Cyclic adenosine monophosphate (cAMP) was the original "second messenger" to be discovered. Its formation is promoted by adenylyl cyclase activation after ligation of G protein-coupled receptors by ligands including hormones, autocoids, prostaglandins, and pharmacologic agents. Increases in intracellular cAMP generally suppress innate immune functions, including inflammatory mediator generation and the phagocytosis and killing of microbes. The importance of the host cAMP axis in regulating antimicrobial defense is underscored by the fact that microbes have evolved virulence-enhancing strategies that exploit it. Many clinical situations that predispose to infection are associated with increases in cAMP, and therapeutic strategies to interrupt cAMP generation or actions have immunostimulatory potential. This article reviews the anatomy of the cAMP axis, the mechanisms by which it controls phagocyte immune function, microbial strategies to dysregulate it, and its clinical relevance.

Prostaglandin E2 (PGE2) is a potent lipid mediator that effects changes in cell functions through ligation of four distinct G protein–coupled E prostanoid (EP) receptors (EP1–EP4). PGE2 inhibits bacterial killing and reactive oxygen intermediate (ROI) production by alveolar macrophages (AMs), although little is known about the operative molecular mechanisms. The aims of this study were to evaluate the molecular mechanisms and the specific EP receptors through which PGE2 inhibits killing of Klebsiella pneumoniae by AMs. The treatment of AMs with PGE2 suppressed the killing of K. pneumoniae, and this effect was blocked by an adenylyl cyclase inhibitor and mimicked by agonists for the stimulatory G protein (Gs)-coupled EP2 and EP4 receptors. Conversely, microbicidal activity was augmented by pretreatment with the cyclooxygenase inhibitor, indomethacin, and antagonists of EP2 and EP4. Similar results were found when ROI production was examined. PGE2 inhibition of killing and ROI generation was associated with its activation of the cAMP effectors, protein kinase A and exchange protein directly activated by cAMP-1, as well as attenuation of the phosphorylation and translocation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component, p47phox, to the phagosomal membrane. We conclude that PGE2 suppresses the microbicidal activity of AMs through the Gs-coupled EP2/EP4 receptors, with increased cAMP inhibiting the assembly and activation of p47phox.

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.

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.

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates survival, proliferation, differentiation, and function of myeloid cells. Recently, GM-CSF has been shown to be important for normal pulmonary homeostasis. We report that GM-CSF is induced in lung leukocytes during infection with Gram-negative bacteria. Therefore, we postulated that deficiencies in GM-CSF would increase susceptibility to Gram-negative infection in vivo. After an intratracheal inoculum with Pseudomonas aeruginosa, GM-CSF−/− mice show decreased survival compared with wild-type mice. GM-CSF−/− mice show increased lung, spleen, and blood bacterial CFU. GM-CSF−/− mice are defective in the production of cysteinyl leukotrienes, prostaglandin E2, macrophage inflammatory protein, and keratinocyte-derived chemokine in lung leukocytes postinfection. Despite these defects, inflammatory cell recruitment is not diminished at 6 or 24 h postinfection, and the functional activity of polymorphonuclear leukocytes from the lung and peritoneum against P. aeruginosa is enhanced in GM-CSF−/− mice. In contrast, alveolar macrophage (AM) phagocytosis, killing, and H2O2 production are defective in GM-CSF−/− mice. Although the absence of GM-CSF has profound effects on AMs, peritoneal macrophages seem to have normal bactericidal activities in GM-CSF−/− mice. Defects in AM function may be related to diminished levels of IFN-γ and TNF-α postinfection. Thus, GM-CSF−/− mice are more susceptible to lung infection with P. aeruginosa as a result of impaired AM function.

Rationale: Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E₂ (PGE₂), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens. Objectives: To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE₂ signaling. Methods: Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE₂ signaling modifiers. Measurements and Main Results: NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3–208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE₂ treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A–dependent manner. Conclusions: Our results suggest blockade of the PGE₂–EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.