Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

Francisella tularensis induces the synthesis of prostaglandin E₂ (PGE₂) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE₂ synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA₂), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE₂ synthesis, cPLA₂ hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE₂. However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA₂ phosphorylation (a marker of activation) and PGE₂ synthesis. These results suggested to us that cPLA₂ is not responsible for the liberation of AA to be converted into PGE₂ by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE₂ biosynthesis. cPLA₂, on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE₂ synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.     

A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB,Encoding a Putative Capsular Biosynthesis Protein,Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD₅₀) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD₅₀ of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.Francisella tularensis is a Gram-negative coccobacillus that causes tularemia, a zoonotic disease spread among small animals such as rabbits and rodents by blood-sucking insects. Humans typically acquire tularemia by handling infected animals or from the bite of infected insects. There are four subspecies of F. tularensis: F. tularensis subsp. tularensis, holarctica, mediasiatica, and novicida (41); of these, F. tularensis subsp. tularensis, found in North America and also known as type A, causes the most severe disease. Following cutaneous exposure, tularemia typically presents as an ulceronodular disease with painful, ulcerated skin lesions and swollen lymph nodes. Following inhalation exposure, tularemia presents with acute flu-like symptoms followed by pleuropneumonic and typhoidal illness. The pneumonic form of tularemia has a high fatality rate (11).Because of its high pathogenicity in humans, especially after respiratory exposure, its low infectious dose, and the relative ease with which it can be cultured and disseminated, F. tularensis is classified as a category A agent of bioterrorism, i.e., among bioterrorist agents thought to pose the greatest risk to the public. Indeed, F. tularensis was previously developed as a bioweapon and stockpiled by Japan during World War II (16) and by the United States and the Soviet Union during the Cold War (1, 6). Although tularemia can be treated with available antibiotics, F. tularensis can be genetically engineered to be antibiotic resistant (30). Moreover, pneumonic tularemia frequently requires hospitalization and intensive care, and even when an infected individual is treated with antibiotics to which the organism is sensitive, the disease may resolve slowly (12); even a moderately sized outbreak could rapidly overwhelm medical facilities (11). Hence, relying on antibiotics to protect against a bioterrorist attack with F. tularensis is not a practical public health approach. A safe and potent vaccine, on the other hand, would appear to offer a much more reliable approach.An unlicensed vaccine known as the live vaccine strain (LVS), an attenuated mutant of F. tularensis subsp. holarctica, was developed in the mid-1900s and is the only vaccine currently available in the United States. The underlying mechanism of attenuation is not fully characterized genetically, although recently, the reintroduction of deleted genes pilA and FTT0918 was shown to restore virulence to the level of virulent type B strains (35). The LVS vaccine has several drawbacks. The vaccine, which retains considerable virulence in animals, shows significant toxicity in humans after both intradermal (i.d.) and aerosol administration (19, 37). Moreover, it provides incomplete protection to humans challenged with type A F. tularensis by aerosol, the route of transmission of greatest concern in a bioterrorist attack (19, 29, 37).In a search for a vaccine that is safer and more potent than LVS, we sought to rationally attenuate LVS and to use the attenuated LVS as both a vaccine and a vector to overexpress immunogenic F. tularensis proteins. We hypothesized that we would render LVS safer by further attenuating it and that we would render it more potent by overexpressing key immunoprotective antigens. This overall strategy mirrors that used successfully to develop the first vaccine against tuberculosis that is more potent than the current Mycobacterium bovis BCG vaccine, rBCG30, a recombinant BCG vaccine overexpressing the Mycobacterium tuberculosis 30-kDa major secretory protein, and to develop the first vaccine both safer and more potent than BCG, rBCG(mbtB)30, an attenuated version of rBCG30 that is engineered and propagated such that it can multiply only a few times in the host (20, 21, 45).In attenuating LVS, we sought a mutation that would greatly reduce virulence but have a minimal impact on immunogenicity and protective efficacy. Transposon mutagenesis studies of F. tularensis subsp. novicida and holarctica (LVS) have shown that mutants with transposon insertions in genes (FTT0806, FTT0805, and FTT0798) encoding proteins putatively involved in capsular biosynthesis, on the basis of partial amino acid sequence homology with capsular biosynthesis proteins of Bacillus anthracis, are highly attenuated (∼100- to 1,000-fold) in mice (43, 47). Consequently, we decided to evaluate the vaccine potential of an LVS mutant with a deletion in one of these genes.In this study, we describe the construction of an antibiotic resistance marker-free FTL_1416/FTT0805 (capB) deletion mutant of F. tularensis LVS (LVS ΔcapB) and show that LVS ΔcapB is resistant to serum killing, outgrown by its parental LVS in human macrophage-like THP-1 cells, and highly attenuated in mice. We demonstrate further that this vaccine, after both i.d. and intranasal (i.n.) administration, induces potent cellular and humoral immune responses and significant protective immunity against respiratory challenge with virulent F. tularensis.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

Prostaglandin E Synthesis and Release by Murine Macrophages and Human Monocytes After in Vitro Treatment with Biological Response Modifiers

Abstract

Prostaglandins of the E series (PGE), produced by mononuclear phagocytes, have many biological activities and are involved in the regulation of myelopoiesis and of the cytotoxic activities of macrophage (M+) and natural killer (NK) cells. Since one of the possible effects of biological response modifiers (BRMs) on immune regulation might be modulation of PGE production, resident peritoneal murine M+ (mMψ) and elutriated human blood mono-cytes (hM) were incubated with BRMs and the supernatants were then assayed for PGE. Control mMψ produced about 4.9 ng PGE/10⁵ mMψ over a 24 hr period. Lipopolysaccharide (LPS, 1-5 μg/ml), polyinosinic-polycytidylic acid complexed with poly-L-lysine (Poly ICLC, 0.1-10 μg/ml) and murine α,β-interferon, (IFN, 10-1000 μ/ml) all caused a highly significant increase in PGE-secretion. BM41.332, a 2–cyanoaziridine (25-50 μg/ml), was found to be less stimulatory, whereas the pyran copolymer MVE-2 (25-50 μg/ml), and Azimexone (25–50 μg/ml) had marginal effects on PGE-production. Kinetic studies showed that the plateau of PGE-production by mMψ occurred during the first 24 hr of incubation, and mMψ which were washed after a 24 hr incubation period could not be restimulated to produce more PGE. PGE release by hM was increased after stimulation with LPS, Poly ICLC and BM41.332, whereas human IFNs (α and β) induced a slight decrease in PGE production. As in the murine studies, Azimexone and MVE-2 had no detectable effect. The ability of some BRMs to augment PGE-secretion by mMψ and hM may contribute to their negative effects on host responses, such as suppression of NK cell activity.     

Rapid Activation of Protein Tyrosine Kinase and Phospholipase C-γ2 and Increase in Cytosolic Free Calcium Are Required by Ehrlichia chaffeensis for Internalization and Growth in THP-1 Cells

