C. Henrique Serezani

MicroRNA 21 Is a Homeostatic Regulator of Macrophage Polarization and Prevents Prostaglandin E2-Mediated M2 Generation

Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE2-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.

Prostaglandin E-2 and the Suppression of Phagocyte Innate Immune Responses in Different Organs

The local and systemic production of prostaglandin E2 (PGE2) and its actions in phagocytes lead to immunosuppressive conditions. PGE2 is produced at high levels during inflammation, and its suppressive effects are caused by the ligation of the E prostanoid receptors EP2 and EP4, which results in the production of cyclic AMP. However, PGE2 also exhibits immunostimulatory properties due to binding to EP3, which results in decreased cAMP levels. The various guanine nucleotide-binding proteins (G proteins) that are coupled to the different EP receptors account for the pleiotropic roles of PGE2 in different disease states. Here, we discuss the production of PGE2 and the actions of this prostanoid in phagocytes from different tissues, the relative contribution of PGE2 to the modulation of innate immune responses, and the novel therapeutic opportunities that can be used to control inflammatory responses.

Prostaglandin E2–Induced Changes in Alveolar Macrophage Scavenger Receptor Profiles Differentially Alter Phagocytosis of Pseudomonas aeruginosa and Staphylococcus aureus Post–Bone Marrow Transplant

The effectiveness of hematopoietic stem cell transplantation as a therapy for malignant and nonmalignant conditions is complicated by pulmonary infections. Using our syngeneic bone marrow transplant (BMT) mouse model, BMT mice with a reconstituted hematopoietic system displayed increased susceptibility to Pseudomonas aeruginosa and Staphylococcus aureus. BMT alveolar macrophages (AMs) exhibited a defect in P. aeruginosa phagocytosis, whereas S. aureus uptake was surprisingly enhanced. We hypothesized that the difference in phagocytosis was due to an altered scavenger receptor (SR) profile. Interestingly, MARCO expression was decreased, whereas SR-AI/II was increased. To understand how these dysregulated SR profiles might affect macrophage function, CHO cells were transfected with SR-AI/II, and phagocytosis assays revealed that SR-AI/II was important for S. aureus uptake but not for P. aeruginosa. Conversely, AMs treated in vitro with soluble MARCO exhibited similar defects in P. aeruginosa internalization as did BMT AMs. The 3′-untranslated region of SR-AI contains a putative target region for microRNA-155 (miR-155), and miR-155 expression is decreased post-BMT. Anti–miR-155–transfected AMs exhibited an increase in SR-AI/II expression and S. aureus phagocytosis. Elevated PGE2 has been implicated in driving an impaired innate immune response post-BMT. In vitro treatment of AMs with PGE2 increased SR-AI/II and decreased MARCO and miR-155. Despite a difference in phagocytic ability, BMT AMs harbor a killing defect to both P. aeruginosa and S. aureus. Thus, our data suggest that PGE2-driven alterations in SR and miR-155 expression account for the differential phagocytosis of P. aeruginosa and S. aureus, but impaired killing ultimately confers increased susceptibility to pulmonary 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.

Macrophage Dectin-1 Expression Is Controlled by Leukotriene B4 via a GM-CSF/PU.1 Axis

Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B4 (LTB4). The influence of G protein-coupled receptor ligands such as LTB4 on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB4 signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB4 production and signaling through its high-affinity G protein-coupled receptor leukotriene B4 receptor 1 (BLT1) direct dectin-1–dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wild-type counterparts. LTB4 increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB4-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB4 effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF−/− macrophages. Our results identify LTB4-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses.

Leukotriene B4 Enhances the Generation of Proinflammatory MicroRNAs To Promote MyD88-Dependent Macrophage Activation

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (LT) B4 (LTB4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that LTB4 control of MyD88 expression involves the generation of microRNAs. Our data show that LTB4, via its receptor B leukotriene receptor 1 (BLT1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. LTB4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the LTB4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and LT-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR BLT1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.

Leukotriene B4–mediated sterile inflammation promotes susceptibility to sepsis in a mouse model of type 1 diabetes

Inhibiting the production of a leukotriene may help type 1 diabetic patients from succumbing to sepsis. Preventing sepsis in type 1 diabetics Patients with type 1 diabetes have chronic systemic inflammation and are more prone to developing sepsis. Filgueiras et al. found that mice that are a model for type 1 diabetes had higher amounts of leukotriene B4, a proinflammatory lipid, and of 5-lipoxygenase, the enzyme that produces leukotriene B4. Mice with type 1 diabetes that were treated with an inhibitor of 5-lipoxygenase survived sepsis and had decreased markers of inflammation, suggesting that targeting 5-lipoxygenase to prevent the production of leukotriene B4 could decrease the susceptibility of type 1 diabetic patients to sepsis. Type 1 diabetes mellitus (T1DM) is associated with chronic systemic inflammation and enhanced susceptibility to systemic bacterial infection (sepsis). We hypothesized that low insulin concentrations in T1DM trigger the enzyme 5-lipoxygenase (5-LO) to produce the lipid mediator leukotriene B4 (LTB4), which triggers systemic inflammation that may increase susceptibility to polymicrobial sepsis. Consistent with chronic inflammation, peritoneal macrophages from two mouse models of T1DM had greater abundance of the adaptor MyD88 (myeloid differentiation factor 88) and its direct transcriptional effector STAT-1 (signal transducer and activator of transcription 1) than macrophages from nondiabetic mice. Expression of Alox5, which encodes 5-LO, and the concentration of the proinflammatory cytokine interleukin-1β (IL-1β) were also increased in peritoneal macrophages and serum from T1DM mice. Insulin treatment reduced LTB4 concentrations in the circulation and Myd88 and Stat1 expression in the macrophages from T1DM mice. T1DM mice treated with a 5-LO inhibitor had reduced Myd88 mRNA in macrophages and increased abundance of IL-1 receptor antagonist and reduced production of IL-β in the circulation. T1DM mice lacking 5-LO or the receptor for LTB4 also produced less proinflammatory cytokines. Compared to wild-type or untreated diabetic mice, T1DM mice lacking the receptor for LTB4 or treated with a 5-LO inhibitor survived polymicrobial sepsis, had reduced production of proinflammatory cytokines, and had decreased bacterial counts. These results uncover a role for LTB4 in promoting sterile inflammation in diabetes and the enhanced susceptibility to sepsis in T1DM.

