Avian Resistance to Campylobacter jejuni Colonization Is Associated with an Intestinal Immunogene Expression Signature Identified by mRNA Sequencing

peer-reviewedThis research was funded by the The Irish Department of Agriculture and Food’s Food Institutional Research Measure (http://www.agriculture.gov.ie/ research/foodinstitutionalresearchmeasurefirm) – Grant No: 06_RDD_486.Campylobacter jejuni is the most common cause of human bacterial gastroenteritis and is associated with several post-infectious manifestations, including onset of the autoimmune neuropathy Guillain-Barré syndrome, causing significant morbidity and mortality. Poorly-cooked chicken meat is the most frequent source of infection as C. jejuni colonizes the avian intestine in a commensal relationship. However, not all chickens are equally colonized and resistance seems to be genetically determined. We hypothesize that differences in immune response may contribute to variation in colonization levels between susceptible and resistant birds. Using high-throughput sequencing in an avian infection model, we investigate gene expression associated with resistance or susceptibility to colonization of the gastrointestinal tract with C. jejuni and find that gut related immune mechanisms are critical for regulating colonization. Amongst a single population of 300 4-week old chickens, there was clear segregation in levels of C. jejuni colonization 48 hours post-exposure. RNAseq analysis of caecal tissue from 14 C. jejuni-susceptible and 14 C. jejuni-resistant birds generated over 363 million short mRNA sequences which were investigated to identify 219 differentially expressed genes. Significantly higher expression of genes involved in the innate immune response, cytokine signaling, B cell and T cell activation and immunoglobulin production, as well as the renin-angiotensin system was observed in resistant birds, suggesting an early active immune response to C. jejuni. Lower expression of these genes in colonized birds suggests suppression or inhibition of a clearing immune response thus facilitating commensal colonization and generating vectors for zoonotic transmission. This study describes biological processes regulating C. jejuni colonization of the avian intestine and gives insight into the differential immune mechanisms incited in response to commensal bacteria in general within vertebrate populations. The results reported here illustrate how an exaggerated immune response may be elicited in a subset of the population, which alters host-microbe interactions and inhibits the commensal state, therefore having wider relevance with regard to inflammatory and autoimmune disease

β-Defensins: Farming the Microbiome for Homeostasis and Health

peer-reviewedDiverse commensal populations are now regarded as key to physiological homeostasis and protection against disease. Although bacteria are the most abundant component of microbiomes, and the most intensively studied, the microbiome also consists of viral, fungal, archael, and protozoan communities, about which comparatively little is known. Host-defense peptides (HDPs), originally described as antimicrobial, now have renewed significance as curators of the pervasive microbial loads required to maintain homeostasis and manage microbiome diversity. Harnessing HDP biology to transition away from non-selective, antibiotic-mediated treatments for clearance of microbes is a new paradigm, particularly in veterinary medicine. One family of evolutionarily conserved HDPs, β-defensins which are produced in diverse combinations by epithelial and immune cell populations, are multifunctional cationic peptides which manage the cross-talk between host and microbes and maintain a healthy yet dynamic equilibrium across mucosal systems. They are therefore key gatekeepers to the oral, respiratory, reproductive and enteric tissues, preventing pathogen-associated inflammation and disease and maintaining physiological normality. Expansions in the number of genes encoding these natural antibiotics have been described in the genomes of some species, the functional significance of which has only recently being appreciated. β-defensin expression has been documented pre-birth and disruptions in their regulation may play a role in maladaptive neonatal immune programming, thereby contributing to subsequent disease susceptibility. Here we review recent evidence supporting a critical role for β-defensins as farmers of the pervasive and complex prokaryotic ecosystems that occupy all body surfaces and cavities. We also share some new perspectives on the role of β-defensins as sensors of homeostasis and the immune vanguard particularly at sites of immunological privilege where inflammation is attenuated

MicroRNA regulation of bovine monocyte inflammatory and metabolic networks in an in vivo infection model.

