Metrics details The gut microbiota exerts profound influence on poultry immunity and metabolism through mechanisms that yet need to be elucidated Here we used conventional and germ-free chickens to explore the influence of the gut microbiota on transcriptomic and metabolic signatures along the gut-lung axis in poultry Our results demonstrated a differential regulation of certain metabolites and genes associated with innate immunity and metabolism in peripheral tissues of germ-free birds we evidenced the gut microbiota’s capacity to regulate mucosal immunity in the chicken lung during avian influenza virus infection by fine-analysing the antiviral pathways triggered by the short-chain fatty acid (SCFA) butyrate in chicken respiratory epithelial cells we found that it regulates interferon-stimulated genes (ISGs) These findings emphasize the pivotal role of the gut microbiota and its metabolites in shaping homeostasis and immunity in poultry offering crucial insights into the mechanisms governing the communication between the gut and lungs in birds the regulation of microbiota composition has well-recognised consequences for immune development and overall health in humans and mammalian model species Understanding the mechanisms through which the GM and SCFAs influence metabolism and immunity in the respiratory tract and other peripheral tissues is an area of active research and would allow the development of probiotics/postbiotics with optimised capacity to enhance immune system functions beyond the gut the gut-lung axis in birds is largely unexplored despite most pathogens in commercial poultry targeting the gut and/or airways To define how the chicken GM regulates metabolism and immunity in the gastrointestinal tract and in the periphery we analysed transcriptomic and metabolic signatures in conventional (CV) and germ-free (GF) chickens GF birds exhibited significant physiological changes in the caecal compartment lacking central GM-derived metabolites like SCFAs SCFA detection in peripheral compartments of CV birds and differential expression of innate immunity genes in GF birds’ peripheral tissues further corroborated the functional existence of a gut-lung axis in birds GF chickens showed different immunoregulatory responses to infection displayed antiviral effects in chicken respiratory epithelial cells likely via the regulation of interferon-stimulated genes The observed antiviral signalling mechanism would involve HDAC inhibition and Sp1-dependent regulation of the OASL promoter shedding light on new mechanisms through which the GM regulates poultry mucosal immunity along the gut-lung axis in the chicken Perturbations in the growth media were visually examined after 18 hours of incubation a drop from the BHI fecal-broth media was placed on a glass slide and examined under a microscope (×40 magnification) for the presence or absence of bacteria If suspicion arose regarding the presence of these microorganisms a sample from the BHI culture was inoculated onto BHI agar plates and incubated at 37 °C for 18 to 48 hours for further analysis Swabs from isolators were periodically tested during hatching and post-hatching periods until the end of the experiment employing the same detection methods for bacteria and fungi described above All experiments were conducted in compliance with the relevant European and national regulations and authorised by the local ethics committee (Comité d’Ethique en Expérimentation Animale Val de Loire) under the reference number APAFIS#30655-2021042114338110 v1 CV and GF birds (n = 8 per group) were sacrificed by cervical dislocation followed by blood withdrawal from the occipital venous plexus Blood samples were stored in heparin-coated tubes for subsequent plasma preparation and lungs were immediately collected from each chicken and stored at −80 °C until further use for metabolomics analysis and/or RNA and DNA extraction and sequencing a group of 24 GF and 24 CV birds were equally inoculated intra-tracheally (i.t.) with 0.2 ml of 106 EID550 of H7N1 and 15 GF and 15 CV control (mock) birds were inoculated i.t and plasma were recovered from a group of animals (n = 5–8) for different downstream analyses Birds were carefully monitored (twice daily) during the course of infection and diseased birds presenting at least 4 of the following symptoms for 24/48 hours were killed by intravenous pentobarbital injection (humane endpoint): ruffled feathers Clinical signs were evaluated according to the following score: 0 (no clinical signs) or 3 (dead or euthanized due to persistent severe clinical signs) A scoring system was used to evaluate macroscopic lung lesions as follows: 1 (mild 2 (moderate oedema and with haemorrhage and fibrinous exudate over ~1/4 of the lung) or 3 (severe haemorrhage and extensive oedema over ~1/2 of the lung) Cells were cultured in Dulbecco’s Modified Eagles’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gibco and 100 UI/ml penicillin and 100 μg/ml streptomycin (PS cells were plated at 5 × 106 cells/ml in 12-well plates Sigma-Aldrich-UK) at varying concentrations and times Some experiments included chicken interferon alpha (Yeast-derived Cell lysates and RNA were analysed for gene expression and metabolomics Cell viability and counts were assessed using Spark® Cyto (Tecan Switzerland) after Trypan Blue (Sigma-Aldrich Primers M52C and M253R span conserved sequences in gene segment 7 of influenza A virus and have no homology to nucleotide sequences from other species available from GenBank Viral load quantification was expressed as PFU/mL or M1 genomic copy numbers Cellular RNA extractions from infection experiments with the H1N1 and H7N1 strains were also used for gene expression analysis UK) was used to measure class I and II HDAC activity in CLEC213 cells according to the manufacture’s protocol the acetylated peptide substrate was added to the cell culture medium in the presence or absence of butyrate and peptide cleavage requiring deacetylated protease activity was quantified by measuring the release of luminescent aminoluciferin using a GloMax plate reader (Promega Reduction in relative light units (RLU) correlated with decreased HDAC activity due to the inhibitory properties of the tested molecule Trichostatin A served as a positive control Data are presented as RLU or as percentage of HDAC activity with untreated cells as the reference (100%) Caecal contents samples (20–50 mg) were homogenised in 1.2 ml of phosphate buffer (0.2 M dried using a SpeedVac Vacuum Concentrator 500 µl of supernatant were transferred into 5 mm NMR tubes and cell lysates were homogenised in 4.85 ml of methanol/water (80/20 2 ml of dichloromethane per gram of tissue were added 2 ml of dichloromethane and 2 ml of water per gram of tissue were added Samples were dried using a SpeedVac Vacuum Concentrator reconstituted in 200 µl of phosphate buffer (0.2 M 150 µL of supernatant were transferred into 3 mm NMR tubes and 50 µL of TSP solution (1 mM) were added to the NMR tube 1H NMR spectra were obtained at 300 K on a Bruker Avance III HD 600 MHz NMR spectrometer The “noesypr1d” pulse sequence was used for water signal suppression with a mixing time of 100 ms A total of 1024 and 256 transients were collected for tissue and caecal samples into 64k data points using a spectral width of 12 ppm an exponential line broadening function of 0.3 Hz was applied to the FID All NMR spectra were phase- and baseline-corrected and referenced to the chemical shift of TSP (0 ppm) using Topspin (V3.2 Metabolite concentrations were calculated using the TSP signal at 0 ppm integrating for 9 protons and with known concentrations of different metabolites with significant logarithmic fold change ratios determined by Wald tests and Benjamini-Hochberg adjustment for multiple testing (P < 0.01) To distinguish monocytes and macrophages in the spleen we utilised an APC-conjugated antibody (clone Kul01 IgG1) targeting the chicken mannose receptor C-type 1 like B (MRCL1-B) of avian monocytes and macrophages we incorporated staining for MHC-II (clone 21-1A6 T lymphocytes were identified based on the expression of their T-cell receptor (TCR) and coreceptors we employed monoclonal antibodies against CD4 (clone CT-4 Chicken thrombocytes were characterised using antibodies recognising the fibrinogen receptor CD41/61 (clone 11C3 we utilised an APC-conjugated antibody recognising the antigen Bu-1 (clone AV20 Quadrant markers were established based on negative populations and isotype controls cell viability was assessed using the fluorescent DNA intercalator 7-aminoactinomycin D (7-AAD Cell acquisition (5 × 105 events) was performed using a BD FACSCanto II cytometer (BD Biosciences) with subsequent analysis conducted using FlowJo 7.5.3 software (TreeStar Inc. The percentage of the analysed population relative to total acquired events was employed in graph construction Unless otherwise specified above or in the figure legends data are presented as the median or mean ± SEM The unpaired Student’s t test was employed to compare the means of two independent groups one-way analysis of variance (ANOVA) was conducted to ascertain statistically significant differences among the means followed by a Tukey post-hoc test utilising a Studentized range statistic to perform pairwise comparisons between groups USA) was employed for statistical analysis Significance levels were set at p < 0.05 for most analyses these data highlight that the isolators employed in this study did not impede the development of a complex caecal microbiota in our chicken line log2 fold change ≥1) from RNAseq data from germ-free (GF) chickens compared to conventionally raised (CV) chickens at 21 days post-hatch were analysed using Ingenuity Analysis Pathway software visualised as a “bubble chart.” Circles in each row represent groups of genes associated with a biological function with size indicating gene count and colour reflecting an intensity gradient ranging from overexpression (orange pattern) to downregulation (blue pattern) in GF chickens using CV chickens as the normalisation controls The software enables tissue or cell selection and provides inferences on unrepresented genes A confidence-boosting maximum adjusted p value threshold was applied: 0.45 for caeca (a) including the analyses of the top 244 genes from the caeca RNAseq analyses; 0.4 for lungs (b) including the analyses of the top 280 genes from the lungs RNAseq analyses; and 0.1 for spleen (c) including the analyses of the top 84 genes from the spleen RNAseq analyses lungs or spleen samples from 5 GF chickens or 5 CV chickens (n = 5 biological replicates) log2 fold change ≥1) associated with immunity and inflammation were identified in the RNAseq analyses of caeca (a) and spleen (c) samples of 21 days-old chickens This selection included standout genes coding for transcription factors enzymes and other molecules with broad functions in innate and adaptive immunity of vertebrates Histograms present the relative expression of each gene in log2 format for germ-free (GF) animals compared to conventionally raised (CV) animals A downregulation trend is particularly pronounced in the caeca and moderately in the spleen contrasting regulatory phenomena with similar intensities coexist d Certain DEGs identified using RNAseq analyses followed by an assessment through the Ingenuity Analysis Pathway software (in which a confidence-boosting maximum adjusted p value threshold was applied) are shared by the three organs studied A Venn diagram illustrates DEG counts per organ reveal OASL as the sole immune-related gene significantly regulated in all three examined organs these findings highlight the GM’s significant impact on the transcriptional regulation of IFN-related genes along the gut-lung axis in the chicken remains largely unaffected by the lack of a GM with resulting p values generating colour scales for the histograms exclusion threshold) were applied to the presented data Each group comprised caecal contents or lung samples from 6 GF chickens or 6 CV chickens (n = 6 biological replicates) Unpaired student’s t test was employed for statistical analyses (a These observed disparities underscore the GM’s profound influence on chicken metabolism and immune response providing solid evidence to further explore these regulatory mechanisms in the context of infectious challenges Germ-free (GF) animals infected via the tracheal route with a 5 × 105 EID50 dose of LPAIV H7N1 exhibited a disease profile indistinguishable from their conventional (CV) counterparts Macroscopic lung lesion scores (haemorrhage oedema) (a) and viral genome copies in the lungs (b) or in the caeca (c) at Days 1–3 post-infection (p.i.) show no significant differences between the two groups d Examination of infection kinetics in GF chickens’ lungs using Medium-Throughput qPCR (Fluidigm) indicates sustained expression of genes associated with inflammation (IL8L1 The heatmap illustrates relative expression levels in GF-infected animals compared to CV-infected animals on the same day Each group comprised caeca or lungs samples from 5 mock and 8 infected GF chickens or 5 mock and 8 infected CV chickens (n = 5–8 biological replicates) each biological replicate is the mean of three technical replicates Statistical analysis employed unpaired student’s t test (a–c) or one-way ANOVA followed by Tukey multiple comparison test (d) Data are represented as the median (a–c) or the mean ± SEM (d These genes showed lower expression at 24 and 48 hours compared to CV animals but a significant elevation at 72 hours indicating a sustained antiviral response beyond 48 hours in GF animals These changes also influenced mucosal immunity as seen in the distinct regulation pattern for IL22 and its receptors (IL22RA1 reduced expression of anti-inflammatory genes like TGFB and SOCS3 at 72 hours p.i in GF animals possibly indicates an unresolved inflammatory state while the absence of GM does not hinder antiviral responses against AIV in chickens it influences the quality and amplitude of the immune response at the transcriptional level The CLEC213 chicken lung epithelial cell line was treated with different concentrations of SCFA for 16 h and acetate was assessed using a Spark® Cyto and expressed as the percentage of cell viability compared to untreated controls (a) qPCR analysis demonstrates that acetate (b) and propionate (d) elicit different responses in terms of selected innate immune gene expression in CLEC213 cells the induction of OASL by butyrate exhibits a concentration-dependent relationship (e) Statistical analysis was performed using One-way ANOVA followed by Tukey multiple comparison test with significance levels indicated as follows: *p < 0.05 in which each biological replicate is the mean of three technical replicates c–e an n = 5–7 biological replicates is shown with colour scales in histograms corresponding to p values c Butyrate induced positive regulation across diverse biological functions in chicken lung epithelial cells as depicted in a ‘bubble chart’ generated from RNAseq data analysis using Ingenuity Analysis Pathway software Circles in each row represent groups of genes associated with a biological function with size indicating gene count and colour reflecting an intensity gradient ranging from overexpression (orange pattern) to downregulation (blue pattern) in butyrate (3 mM) treated cells using untreated cells as the normalisation controls A confidence-boosting maximum adjusted p value threshold of 045 was applied d The log2 relative expression of a group of selected immune-related genes extracted from all DEG from RNAseq data (padj < 0.05 log2 fold change ≥1) in cells receiving butyrate (3 mM) compared to untreated cells at 16 h Data in d are represented as the mean ± SEM Butyrate also exhibits pro-inflammatory actions mirroring observations in GF animals (which lack systemic butyrate) and emphasising the role of SCFA in regulating ISGs in the respiratory mucosa f Identification of potential binding sites for Sp1 in the OASL promoter of selected bird and mammalian species One-way ANOVA followed by Tukey multiple comparison tests (a–d) was employed for statistical analyses b an n = 3 biological replicates are shown Data in e are representative of an n = 6 biological replicates without affecting vital cellular functions in the CLEC213 cell line the function of these genes in the chicken are still not resolved although their conservation among several vertebrate species suggests that they may exhibit overall conserved biological functions Butyrate treatment (3 mM) improves viral clearance in CLEC213 cells infected with an LPAIV H1N1 avian influenza strain Incubation with butyrate prior to infection (MOI 0.1 for 6 hours or MOI 0.01 for 16 hours) significantly reduces viral titre (PFU/ml) (a) and genomic copies (b) compared to untreated cells qPCR analysis showed increased expression of IFNB and OASL in butyrate-treated infected cells (c) Silencing OASL expression abolishes butyrate’s antiviral effects resulting in infectious titre identical to untreated cells concomitantly with reduced OASL expression (d) b) and One-way ANOVA followed by Tukey multiple comparison test (c a an n = 3 biological replicates are shown b–d an n = 3 biological replicates is shown the overall antiviral effects of butyrate would involve enhanced metabolic activity regulatory influence on inflammatory signals suggesting potential compensatory mechanisms for lower nutrient absorption studies linking the GM and SCFA to the regulation of ISG15 or OASL are currently lacking OASL’s regulation in GF animals appears markedly altered both at homoeostasis and infection emphasising its dependence upon positive microbial signals coming from the GM in the chicken Our comparative study using GF and CV chickens thus provides robust evidence of OASL regulation by the microbiota shedding new light on evolutionary biology and comparative immunology its expression decreased by 2 log2 at homoeostasis and surged to a level 60 times higher 24 hours post-infection it appears that a delay in triggering the antiviral innate response in the lungs of GF chickens does not impair the proper control of viral load compared to CV chickens the selected innate immune genes in GF chickens are already at a lower basal level than those in CV counterparts This could potentially attenuate the exacerbated inflammatory response that leads to severe pneumonia and increased mortality as overall lung pathology and clinical scores are similar between CV and GF chickens Further studies with a broader analysis beyond day 3 are necessary to determine if GF animals can sustain viral load control and if their innate immune response will peak and resolve similarly to CV animals This remains undefined at present and could lead to the discovery of new molecular mechanisms through which the chicken GM directs innate immunity toward effective and pro-resolutive viral clearance potentially mitigating inflammation by preventing HIF activation in pulmonary epithelium butyrate may regulate OASL expression through additional metabolic pathways yet to be defined highlighting its dual role in shaping immunity and metabolism in our respiratory epithelial system This unveils a novel mechanism through which a GM-derived SCFA modulates antiviral immunity in peripheral organs of birds Further investigation into the multifaceted regulation of the type I IFN response by butyrate and its varying effects on virus infection in vertebrates is warranted Our research revealed the intricate interaction between caecal microbiota-derived SCFAs and their role in regulating protective immunity and metabolism across the gut-lung axis in chickens These insights have broad implications for understanding the gut-lung axis and developing prophylactic strategies in commercial poultry through microbiota modulation Further exploration of butyrate’s multifaceted effects and its interactions with specific cellular pathways is crucial to harnessing the full potential of SCFAs as next-generation postbiotics for poultry birds The authors declare that all data supporting the findings in this study are available within the article or from the corresponding author on reasonable request All RNAseq data and 16S sequencing data were deposited in the ELIXIR Deposition Database ArrayExpress under the accession numbers E-MTAB-14262 Gut microbiota functions: metabolism of nutrients and other food components Shifting the balance: antibiotic effects on host–microbiota mutualism From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites Butyrate protects mice from clostridium difficile-induced colitis through an HIF-1-dependent mechanism Microbial regulation of host physiology by short-chain fatty acids Principles and clinical implications of the brain–gut–enteric microbiota axis Centennial review: factors affecting the chicken gastrointestinal microbial composition and their association with gut health and productive performance Holistic view of intestinal health in poultry Composition of gut microbiota influences resistance of newly hatched chickens to salmonella enteritidis infection gut health and chicken productivity: what is the connection Possibilities of early life programming in broiler chickens via intestinal microbiota modulation Extensive microbial and functional diversity within the chicken cecal microbiome The role of the gut microbiome in shaping the immune system of chickens Uncovering the core principles of the gut-lung axis to enhance innate immunity in the chicken Highly pathogenic avian influenza viruses at the wild–domestic bird interface in europe: future directions for research and surveillance Guitton, E. et al. Production of germ-free fast-growing broilers from a commercial line for microbiota studies. J. Vis. Exp. e61148 https://doi.org/10.3791/61148 (2020) Broad-range 16S rRNA gene polymerase chain reaction for diagnosis of culture-negative bacterial infections Japanese Pharmacopoeia (JP): the 4.06 Sterility Test as It Appeared in the Partial Revision of the JP 15th Edition Labour and Welfare Ministerial Notification No United States Pharmacopeia (USP): sterility tests as presented in pharmacopeial forum Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy Highly pathogenic or low pathogenic avian influenza virus subtype H7N1 infection in chicken lungs: small differences in general acute responses Major contribution of the RNA-binding domain of NS1 in the pathogenicity and replication potential of an avian H7N1 influenza virus in chickens Modeling immunocompetence development and immunoresponsiveness to challenge in chicks A novel chicken lung epithelial cell line: characterization and response to low pathogenicity avian influenza virus interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition Inhibition of histone deacetylase activity by butyrate Efficient extraction from mice feces for NMR metabolomics measurements with special emphasis on SCFAs Stable suppression of gene expression by RNAi in mammalian cells Role of the chicken oligoadenylate synthase-like gene during in vitro Newcastle disease virus infection Cutadapt removes adapter sequences from high-throughput sequencing reads RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update) An accurate and efficient experimental approach for characterization of the complex oral microbiota Swarm: robust and fast clustering method for amplicon-based studies The SILVA ribosomal RNA gene database project: improved data processing and web-based tools Characterization of chicken leukocyte subsets from lymphatic tissue by flow cytometry Untangling the genetic basis of fibrolytic specialization by Lachnospiraceae and Ruminococcaceae in diverse gut communities Effect of cecal microbiota transplantation between different broiler breeds on the chick flora in the first week of life Contact with adult hen affects development of caecal microbiota in newly hatched chicks The effects of secretory IgA in the mucosal immune system Structure and functions of cellular redox sensor HSCARG/NMRAL1 Amphiregulin promotes intestinal epithelial regeneration: roles of intestinal subepithelial myofibroblasts Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens The avian lung-associated immune system: a review Mzb1 protein regulates calcium homeostasis and integrin activation in innate-like B cells Identification of a novel cytokine-like transcript differentially expressed in avian γδ T cells Type I interferon (IFN)-regulated activation of canonical and non-canonical signaling pathways Pfister S., Kuettel V., Ferrero E. granulator: Rapid benchmarking of methods for *in silico* deconvolution of bulk RNA-seq data. R package version 1.12.0. Granulator. ([object Object]). https://doi.org/10.18129/B9.BIOC.GRANULATOR (2024) Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses Butyrate in energy metabolism: there is still more to learn Butyrate and the intestinal epithelium: modulation of proliferation and inflammation in homeostasis and disease Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells Endothelial aquaporin-1 (AQP1) expression is regulated by transcription factor Mef2c SP1-mediated upregulation of lncRNA LMCD1-AS1 functions a ceRNA for miR-106b-5p to facilitate osteosarcoma progression A genetically engineered waterfowl influenza virus with a deletion in the stalk of the neuraminidase has increased virulence for chickens Roseburia intestinalis: a beneficial gut organism from the discoveries in genus and species Roseburia intestinalis and its metabolite butyrate inhibit colitis and upregulate TLR5 through the SP3 signaling Pathway Roseburia intestinalis generated butyrate boosts anti-PD-1 efficacy in colorectal cancer by activating cytotoxic CD8 + T cells Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis Signals from the gut microbiota to distant organs in physiology and disease The gut microbiota and host innate immunity: regulators of host metabolism and metabolic diseases in poultry?1 1Presented as a part of the Informal Nutrition Symposium “Metabolic Responses to Nutrition and Modifiers” at the Poultry Science Association’s annual meeting in Athens Commensal gut microbiota can modulate adaptive immune responses in chickens vaccinated with whole inactivated avian influenza virus subtype H9N2 Comparative biology of germ-free and conventional poultry Nutritional and physiological characteristics in germ-free chickens The role of short-chain fatty acids in the interplay between diet The short-chain fatty acid acetate in body weight control and insulin sensitivity Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism T cell activation depends on extracellular alanine Dietary fiber confers protection against Flu by shaping Ly6c-patrolling monocyte hematopoiesis and CD8+T cell metabolism The relationship between dietary fiber intake and lung function in the National Health and Nutrition Examination surveys Ayala, A. V. et al. Commensal bacteria promote type I interferon signaling to maintain immune tolerance. https://www.biorxiv.org/content/10.1101/2021.10.21.464743v2 (2022) The Structure and immune regulatory implications of the ubiquitin-like tandem domain within an Avian 2’−5’ oligoadenylate synthetase-like protein Defense genes missing from the flight division Beyond ISGlylation: functions of free intracellular and extracellular ISG15 Commensal bacteria calibrate the activation threshold of innate antiviral Microbiota-driven tonic interferon signals in lung stromal cells protect from influenza virus infection Microbiota regulation of viral infections through interferon signaling Innate immune responses to avian influenza viruses in ducks and chickens Mimicking the passage of avian influenza viruses through the gastrointestinal tract of chickens Microbiota regulates immune defense against respiratory tract influenza A virus infection Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2 Butyrate reprograms expression of specific interferon-stimulated genes Inhibition of interferon gamma signaling by the short chain fatty acid butyrate Functions and mechanisms of non-histone protein acetylation Expanding duplication of free fatty acid receptor-2 (GPR43) genes in the chicken genome Sp1 phosphorylation and its regulation of gene transcription Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights Globaltest and GOEAST: two different approaches for Gene Ontology analysis g:Profiler—interoperable web service for functional enrichment analysis and gene identifier mapping (2023 update) Download references We express our gratitude to the staff of the Plate-forme d’Infectiologie Expérimentale (UE PFIE INRAE) for providing the necessary infrastructure and technical support essential for conducting the animal experiments in this study France (specifically Santé Animale—SA and Microbiologie et Chaine Alimentaire—MICA divisions) by “EuroFéRi” (FEDER-FSE Centre Val de Loire 2014-2020 contributed to data acquisition and analysis provided molecular and bioinformatics tools and participated in data analysis analysed and interpreted data using bioinformatics All authors contributed to writing the article and approved the submitted version The authors declare no competing interests Communications Biology thanks Mohamed Faizal Abdul-Careem reviewer(s) for their contribution to the peer review of this work Primary Handling Editors: Sabina Leanti La Rosa and Tobias Goris Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s42003-024-06815-0 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Clinical Reviews in Allergy & Immunology (2025) Sign up for the Nature Briefing: Anthropocene newsletter — what matters in anthropocene research, free to your inbox weekly. Volume 11 - 2020 | https://doi.org/10.3389/fimmu.2020.613079 This article is part of the Research TopicSensing DNA in Antiviral Innate ImmunityView all 14 articles The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) senses viral DNA as an essential component of the innate response cGAS initiates a range of signaling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING) the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions It is not currently clear in what contexts these disparate signaling outputs are activated by cGAS/STING and to what extent they cross-talk with each other Like other poxviruses the cytoplasmic replication cycle of FWPV exposes large amounts of foreign DNA to intracellular DNA sensing PRRs making cGAS a likely candidate for sensing FWPV infection and making FWPV a potentially useful tool for delineating nucleic acid sensing mechanisms in avian systems The mechanisms by which FWPV is sensed by PRRs during infection have not In this study we show the existence of a cGAS/STING pathway in chicken macrophages and determine its downstream signaling outputs Using cGAS and STING CRISPR/Cas9 knockout HD11 cells and pharmacological inhibitors of STING and TBK1 in primary macrophages we show that the activation of cGAS by intracellular DNA induces an IFN-I response and that this response can be enhanced by priming cells with IFNα we show that cGAS/STING signaling in macrophages can enhance transcription of specific immune recognition molecules including genes encoding the class II major histocompatibility complex (MHC-II) and co-stimulatory proteins Using FWPV mutants that are deficient in specific immunomodulators we are able to overcome the immunosuppression of wild type FWPV and show that this virus is sensed by cGAS resulting in IFN-I and MHC-II transcription These data show that the cGAS/STING/TBK1 pathway senses viral DNA in chicken macrophages and that this pathway regulates not only the antiviral interferon response but also modulates specific components of macrophage effector function machinery polyinosinic-polycytidylic acid (poly(I:C) 2’3’-cGAMP (Invivogen) and chicken interferon alpha (Yeast-derived Recombinant Protein Inc) were diluted in nuclease-free water (Ambion H-151 and BX795 (Invivogen) were diluted in DMSO following the manufacturer’s protocols HD11 cells, an avian myelocytomatosis virus (MC29)-transformed chicken macrophage-like cell line (15) Germany) complemented with 2.5% volume per volume (v/v) heat-inactivated foetal bovine serum (FBS; Sera Laboratories International Ltd) 2.5% volume per volume (v/v) chicken serum (New Zealand origin 10% Tryptose Phosphate Broth solution (Gibco 50 µg/ml of penicillin/streptomycin (P/S; Gibco Chicken embryonic fibroblasts (CEFs) (Pirbright Institute 5% CO2 and were grown in Dulbecco’s Modified Eagle Medium (DMEM) -F12 with Glutamax (Gibco) According to the MB21D1 (cGAS) and TMEM137 (STING) sequences obtained from the Ensembl database (release 94), single guide (sg)RNA sequences (Table 1) were designed targeting the catalytic domain (residues 11-13 and 109) and start of the open reading frame Genome editing of HD11 was performed using ribonucleoprotein (RNP) delivery. tracrRNA was mixed with the target specific sgRNA (Table 1) the tracrRNA/sgRNA mix was incubated with the Cas9 protein (IDT Belgium) and electroporation enhancer at 21°C The successfully edited populations (using guides cGAS sg3 and STING sg1) were diluted to a concentration of 0.5 cell/well and seeded in 96-well plates Individual clones were sequenced by MiSeq and the confirmed knockout clones were expanded for experiments Chicken bone marrow derived macrophages (BMDM) were generated as previously described (17) femurs and tibias of 4 week-old immunologically mature White Leghorn (PA12 line) outbred chickens were removed both ends of the bones were cut and the bone marrow was flushed with RPMI supplemented with P/S Cells were then washed and re-suspended in RPMI loaded onto an equal volume of Histopaque-1077 (Sigma-Aldrich Cells at the interface were collected and washed twice in RPMI were seeded in triplicates at 1×106 cells/ml in sterile 60 mm bacteriological petri dishes in RPMI supplemented with 10% FBS P/S and 25 ng/ml recombinant chicken colony stimulating factor 1 (CSF-1) (Kingfisher Biotech Half of the medium was replaced with fresh medium containing CSF-1 at day 3 adherent cells were harvested and cultured in RPMI supplemented with 10% FBS Procedures were performed in strict compliance with legal dispositions applicable in France which state that animal euthanasia for the only purpose of organ or tissue use is not considered as an experimental procedure with obligation of submission to ethics committee for approval (Ordinance 2013-118 published in the Journal Officiel de la République Française # 0032 of the February 7 cGAS and STING knockouts) were seeded in 12-well plates at a density of 3×105 cells/well the cells were transfected using TransIT-LT1 (Mirus Bio or 5 µg/ml) or Poly(I:C) (1 µg/ml) and harvested 6 or 16 h post-transfection IFNα (200 ng/ml) was added 16 h prior to transfection 2’3’ cGAMP was added at a concentration of 2.5 µg/ml and cells were harvested 6 h post-treatment BMDM were seeded in 6-well plates at 8x105 cells/ml cells were transfected using TransIT-LT1 with HT-DNA (2 µg/ml) IFNα (50 ng/ml) was added 16 h prior transfection to the cells supernatants 2’3’ cGAMP was added to cells supernatants at the concentration of 10 µg/ml and the cells were harvested 6 h post-treatment The presence of IFN-I in supernatants of stimulated BMDM was measured indirectly using a luciferase-based Mx-reporter bioassay (18) cells from the quail fibroblast cell line CEC32 carrying the luciferase gene under the control of chicken Mx promoter (kindly provided by Prof Germany) were seeded at 2.5×105 cells/well in 24-well plates and incubated at 41°C under 5% CO2 cells were incubated for 6 h with the diluted supernatants (1/10 of total volume) Medium was removed and cells were washed twice with PBS Cells were lysed using the Cell Culture Lysis Reagent (Promega according to the manufacturer’s instructions and luciferase activity was measured using the Luciferase assay reagent (Promega USA) and a GloMax-Multi Detection System (Promega BMDM or HD11 viability following different stimuli was assessed using the fluorescent DNA intercalator 7-aminoactinomycin D (7-AAD and the cells were harvested and washed in PBS Cells were stained according to the manufacturer’s protocol and the viability was analyzed by flow cytometry (BD FACS Calibur) Data were expressed as the percentage of 7AAD positive cells over total acquired events (50,000 cells) Cells were lysed by overlaying with 250 µl of lysis buffer containing 4 M guanidine thiocyanate and the solution was transferred to a silica column (Epoch Life Science USA) and centrifuged; all centrifugation steps were performed for 90 s at 16,600 g The bound RNA was washed by centrifugation with 500 µl of buffer containing 1 M guanidine thiocyanate followed by a double washing step with 500 µl of wash buffer 2 [25 mM Tris pH 7 and 70% (v/v) ethanol] RNA was eluted by centrifugation in 30 µl of nuclease-free water and the concentration was measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific Using 500 ng of RNA extracted from HD11 cells, cDNA was produced using SuperScript III reverse transcriptase, following the manufacture’s protocol (Thermo Scientific, Waltham, MA, USA). Samples were diluted in nuclease-free water in a 1:2.5 ratio. One μl of the diluted product was used for quantitative PCR (qPCR) in a final volume of 10 μl. qPCR was performed using SybrGreen Hi-Rox (PCR Biosystems Inc.) using primers described in Table 3 Fold change in mRNA expression was calculated by relative quantification using hypoxanthine phosphoribosyltransferase (HPRT) as endogenous control Total RNA (up to 1 µg per reaction) from BMDM was reverse transcribed with iScript cDNA synthesis kit (Bio-Rad Quantitative PCR was performed using 1 µl of cDNA 5 µl of iQ SYBR Green Supermix (Bio-Rad 0.25 µl of each primer pair and 3.5 µl of nuclease-free water in a total reaction volume of 10 µl Fold-increase in gene expression was calculated by relative quantification using HPRT and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous controls HD11 WT cells were seeded at a confluence of 3x105 cells/ml in 12-well plates The cells were primed with IFNα for 16 h and then with transfected exogenous DNA (HT- and CT-DNA – 2 μg/ml) or treated with 2’3’cGAMP (5 μg/ml) for 6 h the cells were incubated with Zymosan coated beads conjugated with FITC at a ratio of 30 beads to 1 cell for all conditions for 40 min at 37°C The cells were wash two times in PBS and fixed in suspension using the solution (missing ref; BD Biosciences) with 4% PFA Cell populations were counted by analysis on a CytoFLEX cytometer Fowlpox WT (FP9) and mutants [FPV012 (19) and FPV184 (20)] were propagated in primary chicken embryonic fibroblasts (CEFs) and grown in DMEM-F12 (Thermo Fisher Scientific Ten-fold dilutions of cell supernatants were prepared in serum-free DMEM-F12 and used to inoculate confluent monolayers of CEFs for 1.5 h at 37°C Cells were then overlaid with 2xMEM : CMC (1/1 ratio) The foci were counted 7 days later after staining with Toluidine Blue HD11 cells were seeded in 12-well plates in the day prior infection Fowlpox viruses were diluted in serum-free DMEM-F12 at a multiplicity of infection (MOI) of 3 and added in the cells (1 ml per well) Infected cells and supernatants were collected from infections at 8 and 24 h post-infection Prism 7 (GraphPad) was used to generate graphs and perform statistical analysis Data were analyzed using an unpaired t test with Welch’s correction unless stated otherwise Data with P < 0.05 was considered significant and 2-tailed P-value were calculated and presented as: *p < 0.05 Each experiment has at least two biological replicates unless stated indicating that this response is present in both primary macrophages and the transformed monocytic HD11 cell line Figure 1 Intracellular DNA activates an IFN-I response in chicken macrophages (A) HD11 cells were transfected with HT-DNA (1 CT-DNA (5 µg/ml) or Poly(I:C) and transcription of IFNB and ISG12.2 measured by qRT-PCR 6 h later (B) Chicken BMDM were transfected with HT- DNA CT-DNA (2 µg/ml) or Poly(I:C) (1 µg/ml) and transcription of IFNB and ISG12.2 measured by qRT-PCR 6 h later (C) Resting BMDMs or BMDMs primed with IFNα for 6 h were transfected with HT- DNA CT-DNA (2 µg/ml) or Poly(I:C) (1 µg/ml) and interferon activity in the supernatants was measured after 24 h using a bioassay IFNα stimulation is a positive control in this assay (D) HD11 or BMDM were primed with IFNα for 6 h or Poly(I:C) and transcription of IFNB and ISG12.2 measured by qRT-PCR 6 h later (E) HD11 (top) or IFNα-primed BMDM (bottom) were transfected with HT-DNA or Poly(I:C) and cell viability measured by 7AAD staining 24 or 48 h later ***p < 0.001; ****p < 0.0001; ns: no significant difference Data is representative of two or more replicates cell death is likely not a specific output of STING signaling key molecules involved in T cell activation by macrophages are regulated by DNA stimulation but not all macrophage effector functions are equally enhanced by this signal Figure 2 Intracellular DNA stimulates transcription of MHC-II and co-stimulatory molecules (A) BMDMs or (B) HD11 cells were transfected with HT-DNA and CD86 measured by qRT-PCR 6 h later (C) HD11 cells were stimulated with HT-DNA or Poly(I:C) and 6 h later phagocytosis was monitored by FITC-conjugated Histograms of non-treated versus treated cells (left panels) and respective percentages of FITC positive cells for each treatment tested (right panel) are presented Figure 3 STING and TBK1 contribute to DNA-driven transcriptional responses in chicken BMDMs (A) HD11 and (B) BMDM cells were treated with 2’3’cGAMP (10 μg/ml) and qRT-PCR carried out 6 h later for the indicated genes (C) BMDM were treated with the STING inhibitor H-151 (10 uM) or TBK1 inhibitor BX795 (1 uM) for 1 h before transfection with HT-DNA and CT-DNA RNA was extracted and qRT-PCR carried out for the indicated genes (D) BMDM were treated with the STING inhibitor H-151 (10 uM) or TBK1 inhibitor BX795 (1 uM) for 1 h before treatment with 2’3’cGAMP (10 μg/ml) (E) HD11 cells were treated with 2’3’cGAMP (2.5 µg/ml) 6 h later phagocytosis was monitored by FITC-conjugated These data confirm the intracellular DNA PRR function of cGAS in chicken macrophages Figure 4 cGAS is essential for intracellular DNA-dependent IFN-I and MHC-II transcription in HD11 cells (A) Example of identification of indel in clonally selected HD11 cGAS KO using NGS sequencing (B) WT and cGAS KO HD11 cells were transfected with HT-DNA CT-DNA (2 μg/ml) or Poly(I:C) (1 μg/ml) for 6 h and transcription of the indicated genes measured by qRT-PCR (C) cGAS KO HD11 cells were primed with IFNα for 6 h or Poly(I:C) and transcription of IFNB and ISG12.2 measured by qRT-PCR 6 h later (D) WT or cGAS KO cells were treated with 2’3’cGAMP (10 μg/ml) and transcription of IFNB measured by qRT-PCR 6 h later In parallel, using the same methodology, we generated multiple STING knockout HD11 cell lines (Figure 5A). Stimulation of these cells with DNA phenocopied the cGAS knockout lines, and neither STING or cGAS KO altered tonic IFNB transcription, confirming the function of chicken STING downstream of cGAS in the intracellular DNA sensing pathway (Figure 5B, Supplementary Figures 4 and 5) These data are consistent with the presence of a cGAS/STING pathway in HD11 cells and in concert with the data using H151 in BMDMs indicate the function of STING as a critical adaptor protein for intracellular DNA sensing in chicken macrophages Figure 5 STING is essential for intracellular DNA-dependent IFN-I transcription in HD11 cells (A) Example of identification of indel in clonally selected HD11 STING KO using NGS sequencing (B) WT and STING KO HD11 cells were transfected with HT-DNA CT-DNA (2 μg/ml) or Poly(I:C) (1 μg/ml) for 6 h and transcription of the indicated genes measured by qRT-PCR 6 h later Figure 6 Fowlpox triggers a cGAS/STING dependent DNA sensing pathway in HD11 cells (A) HD11 cells were infected with FWPV strain FP9 at a multiplicity of infection of three cGAS or STING KO cells were infected with FP9 (black bars) (D) HD11 cells were infected with FWPV strain FP9 at a multiplicity of infection of three RNA was extracted and qRT-PCR carried out for the indicated FWPV genes cGAS or STING KO cells were infected with FP9 or FPV184 at a multiplicity of infection of three cell supernatants harvested and released FP9 was titrated on CEFs to measure focus forming units (ffu) per ml The ability of innate immune cells to detect virus infection is dependent on a set of PRRs that directly bind viral nucleic acids. Macrophages act in this context as tissue-resident sentinel sensors of infection that express a broad repertoire of PRRs and mount a rapid and robust innate immune response to viruses and other pathogens. Indeed intracellular DNA sensing was first described in macrophages (25) As well as interferon and cytokine production activated macrophages use effector functions for pathogen clearance and for activation of adaptive immunity In mammalian systems the signaling outputs downstream of intracellular DNA detection in macrophages include IRF-dependent IFN and cytokine production and cell death driven by the AIM2 inflammasome we find that intracellular DNA sensing produces IFN but does not result in measurable cell death rather it upregulates a specific set of antigen presentation machinery including the MHC-II gene BLB1 and co-stimulatory molecules providing a direct link between anti-viral innate sensing and the initiation of adaptive immunity Here we show that the cGAS/STING pathway in chicken macrophages can sense FWPV infection and is responsible for the IFN-I response as well as for upregulation of BLB1 but it remains to be seen how individual PRRs like cGAS contribute to protection against FWPV infection in vivo In mice cGAS/STING are essential for the host response to poxvirus infections such as vaccinia and ectromelia viruses and the protective effects are meditated via IFN-I despite the presence of large numbers of immunomodulators targeting PRR signaling including the cGAS/STING systems in these viruses As such it is likely that cGAS/STING mediated FWPV DNA sensing and IFN-I production has a significant contribution to host defence against FWPV infection in chickens although mechanistically it remains to be seen exactify how IFN-I mediates this defence against this avian poxvirus Using CRISPR/Cas9 technology to knockout STING and cGAS in a transformed monocytic cell line (HD11) and complementing these data in primary macrophages with pharmacological inhibitors we have been able to show this cGAS/STING/TBK1 pathway is active in chicken macrophages The use of primary cells in this context is important as transformation or immortalization can significantly alter PRR pathways so as to obscure physiological signaling mechanisms Our data adds to the list of chicken cGAS/STING functions in sensing of avian DNA viruses such as MDV and Adenovirus 4 that replicate in the nucleus or FWPV that replicates in the cytoplasm and in the regulation of macrophage effector functions The ability of this pathway to sense a broad range of DNA viruses that replicate in different compartments in avian innate immune cells indicates that this pathway is a primary DNA sensing mechanism for DNA viruses in chickens The raw data supporting the conclusions of this article will be made available by the authors BF and CB provided the funding and supervised the work and EK performed the experiments and statistical analysis SG and MS generated the mutant fowlpox viruses RG designed the study and wrote the manuscript All authors contributed to the article and approved the submitted version This work was funded by BBSRC grants BB/S001336/1 (BF and CB) BB/H005323/1 & BB/K002465/1 (MS) and by EUROFERI (Région Centre-Val-de-Loire The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest This manuscript has been released as a Pre-print at BioRxiv We thank the experimental facility PFIE (Plateforme d’Infectiologie Expérimentale France) for providing the animals used for the isolation of primary macrophages The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.613079/full#supplementary-material Supplementary Figure1 | Effect of IFNα priming on expression levels of STING and IRF7 in BMDM and HD11 BMDM or HD11 cells were treated with IFNα for 6 h and transcription of STING and IRF7 measured by qRT-PCR 6 h later Supplementary Figure 2 | BLB2 but not BLB1 is an ISG in HD11 cells HD11 cells were treated with IFNα for 6 h and the indicated genes were measured by qRT-PCR Supplementary Figure 3 | cGAS is essential for intracellular DNA-dependent IFN-I transcription in HD11 cells WT or three individual cGAS knockout clones with different indels were stimulated with HT-DNA (2 μg/ml) and IFNB transcription measured by qRT-PCR 6 h later Supplementary Figure 4 | STING is essential for intracellular DNA-dependent IFN-I transcription in HD11 cells Supplementary Figure 5 | STING or cGAS loss does not significantly alter tonic IFN-I transcription IFNB transcription measured by qRT-PCR in WT cGAS KO or STING KO HD11 cells (data shown relative to WT cells) Intracellular sensing of viral DNA by the innate immune system International union of basic and clinical pharmacology Pattern recognition receptors in health and disease The cGAS–cGAMP–STING pathway connects DNA damage to inflammation Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects Can innate immune system targets turn up the heat on “cold” tumours CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Aicardi-Goutieres syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway PubMed Abstract | CrossRef Full Text | Google Scholar The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3 Signalling strength determines proapoptotic functions of STING Host restriction factor SAMHD1 limits human T cell leukemia virus type 1 infection of monocytes via STING-mediated apoptosis Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence Spotlight on avian pathology: fowlpox virus PubMed Abstract | CrossRef Full Text | Google Scholar Type I interferons mediate the innate cytokine response to recombinant fowlpox virus but not the induction of plasmacytoid dendritic cell-dependent adaptive immunity Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation Designer nuclease-mediated generation of knockout THP1 cells CrossRef Full Text | Google Scholar The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge Synthesis of IFN-β by Virus-Infected Chicken Embryo Cells Demonstrated with Specific Antisera and a New Bioassay J Interferon Cytokine Res (2004) 24:179–84 Genetic Screen of a Mutant Poxvirus Library Identifies an Ankyrin Repeat Protein Involved in Blocking Induction of Avian Type I Interferon Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus’ Lateral Body Protein Positive feedback regulation of type I interferon by the interferon-stimulated gene STING What chickens might tell us about the MHC class II system PubMed Abstract | CrossRef Full Text | Google Scholar cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING Recognition of cytosolic DNA activates an IRF3-dependent innate immune response PubMed Abstract | CrossRef Full Text | Google Scholar Induction of interferon and cell death in response to cytosolic DNA in chicken macrophages Inhibition of DNA-Sensing Pathway by Marek’s Disease Virus VP23 Protein through Suppression of Interferon Regulatory Factor 7 Activation Severe histiolymphocytic and heterophilic bronchopneumonia as a reaction to in ovo fowlpox vaccination in broiler chicks Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α) Advances in avian immunology-prospects for disease control: A review PubMed Abstract | CrossRef Full Text | Google Scholar a new member of the interferon regulatory factor (IRF) family that is rapidly and transiently induced by dsRNA Nucleic Acids Res (1995) 23:2137–46 IRF7 Is Involved in Both STING and MAVS Mediating IFN-β Signaling in IRF3-Lacking Chickens The mechanism of transcriptional synergy of an in vitro assembled interferon-β enhanceosome NEMO–IKKβ Are Essential for IRF3 and NF-κB Activation in the cGAS–STING Pathway The herpesviral antagonist m152 reveals differential activation of STING -dependent IRF and NF-κB signaling and STING’s dual role during MCMV infection Avian oncogenic herpesvirus antagonizes the cGAS-STING DNA-sensing pathway to mediate immune evasion Cyclic GMP-AMP synthase is essential for cytosolic double-stranded DNA and fowl adenovirus serotype 4 triggered innate immune responses in chickens Int J Biol Macromol (2020) 146:497–507 Toll-like receptor 3 promotes cross-priming to virus-infected cells Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination Toll-Like Receptor Ligands Modulate Dendritic Cells to Augment Cytomegalovirus- and HIV-1-Specific T Cell Responses Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP Cancer-Cell-Intrinsic cGAS Expression Mediates Tumor Immunogenicity Bryant CE and Ferguson BJ (2021) Chicken cGAS Senses Fowlpox Virus Infection and Regulates Macrophage Effector Functions Received: 01 October 2020; Accepted: 16 December 2020;Published: 01 February 2021 Copyright © 2021 Oliveira, Rodrigues, Guillory, Kut, Giotis, Skinner, Guabiraba, Bryant and Ferguson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Brian J. Ferguson, YmYyMzRAY2FtLmFjLnVr †These authors share senior authorship Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish. The 2 winning projects of the first €1 million Adisseo Research Grant were: The 1st Adisseo Research Grant launched in mid-May and aims to encourage international labs to collaborate on significant challenges facing the feed industry The most recent edition of the Dutch Pork Expo and the Dutch Poultry Expo in the Netherlands presented 250 exhibitors jointly visited by 5,300 people The Smart Animal Farming knowledge platform was launched during the events which will be expanded further for future editions The next Dutch Pork Expo will be held from 14-15 February 2023 in Den Bosch Tyson Foods China’s broiler flock achieved an EPEF of 426.5 while Cargill Animal Protein (Anhui) achieved an EPEF of 413.5 Otto & Sons (Weihai) achieved 151.40 chicks per hen housed at 65 weeks De Heus Vietnam has signed an agreement with Masan to obtain control of 100% of the feed-related business of MNS Feed (100% ANCO and 75.2% Proconco) which covers 13 feed mills and 1 premix plant (total capacity of nearly 4 mmt) This will become the largest animal nutrition company active in the Vietnamese independent feed market Strategic supply agreements will also see De Heus supply pigs and feed to Masan ADM has opened its new animal nutrition laboratory in Rolle the lab has been established to support the development of science-based feed additives for livestock species helping to improve on-farm performance or to help with progressing to the next stage of scale Volume 4 - 2017 | https://doi.org/10.3389/fvets.2017.00226 Lipid mediators are known to play important roles in the onset and resolution phases of the inflammatory response in mammals The phospholipid platelet-activating factor (PAF) is a pro-inflammatory lipid mediator which participates in vascular- and innate immunity-associated processes by increasing vascular permeability by facilitating leukocyte adhesion to the endothelium and by contributing to phagocyte activation PAF exerts its function upon binding to its specific receptor which is abundantly expressed in leukocytes and endothelial cells (ECs) lipid mediators and their functions are still poorly characterized and the role of PAF as an inflammatory mediator has not yet been investigated In the present study we demonstrate that primary chicken macrophages express PAFR and lysophosphatidylcholine acyltransferase 2 (LPCAT2) the latter being essential to PAF biosynthesis during inflammation exogenous PAF treatment induces intracellular calcium increase and increased phagocytosis by primary chicken macrophages in a PAFR-dependent manner We also show that PAF contributes to the Escherichia coli lipopolysaccharide (LPS)-induced pro-inflammatory response and boosts the macrophage response to E coli LPS via phosphatidylinositol 3-kinase/Akt- and calmodulin kinase II-mediated intracellular signaling pathways Exogenous PAF treatment also increases avian pathogenic E coli intracellular killing by chicken macrophages and PAFR and LPCAT2 are upregulated in chicken lungs and liver during experimental pulmonary colibacillosis exogenous PAF treatment increases cell permeability and upregulates the expression of genes coding for proteins involved in leukocyte adhesion to the endothelium in primary chicken endothelial cells (chAEC) PAF boosts the chAEC inflammatory response to bacteria-associated molecular patterns in a PAFR-dependent manner we identified PAF as an inflammation amplifier in chicken macrophages and ECs which suggests that PAF could play important roles in the endothelium-innate immunity interface in birds during major bacterial infectious diseases such as colibacillosis All these physiological or pathological processes have been well characterized in mammals no study has addressed the putative regulatory roles for PAF in chickens during inflammation or infection In line with that, PAF has been implicated in several animal models of lung injury, and treatment with PAF receptor antagonists attenuates endotoxin-induced lung injury (36). During pulmonary Gram-negative bacteria infection, PAF production is important for bacteria clearance (37). However, during experimental influenza A virus infection in mice, PAF plays a deleterious role by amplifying inflammation and tissue damage (38) Despite a growing interest in characterizing early mediators of inflammation in chickens and more notably their crosstalk with innate immunity sentinel cells such as macrophages no previous works have addressed the mechanisms by which lipid mediators may contribute to inflammation or bacterial clearance we aimed to characterize the PAF/PAFR interactions and to shed light on their functions in chicken macrophages and endothelial cells (ECs) especially with respect to how PAF system molecules are modulated during pulmonary colibacillosis Platelet-activating factor (synthetic PAF C16:0 β-acetyl-γ-O-hexadecyl-l-α-phosphatidylcholine hydrate and 2′,7′-dichlorofluorescein diacetate (DCFDA ≥95% HPLC purity tested) were purchased from Sigma-Aldrich >95% HPLC purity tested) and WEB 2086 (Apafant PI3K/Akt inhibitor wortmannin (WMN; SL-2052 and calcium calmodulin kinase II (CaMK II) inhibitor KN62 (C38H35N5O6S2 >98% HPLC purity tested) were purchased from Tocris Biotechne White leghorn chickens from the GB1 Athens inbred line (B13/B13 histocompatible chickens) or from the PA12 outbred line were hatched and raised specific-pathogen-free (SPF) at INRA (Plate-Forme d’Infectiologie Expérimentale B13/B13 histocompatible chickens were housed in BSL2 poultry isolator units HD11, an avian myelocytomatosis virus (MC29)-transformed chicken macrophage-like cell line (39) UK) supplemented with 10% heat-inactivated fetal calf serum (FCS and 100 µg/ml streptomycin (all from Sigma-Aldrich UK) and grown routinely in a 75-cm2 flask (Corning Chicken bone marrow-derived macrophages (BMDM) were outgrown from bone marrow cells using recombinant chicken macrophage colony-stimulating factor 1 (CSF-1) (40) derived from COS-7 cells (fibroblast-like cell line derived from monkey kidney tissue USA) transfected with a pTArget vector (Promega UK) expressing chicken CSF-1 (kindly provided by Prof femurs and tibia of 3–4-week-old B13/B13 histocompatible or PA12 chickens were removed and the marrow flushed with sterile phosphate-buffered saline (PBS loaded onto an equal volume of Histopaque 1.077 gradient (Sigma-Aldrich and centrifuged at 250 g for 30 min without brake and cultured at a final concentration of 1 × 106 cells/ml in sterile 60 mm bacteriological plates (Corning USA) in prewarmed complete RPMI-1640 medium supplemented with COS-7 supernatant containing chicken CSF-1 and 100 µg/ml streptomycin at 40°C under 5% CO2 Half of the medium was replaced with fresh pre-warmed complete medium containing chicken CSF-1 at day 4 adherent cells were washed and harvested in cold PBS containing 2 mM ethylenediaminetetraacetic acid (EDTA and resuspended in complete RPMI-1640 medium All adherent cells presented macrophage-like morphology as evaluated in glass slides prepared in a CytoSpin® Cytocentrifuge (Shandon 97% of these cells were KUL01+ cells (a chicken macrophage marker) as evaluated by Flow Cytometry using a mouse anti-chicken monoclonal antibody (1:200 dilution UK) and a rat-anti mouse phycoerythrin-coupled secondary antibody (1:300 dilution Peripheral blood mononuclear cells (PBMC) were isolated from B13/B13 histocompatible chickens as previously described (41) peripheral blood collected in EDTA-coated tubes was mixed with 1% methylcellulose (Sigma-Aldrich 1:1 v/v) and centrifuged at 25 g for 15 min The upper phase was then diluted with PBS to the original volume of blood/methylcellulose mixture carefully layered onto a Histopaque 1.077 gradient and centrifuged at 250 g for 30 min without brake and resuspended in complete RPMI-1640 medium for further incubation at 40°C under 5% CO2 adherent cells presented monocyte/macrophage-like morphology as evaluated in glass slides prepared in a CytoSpin® Cytocentrifuge (Shandon Primary chicken aortic endothelial cells (chAEC) were prepared from specific-pathogen-free (SPF) 18-day-old PA12 chicken embryos as previously described (42) Primary chAEC were serially passaged (p2–p5) using 0.05% trypsin-EDTA (Gibco UK) and kept in EC growth medium (EGM-2 BulletKit Twelve-well tissue culture plates (Corning USA) were seeded at 7 × 105 cells per well of HD-11 macrophage-like cells or primary chAEC in complete medium and incubated at 40°C under 5% CO2 overnight prior to different stimulation protocols LPS (10 ng/ml) and hPAF-AH (10 µg/ml) were previously diluted in RPMI-1640 and KN62 (1 µM) were previously diluted in DMSO Dead bacteria [DB; APEC strain BEN2908 at 10 multiplicity of infection (MOI) 15 min incubation at 65°C for killing] were previously diluted in LB medium Final concentration of DMSO (the vehicle used for the dilution of PAF and cell signaling inhibitors) in cell culture wells never exceeded 0.1% Treatment with PAF receptor antagonists or intracellular signaling pathway inhibitors was performed 1 h prior to stimulation Cell culture supernatants were recovered 6 h after stimulation (unless otherwise indicated) and stocked at −20 or −80°C for subsequent analyses specified below Cells were washed in sterile PBS and lysed with RNA lysis buffer (Macherey-Nagel Germany) containing 2-mercaptoethanol (Merck Millipore and stocked at −80°C until RNA extraction For total protein quantification and Western blot (WB) analysis cells were washed in PBS followed by cell lysis using RIPA buffer containing a cocktail of protease inhibitors which also included phenylmethylsulfonyl fluoride and sodium orthovanadate (Santa Cruz Biotechnology Primer pairs used in the present study for quantitative real-time PCR analysis The Fluo-4 NW Calcium Assay kit (Molecular Probes USA) was used to measure changes in HD11 cells’ intracellular calcium levels upon different stimuli HD11 cells at 3 × 104 cells per well in a black 96-multiwell plate (Corning USA) were loaded with 100 µl of the dye loading solution containing Fluo-4 NW dye and probenecid according to the manufacturer’s instructions The 96-well plate was incubated at 40°C under 5% CO2 for 30 min in the dark and the stimuli of interest were added to the cells at T = 0 The changes in Fluo-4 NW fluorescence were measured at an excitation wavelength of 494 nm and an emission wavelength of 516 nm in a GloMax®-Multi Detection System plate reader (Promega Calcium mobilization was recorded over time (213 s To evaluate the pro-apoptotic and cytotoxic effects of different stimuli cells were cultured as previously mentioned Apoptotic/late apoptotic cells were identified using annexin V and propidium iodide (PI) double-positive staining strategy and resuspended in an annexin-binding buffer (BD Biosciences) annexin V-fluorescein isothiocyanate (FITC) was added to the cells for 30 min the cells were further incubated with PI (BD Biosciences) for 15 min stained cells (50,000 acquired events over total gated cells) were analyzed immediately using a BD FACSCalibur™ (BD Biosciences) cell viability was determined using the colorimetric methylthiazoletetrazolium bromide (MTT) assay (Sigma-Aldrich UK) that measures metabolic activity as a surrogate for cell viability/cytotoxicity MTT was added to a final concentration of 5 μg/ml per well and cells were incubated for 2 h at 40°C under 5% CO2 After complete solubilization of the dye using DMSO plates were read at 550 nm in a Multiskan Ascent plate reader (Thermo Fisher Scientific Caspase 3/7 activity was carried out using the Caspase-Glo 3/7 assay kit (Promega UK) according to the manufacturer’s protocol BMDM at 3 × 104 cells per well in a black 96-multiwell plate (Corning USA) were incubated with different stimuli for 6 h 100 µl of Caspase-Glo 3/7 reagent was added to the wells Plates were gently shaken and then incubated in the dark at 20°C for 60 min before luciferase activity was recorded using a GloMax®-Multi Detection System plate reader (Promega The cell-penetrating dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) is oxidized by ROS to yield the fluorescent molecule 2′7′-dichlorofluorescein (DCFDA) which is retained intracellularly and visible at the 488 nm excitation laser line To evaluate ROS production upon different