Lisa M. Rogers

Human Intestinal Lamina Propria CD1c+ Dendritic Cells Display an Activated Phenotype at Steady State and Produce IL-23 in Response to TLR7/8 Stimulation

Intestinal dendritic cells (DCs) play key roles in mediating tolerance to commensal flora and inflammatory responses against mucosal pathogens. The mechanisms by which intestinal “conditioning” influences human DC responses to microbial stimuli remain poorly understood. Infections with viruses, such as HIV-1, that target mucosal tissue result in intestinal epithelial barrier breakdown and increased translocation of commensal bacteria into the lamina propria (LP). It is unclear whether innate LP DC responses to concurrent viral and bacterial stimuli influence mucosal HIV-1 pathogenesis. In this study, direct ex vivo phenotype and in vitro constitutive cytokine production of CD1c+ DCs in human intestinal LP were compared with those in peripheral blood (PB). To evaluate innate responses to viral and bacterial stimuli, intracellular cytokine production by LP and PB DCs following stimulation with ligands for TLRs 2, 4, 5, and 7/8 was evaluated. At steady state, LP CD1c+ DCs expressed higher levels of activation markers (CD40, CD83, CD86, HLA-DR, and CCR7) than did PB CD1c+ DCs, and higher frequencies of LP CD1c+ DCs constitutively produced IL-6 and -10 and TNF-α. LP DCs had blunted cytokine responses to TLR4 ligand and TLR5 ligand stimulation relative to PB DCs, yet similarly produced IL-10 in response to TLR2 ligand. Only synthetic TLR7/8 ligand, a mimic of viral ssRNA, induced IL-23 production by LP CD1c+ DCs, and this proinflammatory cytokine response was synergistically enhanced following combined TLR7/8 and TLR4 stimulation. These findings highlight a potential mechanism by which viruses like HIV-1 may subvert homeostatic mechanisms and induce inflammation in the intestinal mucosa.

Leukotriene B4 Enhances Innate Immune Defense against the Puerperal Sepsis Agent Streptococcus pyogenes

Puerperal sepsis is a leading cause of maternal mortality worldwide. Streptococcus pyogenes [group A Streptococcus; (GAS)] is a major etiologic agent of severe postpartum sepsis, yet little is known regarding the pathogenesis of these infections. Tissue macrophages provide innate defense against GAS, and their actions are highly regulated. The intracellular second messenger cAMP can negatively regulate macrophage actions against GAS. Because leukotriene (LT) B4 has been shown to suppress intracellular cAMP in macrophages, we hypothesized that it could enhance innate defenses against GAS. We assessed the capacity of LTB4 to modulate antistreptococcal actions of human macrophages, including placental and decidual macrophages and used a novel intrauterine infection model of GAS in mice lacking the 5-lipoxygenase enzyme to determine the role of endogenous LTs in host defense against this pathogen. Animals lacking 5-lipoxygenase were significantly more vulnerable to intrauterine GAS infection than were wild-type mice and showed enhanced dissemination of bacteria out of the uterus and a more robust inflammatory response than did wild-type mice. In addition, LTB4 reduced intracellular cAMP levels via the BLT1 receptor and was a potent stimulant of macrophage phagocytosis and NADPH oxidase–dependent intracellular killing of GAS. Importantly, interference was observed between the macrophage immunomodulatory actions of LTB4 and the cAMP-inducing lipid PGE2, suggesting that interplay between pro- and anti-inflammatory compounds may be important in vivo. This work underscores the potential for pharmacological targeting of lipid mediator signaling cascades in the treatment of invasive GAS infections.

