive wk and then immunized with P6 plus 17-HDHA or P6 plus vehicle control (Fig. 3A). AT-RvD1 treatment alone resulted in slightly elevated Ab responses when the mice were immunized with P6 plus control vehicle, and this effect was seen in both serum and in lung (BAL) Ab levels. This suggests that AT-RvD1 alone, given after smoke exposure, mitigates the immunosuppressive effects of SHS exposure and primes the mice to make an immune response to the unadjuvanted P6 Ag. The combination of AT-RvD1 treatment after SHS exposure plus immunization with P6 Ag adjuvanted with 17-HDHA markedly elevated the P6-specific Ab titers in serum (systemic) and BAL (mucosal) following vaccination with P6 plus 17-HDHA compared with vaccination with P6 plus vehicle (Fig. 3B, 3D). Additionally, IgG1 and IgG2b Ab subclasses in the serum and BAL were significantly increased (Fig. 3C, 3E). We also observed markedly augmented levels of Ag-specific IgA Abs in the BAL of these mice (Fig. 3F).
17-HDHA– and AT-RvD1–augmented efficacy of P6 vaccination enhanced bacterial clearance in the lungs of SHS-exposed mice
To demonstrate the functional benefit of higher Ab titers, we measured bacterial clearance of an acute NTHI infection following vaccination. Mice exposed to SHS for 8 wk were either immunized with P6 plus 17-HDHA or P6 plus AT-RvD1 or P6 plus vehicle, and then challenged with acute NTHI infection 8 wk later (Fig. 4A). Another group of mice exposed to SHS for 8 wk, were treated with AT-RvD1 for 8 wk and then vaccinated with P6 plus 17-HDHA, and subsequently challenged with an acute NTHI infection at week 24 (Fig. 4B). In both groups of mice, bacterial clearance in the lungs was measured 4 and 24 h following acute challenge with NTHI (Fig. 4A, 4B). We found that SHS-exposed mice that were vaccinated with P6 plus 17-HDHA or P6 plus AT-RvD1 had significantly improved bacterial clearance at both 4 and 24 h following acute NTHI challenge compared with SHS-exposed mice vaccinated with P6 plus vehicle control (Fig. 4C). As pretreatment with AT-RvD1 augmented Ab responses following immunization (Fig. 2), we expected that pretreatment with AT-RvD1 would also enhance bacterial clearance, and this was, indeed, what we found. Pretreatment of the mice with AT-RvD1 markedly improved bacterial clearance, especially at 4 h postinfection, and even in mice immunized with the unadjuvanted P6 Ag (hatched bars versus black bars at 4 h in Fig. 4C). However, use of 17-HDHA as an adjuvant significantly improved clearance in AT-RvD1–treated mice at both 4 and 24 h postinfection.
SPM adjuvant–enhanced immunity reduces SHS-exacerbated inflammatory microenvironment following an acute respiratory infection
We next evaluated whether the augmented adaptive immunity induced by the SPMs had an impact on the SHS-exacerbated pulmonary inflammatory microenvironment induced following an acute bacterial infection with or without AT-RvD1 treatment after SHS exposure (schemas in Fig. 5A, 5B). The total number of pulmonary inflammatory cells was highest in mice exposed to SHS without SPM treatment or immunization. Immunization with P6 plus 17-HDHA or AT-RvD1, but not vehicle, dramatically reduced lung-infiltrating cells. Mice pretreated with AT-RvD1 exhibited reduced numbers of infiltrating leukocytes compared with mice receiving SHS alone, and this was further reduced by immunization with P6 plus 17-HDHA. However, the beneficial effect of immunization with P6 plus 17-HDHA was the same whether the mice were pretreated with AT-RvD1 (Fig. 5C). We also observed that the numbers of neutrophils, CD4+IL-17A+ Th17, and CD4+RoRγ+ Th17 T cells in the lungs of SHS-exposed mice vaccinated with P6 + SPM (17-HDHA or AT-RvD1) were markedly reduced after acute bacterial challenge (measured at both 4 and 24 h) compared with the numbers of these inflammatory cells found in the lungs of mice that were either sham or vaccinated with P6 only (Fig. 6A–F, Supplemental Table I). Moreover, the numbers of these cells were further reduced in the lungs of SHS-exposed mice that were treated with AT-RvD1 for 8 wk prior to vaccination with P6 plus 17-HDHA compared with SHS-exposed, vehicle-treated, sham-vaccinated mice (Fig. 6A–F, Supplemental Table I).
