Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival.

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

Methodological challenges in utilizing miRNAs as circulating biomarkers.

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma- and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

Epigenetic regulation of miR-17~92 contributes to the pathogenesis of pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17~92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression. OBJECTIVES: We investigated the miR-17~92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue. METHODS: Expression and DNA methylation patterns of miR-17~92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17~92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5'-aza-2'-deoxycytidine. MEASUREMENTS AND MAIN RESULTS: Compared with control samples, miR-17~92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17~92 promoter was increased. Several miRNAs from the miR-17~92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17~92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5'-aza-2'-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17~92 cluster expression, and attenuated pulmonary fibrosis. CONCLUSIONS: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17~92 and DNMT-1 in lung fibrosis.

sRAGE induces human monocyte survival and differentiation.

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive, negative regulator of membrane RAGE activation, inhibiting ligand binding. However, some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment, survival, and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE, the recombinant protein was intratracheally administered to mice, which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore, sRAGE binding to cells increased during maturation, which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation, we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages, sRAGE treatment activated Akt, Erk, and NF-kappaB, and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation.

Thrombospondin-1-deficient mice are not protected from bleomycin-induced pulmonary fibrosis.

Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-ß (TGF-ß). Because active TGF-ß is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1-deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1-deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-ß. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.

Passive Immunisation against RHDV2 Induces Protection against Disease but Not Infection.

Rabbit haemorrhagic disease virus 2 (RHDV2) is a lagovirus in the family Caliciviridae. The closely related Rabbit haemorrhagic disease virus (RHDV, termed RHDV1 throughout this manuscript for clarity) has been used extensively as a biocontrol agent in Australia since the mid-1990s to manage wild rabbit populations, a major economic and environmental pest species. Releasing RHDV1 into populations with a high proportion of rabbits less than 8-10 weeks of age leads to non-lethal infection in many of these young animals, with subsequent seroconversion and long-term immunity against reinfection. In contrast, RHDV2 causes lethal disease even in young rabbits, potentially offering substantial benefits for rabbit management programs over RHDV1. However, it is not clear how acquired resistance from maternal antibodies may influence immunity after RHDV2 infection. In this study, we assessed serological responses after RHDV2 challenge in young rabbits of three different ages (5-, 7-, or 9-weeks-old) that were passively immunised with either high- (titre of 2560 by RHDV IgG ELISA; 2.41 mg/mL total protein) or low- (titre of 160-640 by RHDV IgG ELISA; 1.41 mg/mL total protein) dose RHDV2 IgG to simulate maternal antibodies. All rabbits treated with a high dose and 75% of those treated with a low dose of RHDV2 IgG survived virus challenge. Surviving animals developed robust lagovirus-specific IgA, IgM, and IgG responses within 10 days post infection. These findings demonstrate that the protection against RHDV2 conferred by passive immunisation is not sterilising. Correspondingly, this suggests that the presence of maternal antibodies in wild rabbit populations may impede the effectiveness of RHDV2 as a biocontrol.

Age and Infectious Dose Significantly Affect Disease Progression after RHDV2 Infection in Naïve Domestic Rabbits.

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.

Social disruption induces lung inflammation.

Social disruption (SDR) is a well-characterized mouse stressor that causes changes in immune cell reactivity in response to inflammatory stimuli. In this study, we found that SDR in the absence of an immune challenge induced pulmonary inflammation and increased pulmonary myeloperoxidase activity. The percentage of neutrophils within the lungs increased 2-fold after social disruption. Monocyte accumulation in the lungs was also significantly increased. In addition, SDR increased the percentage of neutrophils that expressed CD11b, indicating that more neutrophils were in an activated state. In the lungs, we observed an increased level of the inflammatory cytokine, IL-1beta, as well as higher levels of KC/CXCL1, MIP-2/CXCL2, and MCP-1/CCL2, which are chemokines responsible for neutrophil and monocyte recruitment. Furthermore, social disruption led to increased lung expression of the adhesion molecules P-selectin, E-selectin, and ICAM-1, which localize and recruit immune cells. These data support previous findings of an inflammatory environment induced by SDR. We demonstrate that this effect also occurs in the pulmonary milieu and in the absence of an inflammatory stimulus.

Retrospective serological analysis reveals presence of the emerging lagovirus RHDV2 in Australia in wild rabbits at least five months prior to its first detection.

The lagovirus rabbit haemorrhagic disease virus (RHDV) has been circulating in Australia since the mid-1990s when it was released to control overabundant rabbit populations. In recent years, the viral diversity of different RHDVs in Australia has increased, and currently four different types of RHDV are known to be circulating. To allow for ongoing epidemiological studies and impact assessments of these viruses on Australian wild rabbit populations, it is essential that serological tools are updated. To this end, reference sera were produced against all four virulent RHDVs (RHDV, RHDV2 and two different strains of RHDVa) known to be present in Australia and tested in a series of available immunological assays originally developed for the prototype RHDV, to assess patterns of cross-reactivity and the usefulness of these assays to detect lagovirus antibodies, either in a generic or specific manner. Enzyme-linked immunosorbent assays (ELISAs) developed to detect antibody isotypes IgM, IgA and IgG were sufficiently cross-reactive to detect antibodies raised against all four virulent lagoviruses. For the more specific detection of antibodies to the antigenically more different RHDV2, a competition ELISA was adapted using RHDV2-specific monoclonal antibodies in combination with Australian viral antigen. Archival serum banks from a long-term rabbit monitoring site where rabbits were sampled quarterly over a period of 6 years were re-screened using this assay and revealed serological evidence for the arrival of RHDV2 in this population at least 5 months prior to its initial detection in Australia in a dead rabbit in May 2015. The serological methods and reference reagents described here will provide valuable tools to study presence, prevalence and impact of RHDV2 on Australian rabbit populations; however, the discrimination of different antigenic variants of RHDVs as well as mixed infections at the serological level remains challenging.