peer-reviewedBovine mastitis is an inflammation-driven disease of the bovine mammary gland that costs the global dairy industry several billion dollars per annum. Because disease susceptibility is a multi-factorial complex phenotype, an integrative biology approach is required to dissect the molecular networks involved. Here, we report such an approach, using next generation sequencing combined with advanced network and pathway biology methods to simultaneously profile mRNA and miRNA expression at multiple time-points (0, 12, 24, 36 and 48h) in both milk and blood FACS-isolated CD14+ monocytes from animals infected in vivo with Streptococcus uberis. More than 3,700 differentially expressed (DE) genes were identified in milk-isolated monocytes (MIMs), a key immune cell recruited to the site of infection during mastitis. Up-regulated genes were significantly enriched for inflammatory pathways, while down-regulated genes were enriched for non-glycolytic metabolic pathways. Monocyte transcriptional changes in the blood, however, were more subtle but highlighted the impact of this infection systemically. Genes up-regulated in blood-isolated-monocytes (BIMs) showed a significant association with interferon and chemokine signalling. Furthermore, twenty-six miRNAs were differentially expressed in MIMs and three in BIMs. Pathway analysis revealed that predicted targets of down-regulated miRNAs were highly enriched for roles in innate immunity (FDR < 3.4E-8) in particular TLR signalling, while up-regulated miRNAs preferentially targeted genes involved in metabolism. We conclude that during S. uberis infection miRNAs are key amplifiers of monocyte inflammatory response networks and repressors of several metabolic pathways.This study was funded in part by Teagasc RMIS 6018 and United States Department of Agriculture ARS funding 3625-32000-102-00. NL is supported by a Teagasc Walsh Fellowship

Non-canonical Inflammasome-Mediated IL-1β Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle

Non-canonical Inflammasome-Mediated IL-1β Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent

peer-reviewedInflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle

Human PSC-derived hepatocytes express low levels of viral pathogen recognition receptors, but are capable of mounting an effective innate immune response

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte&ndash;pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on pattern recognition receptor (PRR)-expression and the presence of a metabolic switch. We analysed cytoplasmic PRR and endosomal toll-like receptor (TLR)-expression, as well as activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level levels of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs

Early interferon lambda production is induced by double-stranded RNA in iPS-derived hepatocyte-like cells

Hepatotropic viruses are amongst the most ubiquitous pathogens worldwide, causing significant morbidity and mortality. As hepatocytes are among the primary targets of these viruses, their ability to mount early effective innate defence responses is of major research interest. Interferon lambda (IFNL) is produced early in response to viral stimulation in other cell types, but hepatocyte production of this interferon is little investigated. Due to the difficulty and significant costs in obtaining and culturing human primary hepatocytes, surrogate systems are widely sought. Here we used induced pluripotent stem (iPS)-derived hepatocyte-like cells (HLCs) to investigate hepatic IFNL expression in response to viral-like ligands. We demonstrate that hepatocytes rely on cytoplasmic pattern recognition receptors (PRRs) such as Protein Kinase RNA-dependent (PKR) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) for the detection of double stranded RNA. Stimulation of HLCs by viral-like RNA ligands activating cytosolic RNA sensors resulted in thousand fold increase of type III interferon gene expression. These results are in contrast with type I IFN expression, which was induced to a lower extent. Concomitant induction of interferon stimulated genes, such as interferon-stimulated gene 15 (ISG15) and CXCL10, indicated the ability of HLCs to activate interferon-dependent activity. These results demonstrate that HLCs mount an innate antiviral response upon stimulation with viral-like RNA characterized by the induction of type III IFN

Evidence of Positively Selected Sites in Mammalian a-Defensins

Defensins are a family of mammalian antimicrobial peptides that exhibit variable activity against a panel of microbes, including bacteria, fungi, and enveloped viruses. We have employed a maximum-likelihood approach to detect evidence of positive selection (adaptive evolution) in the evolution of these important molecules of the innate immune response. We have identified 14 amino acid sites that are predicted to be subject to positive selection. Furthermore, we show that all these sites are located in the mature antimicrobial peptide and not in the prepropeptide region of the molecule, implying that they are of functional importance. These results suggest that mammalian a-defensins have been under selective pressure to evolve in response to potentially infectious challenges by fast-evolving microbes

Evidence of Positively Selected Sites in Mammalian a-Defensins

Defensins are a family of mammalian antimicrobial peptides that exhibit variable activity against a panel of microbes, including bacteria, fungi, and enveloped viruses. We have employed a maximum-likelihood approach to detect evidence of positive selection (adaptive evolution) in the evolution of these important molecules of the innate immune response. We have identified 14 amino acid sites that are predicted to be subject to positive selection. Furthermore, we show that all these sites are located in the mature antimicrobial peptide and not in the prepropeptide region of the molecule, implying that they are of functional importance. These results suggest that mammalian a-defensins have been under selective pressure to evolve in response to potentially infectious challenges by fast-evolving microbes