Megan N. Ballinger

Bleomycin-induced E prostanoid receptor changes alter fibroblast responses to prostaglandin E2

Although PGE2 is a potent inhibitor of fibroblast function, PGE2 levels are paradoxically elevated in murine lungs undergoing fibrotic responses. Pulmonary fibroblasts from untreated mice expressed all four E prostanoid (EP) receptors for PGE2. However, following challenge with the fibrogenic agent, bleomycin, fibroblasts showed loss of EP2 expression. Lack of EP2 expression correlated with an inability of fibroblasts from bleomycin-treated mice to be inhibited by PGE2 in assays of proliferation or collagen synthesis and blunted cAMP elevations in response to PGE2. PGE2 was similarly unable to suppress proliferation or collagen synthesis in fibroblasts from EP2−/− mice despite expression of the other EP receptors. EP2−/−, but not EP1−/− or EP3−/− mice, showed exaggerated fibrotic responses to bleomycin administration in vivo as compared with wild-type controls. EP2 loss on fibroblasts was verified in a second model of pulmonary fibrosis using FITC. Our results for the first time link EP2 receptor loss on fibroblasts following fibrotic lung injury to altered suppression by PGE2 and thus identify a novel fibrogenic mechanism.

Critical Role of Prostaglandin E2 Overproduction in Impaired Pulmonary Host Response following Bone Marrow Transplantation

The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.

Synthetic Prostacyclin Analogs Differentially Regulate Macrophage Function via Distinct Analog-Receptor Binding Specificities

PGI2 (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI2, in the regulation of both innate and acquired immunity. As PGI2 is unstable, we sought to define the effects of various PGI2 analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the Gαs protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI2 analogs and more closely resembled the effects of PGE2. Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI2 analogs. These studies are the first to explore the capacity of PGI2 to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.

Cathelicidin-related antimicrobial peptide is required for effective lung mucosal immunity in Gram-negative bacterial pneumonia.

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, cathelicidin-related antimicrobial peptide (CRAMP), to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp−/−) demonstrate impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival in response to intratracheal K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 h in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.

Postinfluenza Bacterial Pneumonia: Host Defenses Gone Awry

Influenza is a common respiratory pathogen causing both seasonal and pandemic disease. Influenza infection predisposes the host to secondary bacterial infection of the respiratory tract, which is a major cause of both morbidity and mortality in flu-related disease. In this review, we will discuss innate and adaptive antiviral responses during influenza infection, and review how these responses modulate protective immunity against secondary bacterial pathogens of the lung. Specific emphasis will be placed on implications of bacterial superinfection and mechanisms involved.

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4lps-d), mice deficient in TLR9 (TLR9−/−) and TLR4/9 double mutant mice (TLR4lps-d/TLR9−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4lps-d/TLR9−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4lps-d/TLR9−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.

A Role for IL-1 Receptor-Associated Kinase-M in Prostaglandin E2-Induced Immunosuppression Post-Bone Marrow Transplantation

Following immune reconstitution, hematopoietic stem cell transplant patients often display reduced immune function and are especially susceptible to lung infections. In a mouse model of syngeneic bone marrow transplantation (BMT), we previously reported that PGE2 is overproduced in lungs of BMT mice, significantly impairing host defense against Pseudomonas aeruginosa. This impairment in host defense post-BMT is also marked by diminished alveolar macrophage (AM) phagocytosis, bacterial killing, and production of TNF-α and cysteinyl leukotrienes. However, a mechanism by which overproduction of PGE2 suppresses pulmonary host defense post-BMT is unknown. As IL-1R–associated kinase (IRAK)-M is a known inhibitor of MyD88-dependent IL-1R/TLR signaling and macrophage function, we sought to determine whether IRAK-M is involved in PGE2-induced immunosuppression post-BMT. We found that IRAK-M expression is elevated 3.5-fold in BMT AMs relative to control AMs, and this is related to AM overproduction of PGE2. Furthermore, genetic ablation of IRAK-M in the bone marrow of BMT mice restores host defense against P. aeruginosa. Despite AM overproduction of PGE2 and elevated E prostanoid 2 receptor expression, AM phagocytosis, killing, and production of cysteinyl leukotrienes and TNF-α are restored in the absence of IRAK-M post-BMT. Also, treatment with PGE2 does not inhibit AM phagocytosis in the absence of IRAK-M. These data suggest that the absence of IRAK-M in the hematopoietic compartment post-BMT enhances pulmonary host defense and mitigates AM sensitivity to the inhibitory effects of PGE2. Therefore, strategies to limit IRAK-M elevation post-BMT may be efficacious in reducing patient susceptibility to infection.

PTEN Directly Activates the Actin Depolymerization Factor Cofilin-1 During PGE2-Mediated Inhibition of Phagocytosis of Fungi

By promoting actin depolymerization, the protein phosphatase activity of PTEN impairs macrophage phagocytosis of a fungal pathogen. Preventing Phagocytosis The fungus Candida albicans is normally a commensal microbe found on mucosal surfaces, including those in the lung. However, C. albicans can cause systemic infections that are a leading cause of morbidity and mortality in immunocompromised individuals. A key innate immune response to C. albicans is its ingestion (phagocytosis) by macrophages, a process that requires polymerization of the actin cytoskeleton. Another component of the macrophage response to fungus is the production of prostaglandin E2 (PGE2), a lipid mediator whose synthesis is initiated by cyclooxygenase (COX) enzymes. Serezani et al. found that infection of alveolar macrophages with C. albicans triggered the production of PGE2, which prevented polymerization of the actin cytoskeleton and inhibited phagocytosis of C. albicans by alveolar macrophages. The authors defined the signaling pathway involved. These results suggest that COX inhibitors, such as aspirin, which are in widespread clinical use, may stimulate innate immune responses. In addition, immunosuppression is associated with increased production of PGE2, which may help to explain how antifungal responses are attenuated in immunocompromised individuals. Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E2 (PGE2) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE2-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE2 suppressed phagocytosis and F-actin formation through the PGE2 receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE2. PGE2-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE2 accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB4 and LTD4 in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB4 enhanced mainly mannose receptor-dependent fungal ingestion, LTD4 enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB4 accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD4 did so exclusively via LIMK-2. Finally, both exogenous LTB4 and LTD4 enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB4 and LTD4 as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

TLR4-dependent GM-CSF protects against lung injury in Gram-negative bacterial pneumonia.

Toll-like receptors (TLRs) are required for protective host defense against bacterial pathogens. However, the role of TLRs in regulating lung injury during Gram-negative bacterial pneumonia has not been thoroughly investigated. In this study, experiments were performed to evaluate the role of TLR4 in pulmonary responses against Klebsiella pneumoniae (Kp). Compared with wild-type (WT) (Balb/c) mice, mice with defective TLR4 signaling (TLR4(lps-d) mice) had substantially higher lung bacterial colony-forming units after intratracheal challenge with Kp, which was associated with considerably greater lung permeability and lung cell death. Reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA and protein was noted in lungs and bronchoalveolar lavage fluid of TLR4 mutant mice postintratracheal Kp compared with WT mice, and primary alveolar epithelial cells (AEC) harvested from TLR4(lps-d) mice produced significantly less GM-CSF in vitro in response to heat-killed Kp compared with WT AEC. TLR4(lps-d) AEC underwent significantly more apoptosis in response to heat-killed Kp in vitro, and treatment with GM-CSF protected these cells from apoptosis in response to Kp. Finally, intratracheal administration of GM-CSF in TLR4(lps-d) mice significantly decreased albumin leak, lung cell apoptosis, and bacteremia in Kp-infected mice. Based on these observations, we conclude that TLR4 plays a protective role on lung epithelium during Gram-negative bacterial pneumonia, an effect that is partially mediated by GM-CSF.