Carlos H. Serezani

Cutting Edge: Macrophage Inhibition by Cyclic AMP (cAMP): Differential Roles of Protein Kinase A and Exchange Protein Directly Activated by cAMP-1

cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcγR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3′,5′-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-α, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.

Efferocytosis impairs pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling

The ingestion of apoptotic cells (ACs; termed “efferocytosis”) by phagocytes has been shown to trigger the release of molecules such as transforming growth factor β, interleukin-10 (IL-10), nitric oxide, and prostaglandin E2 (PGE2). Although the antiinflammatory actions of these mediators may contribute to the restoration of homeostasis after tissue injury, their potential impact on antibacterial defense is unknown. The lung is highly susceptible to diverse forms of injury, and secondary bacterial infections after injury are of enormous clinical importance. We show that ACs suppress in vitro phagocytosis and bacterial killing by alveolar macrophages and that this is mediated by a cyclooxygenase–PGE2–E prostanoid receptor 2 (EP2)–adenylyl cyclase–cyclic AMP pathway. Moreover, intrapulmonary administration of ACs demonstrated that PGE2 generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo. These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses. They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE2 or ligation of EP2.

Differential Stem and Progenitor Cell Trafficking by Prostaglandin E2

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC–niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1–CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Leukotrienes Are Essential for the Control of Leishmania amazonensis Infection and Contribute to Strain Variation in Susceptibility

Leukotrienes (LTs) are known to be produced by macrophages when challenged with Leishmania, but it is not known whether these lipid mediators play a role in host defense against this important protozoan parasite. In this study, we investigated the involvement of LTs in the in vitro and in vivo response to Leishmania amazonensis infection in susceptible (BALB/c) and resistant (C3H/HePAS) mice. Pharmacologic or genetic deficiency of LTs resulted in impaired leishmanicidal activity of peritoneal macrophages in vitro. In contrast, addition of LTB4 increased leishmanicidal activity and this effect was dependent on the BLT1 receptor. LTB4 augmented NO production in response to L. amazonensis challenge, and studies with a NO synthesis inhibitor revealed that NO was critical for the enhancement of macrophage leishmanicidal activity. Interestingly, macrophages from resistant mice produced higher levels of LTB4 upon L. amazonensis challenge than did those from susceptible mice. In vivo infection severity, as assessed by footpad swelling following s.c. promastigote inoculation, was increased when endogenous LT synthesis was abrogated either pharmacologically or genetically. Taken together, these results for the first time reveal an important role for LTB4 in the protective response to L. amazonensis, identify relevant leishmanicidal mechanisms, and suggest that genetic variation in LTB4 synthesis might influence resistance and susceptibility patterns to infection.

Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-κB and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B4 (LTB4) are pivotal components of host defense and inflammatory responses. However, the role of LTB4 in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1β and IL-18) are reduced in mice lacking either 5-LO or the LTB4 receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-κB. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-κB through Stat1-dependent expression of MyD88.

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.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO−/− mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO−/− mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Specific Leukotriene Receptors Couple to Distinct G Proteins to Effect Stimulation of Alveolar Macrophage Host Defense Functions

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB4 and LTD4 also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Gαi and Gαq proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB4 and LTD4. LTB4 but not LTD4 reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcγR-mediated phagocytosis and bacterial killing by LTB4 was also PTX-sensitive, whereas that induced by LTD4 was not. LTD4 and LTB4 induced Ca2+ and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Gαq protein in mediating PTX-insensitive LTD4 enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Gαq/11 and Gαi3 subunits, but not Gαi2 or Gβγ, in LTB4-enhanced phagocytosis. The selective importance of Gαq/11 protein was also demonstrated in LTD4-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Misoprostol Impairs Female Reproductive Tract Innate Immunity against Clostridium sordellii

Fatal cases of acute shock complicating Clostridium sordellii endometritis following medical abortion with mifepristone (also known as RU-486) used with misoprostol were reported. The pathogenesis of this unexpected complication remains enigmatic. Misoprostol is a pharmacomimetic of PGE2, an endogenous suppressor of innate immunity. Clinical C. sordellii infections were associated with intravaginal misoprostol administration, suggesting that high misoprostol concentrations within the uterus impair immune responses against C. sordellii. We modeled C. sordellii endometritis in rats to test this hypothesis. The intrauterine but not the intragastric delivery of misoprostol significantly worsened mortality from C. sordellii uterine infection, and impaired bacterial clearance in vivo. Misoprostol also reduced TNF-α production within the uterus during infection. The intrauterine injection of misoprostol did not enhance mortality from infection by the vaginal commensal bacterium Lactobacillus crispatus. In vitro, misoprostol suppressed macrophage TNF-α and chemokine generation following C. sordellii or peptidoglycan challenge, impaired leukocyte phagocytosis of C. sordellii, and inhibited uterine epithelial cell human β-defensin expression. These immunosuppressive effects of misoprostol, which were not shared by mifepristone, correlated with the activation of the Gs protein-coupled E prostanoid (EP) receptors EP2 and EP4 (macrophages) or EP4 alone (uterine epithelial cells). Our data provide a novel explanation for postabortion sepsis leading to death and also suggest that PGE2, in which production is exaggerated within the reproductive tract during pregnancy, might be an important causal determinant in the pathogenesis of more common infections of the gravid uterus.

Distinct protein kinase a anchoring proteins direct prostaglandin E2 modulation of toll-like receptor signaling in alveolar macrophages

Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E2 (PGE2), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE2/cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE2/cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE2 synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE2 on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE2 potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE2 potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE2 suppression of TNF-α was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.