SOCS1 is a negative regulator of metabolic reprogramming during sepsis

Sepsis can induce an overwhelming systemic inflammatory response, resulting in organ damage and death. Suppressor of cytokine signaling 1 (SOCS1) negatively regulates signaling by cytokine receptors and Toll-like receptors (TLRs). However, the cellular targets and molecular mechanisms for SOCS1 activity during polymicrobial sepsis are unknown. To address this, we utilized a cecal ligation and puncture (CLP) model for sepsis; C57BL/6 mice subjected to CLP were then treated with a peptide (iKIR) that binds the SOCS1 kinase inhibitory region (KIR) and blocks its activity. Treatment with iKIR increased CLP-induced mortality, bacterial burden, and inflammatory cytokine production. Myeloid cell-specific SOCS1 deletion (Socs1Δmyel) mice were also more susceptible to sepsis, demonstrating increased mortality, higher bacterial loads, and elevated inflammatory cytokines, compared with Socs1fl littermate controls. These effects were accompanied by macrophage metabolic reprograming, as evidenced by increased lactic acid production and elevated expression of the glycolytic enzymes hexokinase, lactate dehydrogenase A, and glucose transporter 1 in septic Socs1Δmyel mice. Upregulation was dependent on the STAT3/HIF-1α/glycolysis axis, and blocking glycolysis ameliorated increased susceptibility to sepsis in iKIR-treated CLP mice. These results reveal a role of SOCS1 as a regulator of metabolic reprograming that prevents overwhelming inflammatory response and organ damage during sepsis.

Topical Prostaglandin E Analog Restores Defective Dendritic Cell-Mediated Th17 Host Defense Against Methicillin-Resistant Staphylococcus Aureus in the Skin of Diabetic Mice.

People with diabetes are more prone to Staphylococcus aureus skin infection than healthy individuals. Control of S. aureus infection depends on dendritic cell (DC)–induced T-helper 17 (Th17)–mediated neutrophil recruitment and bacterial clearance. DC ingestion of infected apoptotic cells (IACs) drive prostaglandin E2 (PGE2) secretion to generate Th17 cells. We speculated that hyperglycemia inhibits skin DC migration to the lymph nodes and impairs the Th17 differentiation that accounts for poor skin host defense in diabetic mice. Diabetic mice showed increased skin lesion size and bacterial load and decreased PGE2 secretion and Th17 cells compared with nondiabetic mice after methicillin-resistant S. aureus (MRSA) infection. Bone marrow–derived DCs (BMDCs) cultured in high glucose (25 mmol/L) exhibited decreased Ptges mRNA expression, PGE2 production, lower CCR7-dependent DC migration, and diminished maturation after recognition of MRSA-IACs than BMDCs cultured in low glucose (5 mmol/L). Similar events were observed in DCs from diabetic mice infected with MRSA. Topical treatment of diabetic mice with the PGE analog misoprostol improved host defense against MRSA skin infection by restoring DC migration to draining lymph nodes, Th17 differentiation, and increased antimicrobial peptide expression. These findings identify a novel mechanism involved in poor skin host defense in diabetes and propose a targeted strategy to restore skin host defense in diabetes.

Imbalance between HDAC and HAT activities drives aberrant STAT1/MyD88 expression in macrophages from type 1 diabetic mice

AIMS To investigate the hypothesis that alteration in histone acetylation/deacetylation triggers aberrant STAT1/MyD88 expression in macrophages from diabetics. Increased STAT1/MyD88 expression is correlated with sterile inflammation in type 1 diabetic (T1D) mice. METHODS To induce diabetes, we injected low-doses of streptozotocin in C57BL/6 mice. Peritoneal or bone marrow-differentiated macrophages were cultured in 5mM (low) or 25mM (high glucose). ChIP analysis of macrophages from nondiabetic or diabetic mice was performed to determine acetylation of lysine 9 in histone H3 in MyD88 and STAT1 promoter regions. Macrophages from diabetic mice were treated with the histone acetyltransferase inhibitor anacardic acid (AA), followed by determination of mRNA expression by qPCR. RESULTS Increased STAT1 and MyD88 expression in macrophages from diabetic but not naive mice cultured in low glucose persisted for up to 6days. Macrophages from diabetic mice exhibited increased activity of histone acetyltransferases (HAT) and decreased histone deacetylases (HDAC) activity. We detected increased H3K9Ac binding to Stat1/Myd88 promoters in macrophages from T1D mice and AA in vitro treatment reduced STAT1 and MyD88 mRNA expression. CONCLUSIONS/INTERPRETATION These results indicate that histone acetylation drives elevated Stat1/Myd88 expression in macrophages from diabetic mice, and this mechanism may be involved in sterile inflammation and diabetes comorbidities.