Intrauterine Group A Streptococcal Infections Are Exacerbated by Prostaglandin E2

Streptococcus pyogenes (Group A Streptococcus; GAS) is a major cause of severe postpartum sepsis, a re-emerging cause of maternal morbidity and mortality worldwide. Immunological alterations occur during pregnancy to promote maternofetal tolerance, which may increase the risk for puerperal infection. PGE2 is an immunomodulatory lipid that regulates maternofetal tolerance, parturition, and innate immunity. The extent to which PGE2 regulates host immune responses to GAS infections in the context of endometritis is unknown. To address this, both an in vivo mouse intrauterine (i.u.) GAS infection model and an in vitro human macrophage–GAS interaction model were used. In C57BL/6 mice, i.u. GAS inoculation resulted in local and systemic inflammatory responses and triggered extensive changes in the expression of eicosanoid pathway genes. The i.u. administration of PGE2 increased the mortality of infected mice, suppressed local IL-6 and IL-17A levels, enhanced neutrophilic inflammation, reduced uterine macrophage populations, and increased bacterial dissemination. A role for endogenous PGE2 in the modulation of antistreptococcal host defense was suggested, because mice lacking the genes encoding the microsomal PGE2 synthase-1 or the EP2 receptor were protected from death, as were mice treated with the EP4 receptor antagonist, GW627368X. PGE2 also regulated GAS–macrophage interactions. In GAS-infected human THP-1 (macrophage-like) cells, PGE2 inhibited the production of MCP-1 and TNF-α while augmenting IL-10 expression. PGE2 also impaired the phagocytic ability of human placental macrophages, THP-1 cells, and mouse peritoneal macrophages in vitro. Exploring the targeted disruption of PGE2 synthesis and signaling to optimize existing antimicrobial therapies against GAS may be warranted.

EP4 and EP2 receptor activation of protein kinase A by prostaglandin E2 impairs macrophage phagocytosis of Clostridium sordellii.

Clostridium sordellii causes endometrial infections, but little is known regarding host defenses against this pathogen.

Group B Streptococcus Induces Neutrophil Recruitment to Gestational Tissues and Elaboration of Extracellular Traps and Nutritional Immunity

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a gram-positive bacterial pathogen associated with infection during pregnancy and is a major cause of morbidity and mortality in neonates. Infection of the extraplacental membranes surrounding the developing fetus, a condition known as chorioamnionitis, is characterized histopathologically by profound infiltration of polymorphonuclear cells (PMNs, neutrophils) and greatly increases the risk for preterm labor, stillbirth, or neonatal GBS infection. The advent of animal models of chorioamnionitis provides a powerful tool to study host-pathogen relationships in vivo and ex vivo. The purpose of this study was to evaluate the innate immune response elicited by GBS and evaluate how antimicrobial strategies elaborated by these innate immune cells affect bacteria. Our work using a mouse model of GBS ascending vaginal infection during pregnancy reveals that clinically isolated GBS has the capacity to invade reproductive tissues and elicit host immune responses including infiltration of PMNs within the choriodecidua and placenta during infection, mirroring the human condition. Upon interacting with GBS, murine neutrophils elaborate DNA-containing extracellular traps, which immobilize GBS and are studded with antimicrobial molecules including lactoferrin. Exposure of GBS to holo- or apo-forms of lactoferrin reveals that the iron-sequestration activity of lactoferrin represses GBS growth and viability in a dose-dependent manner. Together, these data indicate that the mouse model of ascending infection is a useful tool to recapitulate human models of GBS infection during pregnancy. Furthermore, this work reveals that neutrophil extracellular traps ensnare GBS and repress bacterial growth via deposition of antimicrobial molecules, which drive nutritional immunity via metal sequestration strategies.

