Deepti Goel

Cigarette Smoke Exposure Impairs Pulmonary Bacterial Clearance and Alveolar Macrophage Complement-Mediated Phagocytosis of Streptococcus pneumoniae

ABSTRACT 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-1β (IL-1β), IL-6, IL-10, and tumor necrosis factor alpha (TNF-α). 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.

Intrapulmonary Administration of Leukotriene B4 Enhances Pulmonary Host Defense against Pneumococcal Pneumonia

ABSTRACT Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation formed by the 5-lipoxygenase (5-LO)-catalyzed oxidation of arachidonic acid. We have previously shown that (i) LTB4 is generated during infection, (ii) its biosynthesis is essential for optimal antimicrobial host defense, (iii) LT deficiency is associated with clinical states of immunocompromise, and (iv) exogenous LTB4 augments antimicrobial functions in phagocytes. Here, we sought to determine whether the administration of LTB4 has therapeutic potential in a mouse model of pneumonia. Wild-type and 5-LO knockout mice were challenged with Streptococcus pneumoniae via the intranasal route, and bacterial burdens, leukocyte counts, and cytokine levels were determined. LTB4 was administered via the intraperitoneal, intravenous, and intranasal routes prior to pneumococcal infection and by aerosol 24 h following infection. Leukocytes recovered from mice given S. pneumoniae and treated with aerosolized LTB4 were evaluated for expression levels of the p47phox subunit of NADPH oxidase. Intrapulmonary but not systemic pretreatment with LTB4 significantly reduced the lung S. pneumoniae burden in wild-type mice. Aerosolized LTB4 was effective at improving lung bacterial clearance when administered postinoculation in animals with established infection and exhibited greater potency in 5-LO knockout animals, which also exhibited greater baseline susceptibility. Augmented bacterial clearance in response to LTB4 was associated with enhanced monocyte recruitment and leukocyte expression of p47phox. The results of the current study in an animal model serve as a proof of concept for the potential utility of treatment with aerosolized LTB4 as an immunostimulatory strategy in patients with bacterial pneumonia.

E-Prostanoid 3 Receptor Deletion Improves Pulmonary Host Defense and Protects Mice from Death in Severe Streptococcus pneumoniae Infection

Prostaglandins (PGs) are potent lipid mediators that are produced during infections and whose synthesis and signaling networks present potential pharmacologic targets for immunomodulation. PGE2 acts through the ligation of four distinct G protein-coupled receptors, E-prostanoid (EP) 1–4. Previous in vitro and in vivo studies demonstrated that the activation of the Gαs-coupled EP2 and EP4 receptors suppresses inflammatory responses to microbial pathogens through cAMP-dependent signaling cascades. Although it is speculated that PGE2 signaling via the Gαi-coupled EP3 receptor might counteract EP2/EP4 immunosuppression in the context of bacterial infection (or severe inflammation), this has not previously been tested in vivo. To address this, we infected wild-type (EP3+/+) and EP3−/− mice with the important respiratory pathogen Streptococcus pneumoniae or injected mice i.p. with LPS. Unexpectedly, we observed that EP3−/− mice were protected from mortality after infection or LPS. The enhanced survival observed in the infected EP3−/− mice correlated with enhanced pulmonary clearance of bacteria; reduced accumulation of lung neutrophils; lower numbers of circulating blood leukocytes; and an impaired febrile response to infection. In vitro studies revealed improved alveolar macrophage phagocytic and bactericidal capacities in EP3−/− cells that were associated with an increased capacity to generate NO in response to immune stimulation. Our studies underscore the complex nature of PGE2 immunomodulation in the context of host-microbial interactions in the lung. Pharmacological targeting of the PGE2-EP3 axis represents a novel area warranting greater investigative interest in the prevention and/or treatment of infectious diseases.

Disruption of Leptin Receptor–STAT3 Signaling Enhances Leukotriene Production and Pulmonary Host Defense against Pneumococcal Pneumonia

The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A2 α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A2 α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.

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 PGE2 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 PGE2 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.

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 CPEfat/fat mice following an intratracheal infection with Streptococcus pneumoniae, the leading cause of community-acquired pneumonia. At 16 weeks of age, CPEfat/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 CPEfat/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 CPEfat/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 CPEfat/fat mice, does not impair innate immunity against pneumococcal pneumonia.