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The capacity for macrophages to polarize into distinct functional activation states (e.g., M1, M2) is critical to tune an inflammatory response to the relevant infection or injury. Alternative or M2 polarization of macrophages is most often achieved in vitro in response to IL-4/IL-13 and results in the transcriptional up-regulation of a constellation of characteristic M2 marker genes. In vivo , additional signals from the inflammatory milieu can further increase or decrease M2 marker expression. Particularly, activation of cAMP-generating G protein-coupled receptors is reported to increase M2 markers, but whether this is strictly dependent upon cAMP production is unclear. We report herein that increased cAMP alone can increase IL-4-dependent M2 marker expression through a PKA/C/EBPβ/CREB dependent pathway in murine macrophages.

The innate inflammatory response to Francisella tularensis (Ft) in macrophages is significantly governed by the expression of type I interferon (IFN). Previously, the proteolytic processing and maturation of pro-IL-1β protein was shown to depend upon type I IFN expression. We show in this report that paracrine type I IFN can profoundly enhance innate recognition and TLR-dependent transcriptional responses to Ft infection upstream of its role in inflammasome regulation in both primary human monocyte-derived macrophages and primary murine peritoneal macrophages but not murine bone marrow-derived macrophages. This type I IFN-enhanced response is synergistic with TLR2 transcriptional responses, partially TLR2-independent, but strictly MyD88-dependent., (© The Author(s) 2016.)

Innate immune inflammatory responses are subject to complex layers of negative regulation at intestinal mucosal surfaces. Although the type I IFN system is critical for amplifying antiviral immunity, it has been shown to play a homeostatic role in some models of autoimmune inflammation. Type I IFN is triggered in the gut by select bacterial pathogens, but whether and how the type I IFN might regulate innate immunity in the intestinal environment have not been investigated in the context of Salmonella enterica serovar Typhimurium (ST). ST infection of human or murine macrophages reveals that IFN-β selectively restricts the transcriptional responses mediated by both the TLRs and the NOD-like receptors. Specifically, IFN-β potently represses ST-dependent innate induction of IL-1 family cytokines and neutrophil chemokines. This IFN-β-mediated transcriptional repression was independent of the effects of IFN-β on ST-induced macrophage cell death, but significantly dependent on IL-10 regulation. We further evaluated ST pathogenesis in vivo following oral inoculation of mice lacking IFN-β. We show that IFN-β(-/-) mice exhibit greater resistance to oral ST infection and a slower spread of ST to distal sterile sites. This work provides mechanistic insight into the relationship between ST and type I IFN, and demonstrates an additional mechanism by which IFN-β may promote spread of enteric pathogens., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars.

Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα(-/-) and STAT6(-/-) macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ-associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13(-/-) macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα-deficient mice survived LPS + d-galactosamine-induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ-associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.

Bordetella pertussis causes a profound inflammatory response in lungs of infected individuals. The adenylate cyclase toxin (ACT) of B. pertussis is a potent enzyme that converts cytosolic ATP into cAMP, and is required for virulence in vivo. During infection, secreted ACT binds to macrophages utilizing the beta2 integrin, Mac-1 (CR3, CD11b/CD18), and subsequent intoxication by ACT inhibits essential antibacterial activities of macrophages. Additionally, Mac-1 has been reported to be a co-receptor for TLR4 required for the full induction of some LPS-responsive genes, including pro-inflammatory cyclooxygenase 2 (COX-2). We have examined the effect of ACT on COX-2 expression in HEK293T cells expressing Mac-1 and in murine macrophages. We report that ACT induces COX-2 in a manner that absolutely requires the catalytic activity of this enzyme and Mac-1 expression dramatically enhanced the sensitivity of cells to ACT-dependent COX-2 induction. The mechanism of COX-2 induction by ACT utilizes the cAMP-PKA-CREB-dependent pathway. Finally, ACT and TLR2 or TLR4 act synergistically to increase COX-2 expression. These data suggest that ACT contributes significantly to the inflammatory response induced by B. pertussis infection by augmenting COX-2 expression and provides evidence against the concept that ACT functions exclusively via its inhibitory effects on phagocytic leucocytes.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum. [ABSTRACT FROM AUTHOR]

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1β and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of TLR (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non- TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IFN regulatory factor- 3 (IRF-3) in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3-/- macrophages and TBK-l-/- mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3 dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-&bgr;-/- macrophages, indicating that IL-33 is not dependent on IFN-&bgr; as an intermediate. Epinephrine and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergize with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the protein kinase A inhibitor H-89 but not by tyrosine kinase or protein kinase C inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter "A" species is preferentially induced by all IL-33-inducing agonists except Newcastle disease virus, a RIG-I agonist that induced expression of both "A" and "B" transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways. [ABSTRACT FROM AUTHOR]