Differing mechanisms of surviving phagosomal stress among group B Streptococcus strains of varying genotypes

ABSTRACT Group B Streptococcus (GBS), a leading cause of neonatal sepsis and meningitis, asymptomatically colonizes up to 30% of women and can persistently colonize even after antibiotic treatment. Previous studies have shown that GBS resides inside macrophages, but the mechanism by which it survives remains unknown. Here, we examined the ability of 4 GBS strains to survive inside macrophages and then focused on 2 strains belonging to sequence type (ST)-17 and ST-12, to examine persistence in the presence of antibiotics. A multiple stress medium was also developed using several stressors found in the phagosome to assess the ability of 30 GBS strains to withstand phagosomal stress. The ST-17 strain was more readily phagocytosed and survived intracellularly longer than the ST-12 strain, but the ST-12 strain was tolerant to ampicillin unlike the ST-17 strain. Exposure to sub-inhibitory concentrations of ampicillin and erythromycin increased the level of phagocytosis of the ST-17 strain, but had no effect on the ST-12 strain. In addition, blocking acidification of the phagosome decreased the survival of the ST-17 strain indicating a pH-dependent survival mechanism for the ST-17 strain. Congruent with the macrophage experiments, the ST-17 strain had a higher survival rate in the multiple stress medium than the ST-12 strain, and overall, serotype III isolates survived significantly better than other serotypes. These results indicate that diverse GBS strains may use differing mechanisms to persist and that serotype III strains are better able to survive specific stressors inside the phagosome relative to other serotypes.

Prostaglandin E2 Regulation of Macrophage Innate Immunity

Globally, maternal and fetal health is greatly impacted by extraplacental inflammation. Group B Streptococcus (GBS), a leading cause of chorioamnionitis, is thought to take advantage of the uterine environment during pregnancy in order to cause inflammation and infection. In this study, we demonstrate the metabolic changes of murine macrophages caused by GBS exposure. GBS alone prompted a delayed increase in lactate production, highlighting its ability to redirect macrophage metabolism from aerobic to anaerobic respiration. This production of lactate is thought to aid in the development and propagation of GBS throughout the surrounding tissue. Additionally, this study shows that PGE2 priming was able to exacerbate lactate production, shown by the rapid and substantial lactate increases seen upon GBS exposure. These data provide a novel model to study the role of GBS exposure to macrophages with and without PGE2 priming.

Lipid profiling of polarized human monocyte-derived macrophages

The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.

Staphylococcus aureus Infection of Human Gestational Membranes Induces Bacterial Biofilm Formation and Host Production of Cytokines.

Staphylococcus aureus, a metabolically flexible gram-positive pathogen, causes infections in a variety of tissues. Recent evidence implicates S. aureus as an emerging cause of chorioamnionitis and premature rupture of membranes, which are associated with preterm birth and neonatal disease. We demonstrate here that S. aureus infects and forms biofilms on the choriodecidual surface of explanted human gestational membranes. Concomitantly, S. aureus elicits the production of proinflammatory cytokines, which could ultimately perturb maternal-fetal tolerance during pregnancy. Therefore, targeting the immunological response to S. aureus infection during pregnancy could attenuate disease among infected individuals, especially in the context of antibiotic resistance.

Macrophage Extracellular Traps: A Scoping Review

Tissue macrophages are derived from either circulating blood monocytes that originate in the bone marrow, or embryonic precursors that establish residence in tissues and are maintained independent of bone marrow progenitors. Macrophages perform diverse functions including tissue repair, the maintenance of homeostasis, and immune regulation. Recent studies have demonstrated that macrophages produce extracellular traps (ETs). ETs are an immune response by which a cell undergoes “ETosis” to release net-like material, with strands composed of cellular DNA that is studded with histones and cellular proteins. ETs are thought to immobilize and kill microorganisms, but also been implicated in disease pathology including aseptic inflammation and autoimmune disease. We conducted a scoping review to define what is known from the existing literature about the ETs produced by monocytes or macrophages. The results suggest that macrophage ETs (METs) are produced in response to various microorganisms and have similar features to neutrophil ETs (NETs), in that METs are produced by a unique cell death program (METosis), which results in release of fibers composed of DNA and studded with cellular proteins. METs function to immobilize and kill some microorganisms, but may also play a role in disease pathology.