The Effect of Retinoic Acid on Neutrophil Innate Immune Interactions With Cutaneous Bacterial Pathogens.

Vitamin A and its biologically active derivative, retinoic acid (RA), are important for many immune processes. RA, in particular, is essential for the development of immune cells, including neutrophils, which serve as a front-line defense against infection. While vitamin A deficiency has been linked to higher susceptibility to infections, the precise role of vitamin A/RA in host-pathogen interactions remains poorly understood. Here, we provided evidence that RA boosts neutrophil killing of methicillin-resistant Staphylococcus aureus (MRSA). RA treatment stimulated primary human neutrophils to produce reactive oxygen species, neutrophil extracellular traps, and the antimicrobial peptide cathelicidin (LL-37). Because RA treatment was insufficient to reduce MRSA burden in an in vivo murine model of skin infection, we expanded our analysis to other infectious agents. RA did not affect the growth of a number of common bacterial pathogens, including MRSA, Escherichia coli K1 and Pseudomonas aeruginosa; however, RA directly inhibited the growth of group A Streptococcus (GAS). This antimicrobial effect, likely in combination with RA-mediated neutrophil boosting, resulted in substantial GAS killing in neutrophil killing assays conducted in the presence of RA. Furthermore, in a murine model of GAS skin infection, topical RA treatment showed therapeutic potential by reducing both skin lesion size and bacterial burden. These findings suggest that RA may hold promise as a therapeutic agent against GAS and perhaps other clinically significant human pathogens.

Efficient G protein coupling is not required for agonist-mediated internalization and membrane reorganization of the adenosine A3 receptor.

Organization of G protein-coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3 AR) to that of the wild-type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA-X-BY630) demonstrated that both wild-type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist-mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3 AR and A3 AR R108A underwent similar agonist-induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit ß-arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild-type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist-stimulated ß-arrestin recruitment and membrane reorganization of the A3 AR.

How Neutrophils Meet Their End.

Neutrophil death can transpire via diverse pathways and is regulated by interactions with commensal and pathogenic microorganisms, environmental exposures, and cell age. At steady state, neutrophil turnover and replenishment are continually maintained via a delicate balance between host-mediated responses and microbial forces. Disruptions in this equilibrium directly impact neutrophil numbers in circulation, cell trafficking, antimicrobial defenses, and host well-being. How neutrophils meet their end is physiologically important and can result in different immunologic consequences. Whereas nonlytic forms of neutrophil death typically elicit anti-inflammatory responses and promote healing, pathways ending with cell membrane rupture may incite deleterious proinflammatory responses, which can exacerbate local tissue injury, lead to chronic inflammation, or precipitate autoimmunity. This review seeks to provide a contemporary analysis of mechanisms of neutrophil death.

E-cigarette use increases susceptibility to bacterial infection by impairment of human neutrophil chemotaxis, phagocytosis, and NET formation.

E-cigarettes are portrayed as safer relative to conventional tobacco. However, burgeoning evidence suggests that E-cigarettes may adversely affect host defenses. However, the precise mechanisms by which E-cigarette vapor alters innate immune cell function have not been fully elucidated. We determined the effects of E-cigarette exposure on the function and responses to infectious challenge of the most abundant innate immune cell, the neutrophil, using isolated human neutrophils and a mouse model of gram-negative infection. Our results revealed that human neutrophils exposed to E-cigarette vapor had 4.2-fold reductions in chemotaxis toward the bacterial cell-well component f-Met-Leu-Phe (P < 0.001). F-actin polarization and membrane fluidity were also adversely affected by E-cigarette vapor exposure. E-cigarette-exposed human neutrophils exhibited a 48% reduction in production of reactive oxygen species (ROS; P < 0.001). Given the central role of ROS in neutrophil extracellular trap (NET) production, NET production was quantified, and E-cigarette vapor exposure was found to reduce NETosis by 3.5-fold (P < 0.01); formulations with and without nicotine containing propylene glycol exhibiting significant suppressive effects. However, noncanonical NETosis was unaffected. In addition, exposure to E-cigarette vapor lowered the rate of phagocytosis of bacterial bioparticles by 47% (P < 0.05). In our physiological mouse model of chronic E-cigarette exposure and sepsis, E-cigarette vapor inhalation led to reduced neutrophil migration in infected spaces and a higher burden of Pseudomonas. These findings provide evidence that E-cigarette use adversely impacts the innate immune system and may place E-cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.

GPCRs show widespread differential mRNA expression and frequent mutation and copy number variation in solid tumors.

G protein-coupled receptors (GPCRs) are the most widely targeted gene family for Food and Drug Administration (FDA)-approved drugs. To assess possible roles for GPCRs in cancer, we analyzed The Cancer Genome Atlas (TCGA) data for mRNA expression, mutations, and copy number variation (CNV) in 20 categories and 45 subtypes of solid tumors and quantified differential expression (DE) of GPCRs by comparing tumors against normal tissue from the Gene Tissue Expression Project (GTEx) database. GPCRs are overrepresented among coding genes with elevated expression in solid tumors. This analysis reveals that most tumor types differentially express >50 GPCRs, including many targets for approved drugs, hitherto largely unrecognized as targets of interest in cancer. GPCR mRNA signatures characterize specific tumor types and correlate with expression of cancer-related pathways. Tumor GPCR mRNA signatures have prognostic relevance for survival and correlate with expression of numerous cancer-related genes and pathways. GPCR expression in tumors is largely independent of staging, grading, metastasis, and/or driver mutations. GPCRs expressed in cancer cell lines largely parallel GPCR expression in tumors. Certain GPCRs are frequently mutated and appear to be hotspots, serving as bellwethers of accumulated genomic damage. CNV of GPCRs is common but does not generally correlate with mRNA expression. Our results suggest a previously underappreciated role for GPCRs in cancer, perhaps as functional oncogenes, biomarkers, surface antigens, and pharmacological targets.

Inhibition of Human Neutrophil Extracellular Trap (NET) Production by Propofol and Lipid Emulsion.

Uncontrolled bacteremia is a common and life threatening condition that can lead to sepsis and septic shock with significant morbidity and mortality. Neutrophil granulocytes, the most abundant phagocytic leukocyte of the innate immune system, play an essential role in capturing and killing invading pathogens. Their antimicrobial repertoire includes the formation of Neutrophil Extracellular Traps (NETs), chromatin-based, web-like structures of DNA that facilitate the capture and killing of bacteria. In sepsis, however, it has been suggested that the uncontrolled release of NETs worsens disseminated coagulation and promotes venous thrombosis. Here, we describe how clinically relevant concentrations of the commonly used sedative propofol as well as a lipid composition similar to the propofol carrier impair NET production by human neutrophils. Drugs commonly administered in the Intensive Care Unit (ICU) may impact the inflammatory response to either worsen or improve clinical outcomes and may therefore be considered for additional therapeutic effects if clinical studies confirm such findings.

Alterations in human neutrophil function caused by bisphenol A.

Bisphenol A (BPA) is a synthetic, organic compound frequently present in consumer plastics, including plastic-lined cans, water bottles, toys, and teeth sutures. Previous studies have shown that BPA can produce adverse health effects that include defects in reproductive function and altered prenatal/childhood development. However, little is known regarding the effects of BPA on immune function. In this study, we assessed the effect of BPA on human neutrophils, a critical component of the innate immune system's defense against pathogens. We found that BPA induces a concentration-dependent increase in reactive oxygen species (ROS) generation by neutrophils, which is inhibited by the estrogen receptor-ß antagonist PHTPP. Furthermore, incubation with the membrane-permeable calcium chelator BAPTA-AM and/or removal of extracellular calcium inhibited BPA-induced ROS production, indicating that the process is calcium dependent. Transwell chemotaxis assays revealed that BPA exposure reduces the chemotactic capacity of neutrophils in a gradient of the bacterial cell wall component f-Met-Leu-Phe, a potent chemoattractant. Exposure to BPA also inhibits the ability of neutrophils to kill methicillin-resistant Staphylococcus aureus, a leading human pathogen. Our findings reveal that BPA alters the in vitro function of neutrophils, including ROS production, chemotaxis, and bacterial killing, and raises the possibility of altered innate immunity in vivo, especially in those with compromised immune function and who can be exposed to BPA in a wide variety of products.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity.

Objectives: The role of protease-activated receptor 1 (PAR1) in the pathogenesis of pneumonia and sepsis is ambiguous given the existing literature. As PAR1 is classically activated by the coagulation-based protease thrombin and leads to vascular leakage, our hypothesis was that PAR1 blockade with SCH79797 would be therapeutically beneficial in an experimental model of murine Gram-negative pneumonia. Methods: In this study, we administered SCH79797 via the intrapulmonary route 6 h after the establishment of Escherichia coli pneumonia and observed a significant improvement in survival, lung injury, bacterial clearance and inflammation. We focused on neutrophils as a potential target of the PAR1 antagonist, since they are the predominant inflammatory cell type in the infected lung. Results: Neutrophils appear to express PAR1 at low levels and the PAR1 antagonist SCH79797 enhanced neutrophil killing. Part of this effect may be explained by alterations in the generation of reactive oxygen species (ROS). SCH79797 also led to robust neutrophil extracellular trap (NET) generation and cathelicidin-related antimicrobial peptide (CRAMP) release by neutrophils. Surprisingly, SCH79797 also had a potent, direct antibiotic effect with disruption of the E. coli cell membrane. However, the newer-generation PAR1 antagonist, vorapaxar (SCH530348), had no appreciable effect on neutrophil activity or direct bacterial killing, which suggests the effects seen with SCH79797 may be PAR1 independent. Conclusions: In summary, we observed that intrapulmonary treatment with SCH79797 has significant therapeutic effects in a model of E. coli pneumonia that appear to be due, in part, to both neutrophil-stimulating and direct antibacterial effects of SCH79797.