Ehrlichia chaffeensis, a bacterium that cannot survive outside the eukaryotic cell, proliferates exclusively in human monocytes and macrophages. In this study, signaling events required for ehrlichial infection of human monocytic cell line THP-1 were characterized. Entry and proliferation of E. chaffeensis in THP-1 cells were significantly blocked by various inhibitors that can regulate calcium signaling, including 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate and 2-aminoethoxydiphenyl borate (intracellular calcium mobilization inhibitors), verapamil and 1-[beta-[3-(4-methoxyphenyl)propyl]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) (calcium channel inhibitors), neomycin and 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C [PLC] inhibitors), monodansylcadaverine (a transglutaminase [TGase] inhibitor), and genistein (a protein tyrosine kinase [PTK] inhibitor). Addition of E. chaffeensis resulted in rapid increases in the level of inositol 1,4,5-trisphosphate (IP(3)) and the level of cytosolic free calcium ([Ca(2+)](i)) in THP-1 cells, which were prevented by pretreatment of THP-1 cells with inhibitors of TGase, PTK, and PLC. E. chaffeensis induced rapid tyrosine phosphorylation of PLC-gamma2, and the presence of a PLC-gamma2 antisense oligonucleotide in THP-1 cells significantly blocked ehrlichial infection. Furthermore, tyrosine-phosphorylated proteins and PLC-gamma2 were colocalized with ehrlichial inclusions, as determined by double-immunofluorescence labeling. The heat-sensitive component of viable E. chaffeensis cells was essential for these signaling events. E. chaffeensis, therefore, can recruit interacting signal-transducing molecules and induce the following signaling events required for the establishment of infection in host cells: protein cross-linking by TGase, tyrosine phosphorylation, PLC-gamma2 activation, IP(3) production, and an increase in [Ca(2+)](i).     

Production of Prostaglandin E2 and Interleukin 1 by Mouse Peritoneal Macrophages Stimulated with β-l, 3-D-Glucan Derivatized Plastic Beads

Fluorescein-labelled plastic microbeads, with or without covalently attached beta-1,3-D-glucan, were injected into the peritoneal cavity of mice. Peritoneal cells were subsequently analysed by flow cytometry according to fluorescence and light scatter and separated into fluorescence positive and negative cells. We report that cells from animals treated with glucan-plastic beads produced large amounts of prostaglandin E2 (PGE2) whether the cells actually contained beads or not. On the other hand, cells from animals treated with glucan-plastic beads produced less thymocyte-stimulatory activity--presumably corresponding to interleukin 1 (IL-1)--than cells from control animals treated with commercial latex beads. However, when indomethacin was added, either in vivo or in vitro, cells from animals treated with glucan-plastic beads produced more thymocyte-stimulatory activity than controls. We interpret this to mean that glucan-plastic beads stimulate both IL-1 and PGE2, but that under circumstances where the cellular cyclo-oxygenase is not inhibited, the PGE2 will block IL-1 production.     

Vaccination with a Porcine Reproductive and Respiratory Syndrome (PRRS) Modified Live Virus Vaccine Followed by Challenge with PRRS Virus and Porcine Circovirus Type 2 (PCV2) Protects against PRRS but Enhances PCV2 Replication and Pathogenesis Compared to Results for Nonvaccinated Cochallenged Controls

Coinfections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) contribute to a group of disease syndromes known as porcine circovirus-associated disease (PCVAD). Presumably, PRRSV infection enhances PCV2 replication as a result of modulation of host immunity. The purpose of this study was to evaluate PCV2 replication and pathogenesis in pigs vaccinated with a PRRS modified live virus (MLV) vaccine and subsequently challenged with a combination of PRRSV and PCV2. During the early postchallenge period, the number of pigs with PRRSV-associated clinical signs was decreased, and average daily gain (ADG) was increased, in the vaccinated group, demonstrating the protective effect of PRRS vaccination. However, during the later postchallenge period, more pigs in the vaccinated group showed increased PCV2 viremia, decreased ADG, increased PCVAD clinical signs, and increased mortality. In this disease model, the early benefits of PRRSV vaccination were outweighed by the later amplification of PCVAD.     

Novel KCNQ2 and KCNQ3 Mutations in a Large Cohort of Families with Benign Neonatal Epilepsy: First Evidence for an Altered Channel Regulation by Syntaxin‐1A

Mutations in the KCNQ2 and KCNQ3 genes encoding for K_v7.2 (KCNQ2; Q2) and K_v7.3 (KCNQ3; Q3) voltage‐dependent K⁺ channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug‐resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression‐burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named “KCNQ2 encephalopathy.” In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin‐1A, highlighting a novel pathogenetic mechanism for KCNQ2‐related epilepsies.