SPM-enhanced efficacy of P6 vaccination diminished proinflammatory cytokine induction in the BAL and translated into reduced lung-epithelial damage in response to acute infection
We also evaluated the effects of 17-HDHA and AT-RvD1 on the levels of pulmonary proinflammatory cytokines. We observed lower levels of proinflammatory cytokines IL-17A, IL-6, and TNF-α measured in the BAL at both 4 and 24 h after acute infection in mice, immunized with P6 plus 17-HDHA or AT-RvD1, compared with sham or P6 plus vehicle–immunized mice (Fig. 7A–C). However, IL-1β levels were reduced only 24 h after acute challenge with NTHI (Fig. 7D). Pretreatment with AT-RvD1 alone also reduced levels of inflammatory cytokines to a similar degree, consistent with our previous finding that this SPM accelerated resolution of inflammation after smoke exposure (24). The lowest levels of proinflammatory cytokines were seen 24 h postinfection in mice that were pretreated with AT-RvD1 and immunized with P6 plus 17-HDHA.
Finally, we measured the levels of mouse serum albumin in the BAL fluid, a surrogate measure for alveolar epithelial barrier function and integrity. SHS-exposed mice either vaccinated with P6 plus SPM (17-HDHA or AT-RvD1) or pretreated with AT-RvD1 for 8 wk and then vaccinated with P6 plus 17-HDHA, had markedly lower levels of albumin in the BAL than found in SHS-exposed mice that were sham-vaccinated or immunized with P6 + vehicle (Fig. 7E).
Tobacco smoke, including involuntary SHS exposure, is associated with many human disorders including respiratory diseases like COPD (1, 6, 13–15, 41). In response to toxic exogenous insults like SHS, pulmonary homeostasis is maintained via an array of immune cells, including recruited neutrophils and resident macrophages, and secreted cytokines (42). However repeated stimulation with toxic insults results in the persistence of immune mediators that disrupt pulmonary homeostasis and induce chronic lung inflammation (43). These physiological changes induced by tobacco smoke account for the exacerbated pulmonary inflammation observed in smokers with COPD that results in pulmonary damage and impaired lung function (1, 16, 17). Many studies have established that tobacco smoke exacerbates infection-associated pulmonary inflammation, dysregulates immune function, and augments susceptibility to respiratory infections, thereby further worsening lung inflammation and function particularly in susceptible individuals like COPD patients (1, 8, 9, 18). Smokers, ex-smokers, children, and other vulnerable populations exposed to SHS are shown to be at increased risk for pneumonia and other infections, and this has been replicated in mouse models of smoke exposure and infection (1, 6, 8, 9, 13–15). For instance, SHS exposure in children is known to increase the risk of ear infections, cause acute lower–respiratory tract infections like bronchitis and pneumonia in infants and young children, and trigger respiratory symptoms like cough, phlegm, wheezing, and breathlessness in school-going children (6, 13–15). Viral and bacterial infections account for up to 75% of acute exacerbations of COPD, and account for much of the morbidity experienced by these patients (19, 20, 44). Various studies have clearly shown that tobacco smoke inhibits innate and adaptive immunity by suppressing functions of immune cells ranging from macrophages, dendritic cells, NK cells, and neutrophils to B and T cells (1, 18). Tobacco smoke exposure, both mainstream and secondhand, also reduces vaccine efficacy (8, 9). Therefore, mitigating the effect of chronic tobacco smoke exposure on the immune system, and improving vaccine efficacy in people exposed to tobacco smoke, is a critical unmet clinical problem.
In the current study, we demonstrate that SPMs 17-HDHA and AT-RvD1 as vaccine adjuvants potently overcome the SHS-induced suppression of protective immune responses to vaccination by augmenting systemic as well as mucosal Ag-specific Ab responses. These SPM adjuvant–augmented Ab responses were functionally relevant as they diminished the bacterial burden and reduced proinflammatory cytokines and alveolar leakage following an acute respiratory infection. Additionally, treatment with AT-RvD1 before vaccination of SHS-exposed mice further enhanced SPM adjuvant–augmented Ab responses that translated into improved protection against acute NTHI pulmonary infection. Our study establishes the utility of SPMs to diminish SHS-exacerbated proinflammatory milieu and enhance Ag-specific systemic and mucosal immunity. As a first step, we revalidated our previously published findings that chronic SHS exposure suppresses Ag-specific Ab responses to both chronic NTHI infection as well as NTHI P6 Ag vaccination. Importantly, we found that SPM AT-RvD1 treatment in SHS-exposed mice significantly augmented systemic Ag-specific Ab titers following chronic infection and vaccination, thus highlighting the physiological effects of AT-RvD1 treatment in SHS-exposed, -infected, or -vaccinated mice.
Chronic cigarette smoke exposure–induced immune suppression worsens proinflammatory pulmonary microenvironment to increase bacterial burden in the lungs following infection (8, 9), a phenomenon commonly observed in smokers with COPD during disease exacerbations and associated with increased morbidity and mortality (1, 16–20). SPMs, naturally occurring proresolving and anti-inflammatory bioactive agents, are involved in inflammation resolution without causing immunosuppression (22–26). Additionally, SPMs augment adaptive immunity via mechanisms that include upregulation of CD80 and CD86 expression on B cells and promoting differentiation of B cells into Ab-secreting plasma cells to augment Ab responses (29, 30). We thus hypothesized that if SPMs could be used to enhance efficacy of vaccination using a candidate vaccine Ag, SHS-suppressed antibacterial responses could potentially be restored. Furthermore, we posited that SPM adjuvant–enhanced antibacterial responses might have translational relevance in diminishing SHS-exacerbated, infection-induced pulmonary proinflammatory microenvironment, thereby reducing lung-epithelial damage. It has been reported that RvD1 treatment reduces inflammatory cytokines and leads to diminution of bacterial burden (26). We therefore sought to evaluate the beneficial impact of AT-RvD1 treatment in combination with SPM adjuvant plus Ag vaccination, on Ag-specific immune responses and its impact on SHS-exacerbated, infection-induced pulmonary proinflammatory microenvironment and damage.
It is known that cigarette smoke–suppressed immunity delays resolution of bacterial infection in the lungs of mice (1, 8, 9). By exploiting 17-HDHA and AT-RvD1 as vaccine adjuvants to enhance systemic and mucosal P6 Ag–specific Ab responses, we observed decreased NTHI burden in the lungs of SHS-exposed mice following acute infection. Because cigarette smoke–augmented pulmonary bacterial burden correlates with exacerbated lung inflammation (8, 9, 45), we thus speculated that diminished bacterial burden in the lungs of SHS-exposed mice would lead to diminution in the proinflammatory pulmonary milieu. Indeed, we observed that SPM adjuvant–enhanced immunity by reducing bacterial burden in the lungs of SHS-exposed mice, also helped diminish SHS-exacerbated pulmonary inflammatory microenvironment by decreasing proinflammatory immune cell accumulation, which correlated with diminished levels of proinflammatory cytokines following infection.
The translational relevance of our study is depicted by a rapid clearance of bacteria from the lungs of SHS-exposed, SPM adjuvant plus P6–vaccinated mice with a concomitant reduction in lung-epithelial damage. This beneficial effect of SPM adjuvants is clinically relevant because in susceptible individuals like smokers with COPD, elderly, or children exposed to environmental tobacco smoke, increased pulmonary bacterial burden associates with augmented inflammation, enhanced lung damage, and impaired lung function (1, 6, 8, 9, 13–20). An important observation emerging from our study was seen when SHS-exposed mice were treated with AT-RvD1 before P6 + SPM adjuvant vaccination, resulting in enhanced vaccination efficacy that translated into reduced pulmonary bacterial burden, diminished pulmonary inflammatory responses, and decreased lung-epithelial damage following acute bacterial infection. However, we cannot rule out the involvelement of other mechanisms associated with resolvin-mediated bacterial clearance including reduction of chronic smoke-induced inflammation (46) and improved macrophage function (26). However, our previously published studies on the use of conventional adjuvants with outer membrane NTHI lipoprotein P6 Ag to induce protective immunity to NTHI amply demonstrates that an appropriate humoral response can have a significant beneficial effect in this model (8, 9). We now clearly demonstrate the beneficial effects of resolvins in the comparison between P6 plus vehicle immunization and P6 immunization with either 17-HDHA or AT-RvD1. It is also of interest that the maximum benefit of immunization with P6 plus 17-HDHA was observed in mice that had also been treated with AT-RvD1 prior to immunization. The results suggest that the beneficial effects of modifying the chronic inflammatory milieu induced by smoke exposure and of improving Ab responses were additive, and resulted in the lowest inflammatory response and most improved bacterial clearance, which is an eventual therapeutic goal in patients with chronic lung inflammation who experience infectious exacerbations. Indeed, using SPMs to treat inflammatory diseases has shown beneficial results in various models including mouse models of COPD, asthma, arthritis, periodontitis, viral infection, and cancer (24, 26, 30, 46–52).