Constitutive AKT Activity Predisposes Lung Fibrosis by Regulating Macrophage, Myofibroblast and Fibrocyte Recruitment and Changes in Autophagy.

The etiology and pathogenesis of pulmonary fibrosis is poorly understood. We and others reported that M-CSF/CSF-1, M-CSF-R and downstream AKT activation plays an important role in lung fibrosis in mice models and in IPF patients. To understand potential molecular pathways used by M-CSF-R activation to direct lung fibrosis, we used a novel transgenic mouse model that expresses a constitutively-active form of AKT, myristoylated AKT (Myr-Akt), driven by the c-fms (M-CSF-R) promoter. We were particularly interested in the basal immune state of the lungs of these Myr-Akt mice to assess M-CSF-R-related priming for lung fibrosis. In support of a priming effect, macrophages isolated from the lungs of unchallenged Myr-Akt mice displayed an M2-tropism, enhanced co-expression of M-CSF-R and α-SMA, reduced autophagy reflected by reduced expression of the key autophagy genes Beclin-1, MAP1-Lc3a(Lc3a), and MAP1-Lc3b(Lc3b), and increased p62/STSQM1 expression compared with littermate WT mice. Furthermore, Myr-Akt mice had more basal circulating fibrocytes than WT mice. Lastly, upon bleomycin challenge, Myr-Akt mice showed enhanced collagen deposition, increased F4/80+ and CD45+ cells, reduced autophagy genes Beclin-1, Lc3a, and Lc3b expression, and a shorter life-span than WT littermates. These data provide support that M-CSF-R/AKT activation may have a priming effect which can predispose lung tissue to pulmonary fibrosis.

Mitochondrial DNA and trade data support multiple origins of Helicoverpa armigera (Lepidoptera, Noctuidae) in Brazil.

The Old World bollworm Helicoverpa armigera is now established in Brazil but efforts to identify incursion origin(s) and pathway(s) have met with limited success due to the patchiness of available data. Using international agricultural/horticultural commodity trade data and mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) gene markers, we inferred the origins and incursion pathways into Brazil. We detected 20 mtDNA haplotypes from six Brazilian states, eight of which were new to our 97 global COI-Cyt b haplotype database. Direct sequence matches indicated five Brazilian haplotypes had Asian, African, and European origins. We identified 45 parsimoniously informative sites and multiple substitutions per site within the concatenated (945 bp) nucleotide dataset, implying that probabilistic phylogenetic analysis methods are needed. High diversity and signatures of uniquely shared haplotypes with diverse localities combined with the trade data suggested multiple incursions and introduction origins in Brazil. Increasing agricultural/horticultural trade activities between the Old and New Worlds represents a significant biosecurity risk factor. Identifying pest origins will enable resistance profiling that reflects countries of origin to be included when developing a resistance management strategy, while identifying incursion pathways will improve biosecurity protocols and risk analysis at biosecurity hotspots including national ports.

Complete mitochondrial genome of the European Grapevine moth (EGVM) Lobesia botrana (Lepidoptera: Tortricidae).

The Lobesia botrana larvae feed on grapevine (Vitis vinifera L.), thereby reducing crop yield and increasing crop susceptibility to fungal and bacterial attacks. We determined the circular mitochondrial genome of L. botrana as 15 229 bp (GenBank KP677508) and contained 13 protein coding genes (PCG's), 22 transfer RNAs (tRNA), and two ribosomal RNAs. All tRNAs have the "clover-shaped" 2-D structures, while the tRNA-Ile which has the TψC-stem but lacked the TψC-loop. Knowledge of L. botrana mitochondrial genome represents a valuable molecular resource for developing effective DNA identification tools for biosecurity purposes and will contribute to better understanding of its evolutionary and population genetics.

MicroRNA 17-92 cluster mediates ETS1 and ETS2-dependent RAS-oncogenic transformation.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation.

Analyzing the circulating microRNAs in exosomes/extracellular vesicles from serum or plasma by qRT-PCR.

Small extracellular vesicles are released from both healthy and disease cells to facilitate cellular communication. They have a wide variety of names including exosomes, microvesicles and microparticles. Depending on their size, very small extracellular vesicles originating from the endocytic pathway have been called exosomes and in some cases nanovesicles. Collectively, extracellular vesicles are important mediators of a wide variety of functions including immune cell development and homeostasis. Encapsulated in the extracellular vesicles are proteins and nucleic acids including mRNA and microRNA molecules. MicroRNAs are small, non-coding RNA molecules implicated in the post-transcriptional control of gene expression that have emerged as important regulatory molecules and are involved in disease pathogenesis including cancer. In some diseases, not only does the quantity and the subpopulations of extracellular vesicles change in the peripheral blood but also microRNAs. Here, we described the analysis of peripheral blood extracellular vesicles by flow cytometry and the RNA extraction from extracellular vesicles isolated from the plasma or serum to profile microRNA expression.

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the "ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)", 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

M-CSF induces monocyte survival by activating NF-κB p65 phosphorylation at Ser276 via protein kinase C.

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/ß inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes.