Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice.

The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections.

Ablation of leptin receptor-mediated ERK activation impairs host defense against Gram-negative pneumonia.

The adipocyte-derived hormone leptin plays an important role in regulation of energy homeostasis and the innate immune response against bacterial infections. Leptin's actions are mediated by signaling events initiated by phosphorylation of tyrosine residues on the long form of the leptin receptor. We recently reported that disruption of leptin receptor-mediated STAT3 activation augmented host defense against pneumococcal pneumonia. In this report, we assessed leptin receptor-mediated ERK activation, a pathway that was ablated in the l/l mouse through a mutation of the tyrosine 985 residue in the leptin receptor, to determine its role in host defense against bacterial pneumonia in vivo and in alveolar macrophage (AM) antibacterial functions in vitro. l/l mice exhibited increased mortality and impaired pulmonary bacterial clearance after intratracheal challenge with Klebsiella pneumoniae. The synthesis of cysteinyl-leukotrienes was reduced and that of PGE(2) enhanced in AMs in vitro and the lungs of l/l mice after infection with K. pneumoniae in vivo. We also observed reduced phagocytosis and killing of K. pneumoniae in AMs from l/l mice that was associated with reduced reactive oxygen intermediate production in vitro. cAMP, known to suppress phagocytosis, bactericidal capacity, and reactive oxygen intermediate production, was also increased 2-fold in AMs from l/l mice. Pharmacologic blockade of PGE(2) synthesis reduced cAMP levels and overcame the defective phagocytosis and killing of bacteria in AMs from l/l mice in vitro. These results demonstrate that leptin receptor-mediated ERK activation plays an essential role in host defense against bacterial pneumonia and in leukocyte antibacterial effector functions.

E-prostanoid 2 receptor signaling suppresses lung innate immunity against Streptococcus pneumoniae.

Pneumonia is a major global health problem. Prostaglandin (PG) E(2) is an immunomodulatory lipid with anti-inflammatory, immunosuppressive, and pro-resolving actions. Data suggest that the E-prostanoid (EP) 2 receptor mediates immunomodulatory effects of PGE(2), but the extent to which this occurs in Streptococcus pneumoniae infection is unknown. Intratracheal lung infection of C57BL/6 mice possessing (EP2(+/+)) or lacking (EP2(-/-)) the EP2 receptor was performed, as were in vitro studies of alveolar macrophage (AM) host defense functions. Bacterial clearance and survival were significantly improved in vivo in EP2(-/-) mice and it correlated with greater neutrophilic inflammation and higher lung IL-12 levels. Upon ex vivo challenge with pneumococcus, EP2(-/-)cells expressed greater amounts of TNF-α and MIP-2 than did EP2(+/+) AMs, and had improved phagocytosis, intracellular killing, and reactive oxygen intermediate generation. These data suggest that PGE(2)-EP2 signaling may provide a novel pharmacological target for treating pneumococcal pneumonia in combination with antimicrobials.

Cigarette smoke exposure impairs pulmonary bacterial clearance and alveolar macrophage complement-mediated phagocytosis of Streptococcus pneumoniae.

Cigarette smoke exposure increases the risk of pulmonary and invasive infections caused by Streptococcus pneumoniae, the most commonly isolated organism from patients with community-acquired pneumonia. Despite this association, the mechanisms by which cigarette smoke exposure diminishes host defense against S. pneumoniae infections are poorly understood. In this study, we compared the responses of BALB/c mice following an intratracheal challenge with S. pneumoniae after 5 weeks of exposure to room air or cigarette smoke in a whole-body exposure chamber in vivo and the effects of cigarette smoke on alveolar macrophage phagocytosis of S. pneumoniae in vitro. Bacterial burdens in cigarette smoke-exposed mice were increased at 24 and 48 h postinfection, and this was accompanied by a more pronounced clinical appearance of illness, hypothermia, and increased lung homogenate cytokines interleukin-1beta (IL-1beta), IL-6, IL-10, and tumor necrosis factor alpha (TNF-alpha). We also found greater numbers of neutrophils in bronchoalveolar lavage fluid recovered from cigarette smoke-exposed mice following a challenge with heat-killed S. pneumoniae. Interestingly, overnight culture of alveolar macrophages with 1% cigarette smoke extract, a level that did not affect alveolar macrophage viability, reduced complement-mediated phagocytosis of S. pneumoniae, while the ingestion of unopsonized bacteria or IgG-coated microspheres was not affected. This murine model provides robust additional support to the hypothesis that cigarette smoke exposure increases the risk of pneumococcal pneumonia and defines a novel cellular mechanism to help explain this immunosuppressive effect.

Inhalation of the reactive aldehyde acrolein promotes antigen sensitization to ovalbumin and enhances neutrophilic inflammation.

Acrolein (ACR), an α,ß-unsaturated aldehyde and a major component of tobacco smoke, is a highly reactive electrophilic respiratory irritant implicated in asthma pathogenesis and severity. However, few studies have directly investigated the influence of ACR exposure on allergen sensitization and pulmonary inflammation. The present study was designed to examine the impact of ACR inhalation on allergic sensitization to the inhaled antigen ovalbumin (OVA), as well as pulmonary inflammation during subsequent OVA challenge. Adult male C57BL/6 mice were exposed to inhaled OVA (1%, 30 min/day, 4 days/week) and/or ACR (5 ppm, 4 h/day, 4 days/week) over 2 weeks and subsequently challenged with aerosolized OVA (1%, 30 min/day) over three consecutive days. Serum anti-OVA IgG1 levels were increased significantly in animals exposed to both OVA and ACR, compared to animals exposed to either OVA or ACR alone. In addition, differential cell counts and histological analysis revealed an increase in BAL neutrophils in animals exposed to both OVA and ACR. However, exposure to both OVA and ACR did not influence mRNA expression of the cytokines il5, il10, il13 or tnfa, but significantly increased mRNA expression of ccl20. Moreover, ACR exposure enhanced lung mRNA levels of il17f and tgfb1, suggesting development of enhanced inhalation tolerance to OVA. Overall, the findings indicate that ACR inhalation can promote airway-mediated sensitization to otherwise innocuous inhaled antigens, such as OVA, but also enhances immune tolerance, thereby favoring neutrophilic airway inflammation.