The present study confirms that chronic SHS suppresses systemic and mucosal NTHI P6 Ag–specific Ab responses. Long-term immunity is a precondition for durable protection against infections in susceptible individuals. Because alum is the only Food and Drug Administration–approved adjuvant currently in use in the United States (53), evaluation of a new class of adjuvants is deemed important to improve the efficacy of human vaccines. Our results establish that SPMs 17-HDHA and AT-RvD1 are potent vaccine adjuvants that can significantly restore SHS-suppressed immunity by augmenting protective Ag-specific systemic and mucosal Ab responses. It is also well understood that some populations, particularly the elderly and immune-compromised indivuduals, are at increased risk of morbidity and mortality from respiratory infections while also being less able to generate protective Ab responses following immunization. Toward this endeavor, SPMs might also be an important new way to augment vaccine responses in the elderly and immune-compromised individuals, irrespective of their smoking history (54, 55). Indeed, the current coronavirus pandemic highlights the critical problem of respiratory infections in the elderly, with estimated patient fatality rates 10- to 100-fold higher among the over-60 age group compared with the under-60 age group(56, 57). SPMs have already been theorized to be able to dampen cytokine storm in COVID-19 patients (58); our results suggest that SPMs might also have beneficial effects as adjuvants when a COVID-19 vaccine is developed.
Our present results as well as earlier findings by us and other groups clearly indicate that immunoenhancing effects of SPMs are not restricted to reducing the immunosuppressive effects of cigarettte smoke on the immune responses to NTHI infection in our murine model of infection and immunity. We have previously reported that 17-HDHA protects against lethal challenge by H1N1 influenza A infection in mice when used as an adjuvant mixed with the HA Ag (30). 17-HDHA adjuvant–mediated immunity provided long-term protection against live influenza viral infection in mice and markedly increased their survival with minimum weight loss compared with nonadjuvanted, immunized mice. Studies in the literature also show that resolvins have protective effects against various infections including Escherichia coli and Staphylococcus aureus via mechanisms like lowering the antibiotic requirements for clearing bacterial infections, thus leading to reduction in local and systemic bacterial loads and enhancing the survival in mice (59, 60). Furtheremore, data from human studies show a benefical association between SPM resolvins and controlling infections including parasites (Trypanosoma cruzi) (61), acute exacerbations of COPD (62), and sepsis (63).
A limitation of our study regarding the adjuvant effect of 17-HDHA and AT-RvD1 with P6 immunization is that we did not include air-only controls in every experiment presented. However, we have previously reported that 17-HDHA and AT-RvD1 are effective adjuvants in mice that were not exposed to smoke when immunized with influenza HA Ag (30). The key goal of the current study was to demonstrate that SPMs can also augment immune responses in a mouse model of immune suppression following 8 wk of cigarette smoke exposure. Thus, the results from Figs. 2, 3 illustrate the beneficial effects of using SPMs as adjuvants on P6 Ab titers. The data from the experiment reported in Figs. 4–7 show that SPMs clearly rescue the immune impairment caused by chronic exposure to cigarette smoke. For example, in Fig. 4C, the decreased NTHI load between column 1 and 5 at the 4-h time point are attributable to the rest period with inhaled AT-RvD1 treatment, whereas the decrease between column 5 and 6 represents the effect of immunization with P6 plus 17-HDHA as an i.m. injection. The same effect is seen at 24 h. A consistent effect pattern is seen with lung cell counts (Fig. 5C), neutrophils (Fig. 6C), Th17 cells (Fig. 6D), and cytokines (Fig. 7). However, we cannot rule out the possibility that some relief from the smoke-induced immune suppression in the experiment reported in Figs. 4–7 may be attributable to time lapsed, rather than the effects of inhaled AT-RvD1 and/or adjuvanted immunization. However, our earlier investigations of cigarette smoke–mediated immune suppression in the NHTI model argue that time alone does not restore immune function (9), as does our results in Fig. 1E–H. Overall, our current results and previous findings by us and others allow us to conclude that SPMs 17-HDHA and AT-RvD1 show multiple beneficial effects to restore immune function after smoke exposure, and thus indicate that immune enhancing effects of 17-HDHA/AT-RvD1 are physiologically significant.
Collectively, our results establish that SPMs 17-HDHA and AT-RvD1 by acting as proresolving mediators and vaccine adjuvants rescue SHS-suppressed immunity to vaccination and infection.
This work was supported by a Flight Attendant Medical Research Institute Clinical Innovators Award to Y.T. and P.J.S. This work was supported in part by National Institutes of Health Grant R01 HL120908 (to P.J.S.). This work also was supported by National Cancer Institute Grant P30CA016056 involving the use of Roswell Park Core facilities: Division of Laboratory Animal Resources, Mouse Tumor Model Resource, and Flow Cytometry.
The online version of this article contains supplemental material.
Abbreviations used in this article:
The authors have no financial conflicts of interest.