Bisphenol A at concentrations relevant to human exposure enhances histamine and cysteinyl leukotriene release from bone marrow-derived mast cells.

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxide resin, acts as an endocrine-active compound and has been shown to enhance the inflammatory response to allergen challenge. Previous reports in rodents have demonstrated that perinatal BPA exposure alters airway inflammation following sensitization and challenge to ovalbumin in juvenile and adult offspring. Additionally, a high concentration of BPA has been shown to enhance mediator release in mast cell lines. This study aimed to determine if short-term BPA exposure, at levels relevant to human exposure, enhances mast cell release of histamine and cysteinyl leukotrienes (CysLTs). Primary murine bone marrow-derived mast cells (BMMC) produced from the femurs of female C57BL/6 mice were stimulated with BPA or estradiol (E2) in vitro. It was observed that both BPA and E2 increased BMMC histamine release over a range of nanomolar concentrations (1-1000 nM). The estrogen receptor (ER) antagonist ICI 182,780 partially blocked the ability of E2, but not BPA, to elevate histamine release. BPA also increased CysLT release, which was not abrogated by ER inhibition. It was also observed that the ability of BPA to enhance histamine and CysLT release was inhibited by blocking the extracellular signal-regulated kinase (ERK) pathway with U0126 or by chelating extracellular calcium (Ca(2+)) using EGTA. In summary, these experiments are the first to demonstrate that acute BPA exposure enhances mast cell histamine and CysLT release in vitro--an effect that is not dependent on an ER-mediated mechanism. Instead, BPA-induced mast cell histamine and CysLT release may be mediated, in part, by the ERK pathway and extracellular Ca(2+) concentrations. These data suggest that exposure to BPA at levels relevant to human exposure may provoke an acute inflammatory response in atopic individuals via enhanced mast cell activation.

Perinatal bisphenol A exposures increase production of pro-inflammatory mediators in bone marrow-derived mast cells of adult mice.

Bisphenol A (BPA) is a widely used monomer of polycarbonate plastics and epoxide resin that has been implicated in asthma pathogenesis when exposure occurs to the developing fetus. However, few studies have examined the relationship between perinatal BPA exposure and asthma pathogenesis in adulthood. This study used an isogenic mouse model to examine the influence of perinatal BPA exposure via maternal diet on inflammatory mediators associated with asthma in 6-month-old adult offspring by measuring bone marrow-derived mast cell (BMMC) production of lipid mediators (cysteinyl leukotrienes and prostaglandin D2), cytokines (interleukin [IL]-4, IL-5, IL-6, IL-13, and tumor necrosis factor [TNF]-α), and histamine. Global DNA methylation levels in BMMCs from adult offspring were determined to elucidate a potential regulatory mechanism linking perinatal exposure to mast cell phenotype later in life. Four BPA exposure doses were tested: low (50 ng BPA/kg diet, n = 5), medium (50 µg BPA/kg diet, n = 4), high (50 mg BPA/kg diet, n = 4), and control (n = 3). Following BMMC activation, increases in cysteinyl leukotriene (p < 0.01) and TNFα (p < 0.05) production were observed in all BPA-exposure groups, and increases in prostaglandin D2 (p < 0.01) and IL-13 (p < 0.01) production were observed in the high exposure group. Additionally, BMMCs from adult mice in all exposure groups displayed a decrease in global DNA methylation compared to control animals. Thus, perinatal BPA exposure displayed a long-term influence on mast cell-mediated production of pro-inflammatory mediators associated with asthma and global DNA methylation levels, suggesting a potential for mast cell dysregulation, which could affect pulmonary inflammation associated with allergic airway disease into adulthood.

No Impairment in host defense against Streptococcus pneumoniae in obese CPEfat/fat mice.

In the US and globally, dramatic increases in the prevalence of adult and childhood obesity have been reported during the last 30 years. In addition to cardiovascular disease, type II diabetes, and liver disease, obesity has recently been recognized as an important risk factor for influenza pneumonia. During the influenza pandemic of 2009, obese individuals experienced a greater severity of illness from the H1N1 virus. In addition, obese mice have also been shown to exhibit increased lethality and aberrant pulmonary inflammatory responses following influenza infection. In contrast to influenza, the impact of obesity on bacterial pneumonia in human patients is controversial. In this report, we compared the responses of lean WT and obese CPE(fat/fat) mice following an intratracheal infection with Streptococcus pneumoniae, the leading cause of community-acquired pneumonia. At 16 weeks of age, CPE(fat/fat) mice develop severe obesity, hyperglycemia, elevated serum triglycerides and leptin, and increased blood neutrophil counts. There were no differences between lean WT and obese CPE(fat/fat) mice in survival or lung and spleen bacterial burdens following intratracheal infection with S. pneumoniae. Besides a modest increase in TNF-α levels and increased peripheral blood neutrophil counts in CPE(fat/fat) mice, there were not differences in lung or serum cytokines after infection. These results suggest that obesity, accompanied by hyperglycemia and modestly elevated triglycerides, at least in the case of CPE(fat/fat) mice, does not impair innate immunity against pneumococcal pneumonia.