stimuli BMDM were cultured as previously described and incubated with 10 µM H2DCFDA (Sigma-Aldrich and cells receiving different stimuli were kept at 40°C under 5% CO2 for 2 h Cells were then collected and washed in ice-cold PBS prior to flow cytometry analysis (50,000 acquired events over total gated cells) using a BD FACSCalibur™ (BD Biosciences For the identification of PAFR expression in BMDM and incubated for 2 h at 40°C under 5% CO2 washed in FACS buffer (2 mM EDTA and 2% FCS in PBS) and stained with a rabbit polyclonal antibody raised against amino acids 1–300 of PAFR of human origin (Santa Cruz Biotechnology and stained with a goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology and analyzed by flow cytometry (100,000 acquired events over total gated cells) using a BD FACSCalibur™ (BD Biosciences Bone marrow-derived macrophages or HD11 cells were grown at 2 × 105 cells per well on sterile glass coverslips placed in 24-well plates containing complete medium and incubated at 40°C under 5% CO2 overnight After adding the stimuli of interest for 2 h cells were washed in PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich permeabilized with 0.5% Triton X-100 (Sigma-Aldrich and 2% bovine serum albumin (Sigma-Aldrich Immunostaining was performed with a rabbit polyclonal antibody raised against amino acids 1–300 of PAFR of human origin or rabbit polyclonal IgG as an isotype control (Santa Cruz Biotechnology Secondary antibody goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology Cell nuclei were counterstained with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Cells were observed under an Axiovert 200 M inverted epifluorescence microscope equipped with the Apotome imaging system (Zeiss Images were captured with an Axiocam MRm camera and analyzed using the Axiovision software (Zeiss Total protein from BMDM cell lysates was quantified using a Quick Start™ Bradford Protein Assay (Bio-Rad Thirty micrograms of protein from whole cell lysates were mixed with 2× electrophoresis sample buffer (Santa Cruz Biotechnology proteins were separated using 4–15% Mini-PROTEAN TGX™ Precast gels (Bio-Rad USA) and transferred onto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot® Turbo™ Mini PVDF Transfer pack (Bio-Rad USA) in a Trans-Blot® Turbo™ Transfer system (Bio-Rad The membranes were blocked in a buffer containing 5% non-fat dry milk in PBS for 1 h Primary antibody targeting PAFR was the same as utilized for immunofluorescence (IF) staining at 1:500 dilution a rabbit anti-Akt IgG polyclonal antibody (detecting endogenous levels of total Akt1 and Akt3 proteins—Cell Signaling Technology a rabbit anti-CaMK II IgG polyclonal antibody (epitope corresponding to amino acids 1–300 mapping at the N-terminus of CaMK IIα of human origin Mouse anti-glyceraldehyde-3-phosphate dehydrogenase IgG1 monoclonal antibody (GAPDH Germany) was utilized as internal loading control at 1:1,000 dilution primary antibodies diluted in 5% non-fat dry milk in PBS were added to the membranes for incubation overnight at 4°C under agitation Following reaction with the primary antibodies membranes were thoroughly washed in wash buffer containing 0.1% Tween 20 (Sigma-Aldrich UK) in PBS and labeled with HRP-conjugated secondary antibodies (goat anti-rabbit or rabbit anti-mouse the antigens were visualized using a Clarity™ ECL Western Blotting Substrate (Bio-Rad USA) in a chemiluminescence FUSION FX apparatus (Vilber Lourmat Bone marrow-derived macrophages were seeded at 2 × 105 cells per well in 24-well tissue culture plates treated with different stimuli and immediately incubated with 10 µg/ml of Zymosan A (Saccharomyces cerevisiae) BioParticles™ Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific USA) in complete medium at 4°C or 40°C under 5% CO2 for 1 h washed twice with ice-cold EDTA-containing PBS Data were expressed as the percentage difference (delta) between fluorescence positive cells and cells incubated at 40°C (active phagocytosis) and 4°C (limited phagocytosis) Nitrite (NaNO2) content was used as an index of nitric oxide (NO) production by chicken macrophages Nitrite levels were determined in supernatants of BMDM or HD11 cell cultures by spectrophotometry using the Griess reagent system (Promega 50 µl of the samples were added to 50 µl of freshly prepared Griess reagent in a 96-well plate The absorbance was read at 550 nm in a Multiskan Ascent plate reader (Thermo Fisher Scientific The nitrite concentration was calculated using a sodium nitrite standard curve HD11 cells were seeded in 12-well plates at 5 × 105 cells per well and incubated at 40°C under 5% CO2 overnight. APEC strains BEN2908 [O2:K1:H5, a nalidixic acid-resistant derivative of strain MT78 which was isolated from the trachea of a chicken with respiratory infection (44)] and BEN3421 (O2:K1:H4 a nalidixic acid- and tetracyclin-resistant strain isolated from a chicken presenting colibacillosis in 2009 in France) were utilized for infection Cell monolayers were infected with mid-log-phase bacteria at an MOI of 10 and incubated for 1 h in cell culture medium without FCS Cells were washed and remaining extracellular bacteria were killed by incubation in complete medium containing gentamicin (100 µg/ml) for 1 h 30 min A group of cells were then washed and lysed with sterile water for 30 min at 4°C to evaluate the percentage of invasion through bacterial enumeration by viable counts on LB agar plates Percentage of invasion (T = 0) was calculated as the number of intracellular bacteria divided by the number of bacteria in the inoculum and gentamicin concentration was reduced to 10 µg/ml (a concentration known to kill BEN2908 and BEN3421) to avoid putative accumulation within cells during prolonged incubation PAF (1 or 10 µM) was added together with gentamicin supernatant was recovered for nitrite dosage and stocked at −20°C Cells were washed and lysed with RNA lysis buffer or sterile water for 30 min at 4°C Intracellular bacteria were enumerated by viable counts on LB agar plates Percentage of growth was calculated as the number of intracellular bacteria at a given time point divided by the number of bacteria at T = 0 Four-week-old B13/B13 histocompatible chickens were infected intratracheally with 109 colony-forming units of the highly adhesive/invasive APEC strain BEN2908 in 200 µl of sterile endotoxin-free NaCl 0.9% solution The inoculum was prepared with bacteria in the mid-log-phase of growth The control group was inoculated with sterile NaCl 0.9% solution Lung and liver samples were collected aseptically from each chicken 6 h post bacterial inoculation Tissue samples for RNA extraction were snap frozen in liquid nitrogen and stocked at −80°C placed in gentleMACS™ C tubes containing sterile PBS and homogenized using a gentle MACS™ Dissociator (Miltenyi Biotec Dilutions of lung homogenates were plated onto Drigalski agar plates supplemented with nalidixic acid (30 µg/ml) for bacterial quantification and 1 ml was incubated in brain heart infusion for qualitative detection of E Primary chAEC were seeded at a density of 5 × 104 cells on fibronectin-coated Falcon® Permeable Support (8.0 µm pore) PET membranes (Corning placed in 24-well plates containing EC medium and cultured for 72 h at 40°C under 5% CO2 chAEC monolayers in the upper chamber received different stimuli and EC growth medium containing 1 mg/ml FITC-dextran (40,000 MW) (Sigma-Aldrich UK) was added followed by further incubation for 30 min at 40°C under 5% CO2 50 µl were collected from the lower chamber and fluorescence signals relating to FITC-dextran passage from the upper to the lower chamber were measured using a GloMax®-Multi Detection System plate reader (Promega Cell layer permeability was expressed as fold increase in FITC-dextran fluorescence as compared to the control group (vehicle) and Gallus gallus: XP_004947758) and LPCAT2 (H Comparisons between two groups were performed using a two-tailed unpaired Student’s t test Multiple groups were compared using a one-way analysis of variance followed by a Newman–Keuls multiple comparison post hoc test Values for all measurements are expressed as mean ± SEM P < 0.05 was considered statistically significant Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Data are representative of at least two independent experiments Chicken PAFR also contains two conserved predicted sites for N-linked glycosylation and nine threonine (T) or serine (S) residues identified as potential sites for phosphorylation by protein kinase A or C (Figure S1 in Supplementary Material) Components of the platelet-activating factor (PAF) system are expressed in chickens and exogenous PAF induces intracellular calcium increase in chicken macrophages (A) The gene expressions of PAF receptor (PAFR)/PTAFR and (B) LPCAT2/LPCAT2 were evaluated by quantitative real-time PCR (qRT-PCR) in unstimulated chicken tissues and macrophages [HD11 macrophage-like cells bone marrow-derived macrophages (BMDM) and peripheral blood mononuclear cells (PBMC)] (C) PAFR/PTAFR gene expression was evaluated by qRT-PCR in purified bone marrow cells following chCSF-1 medium complementation for BMDM differentiation from days 0 to 7 Data are expressed as relative normalized expression (as compared to two housekeeping genes) In (D,E) the HD11 cell line was stimulated with PAF and/or PAFR antagonists (PCA 4248 and WEB 2086) and the increase in intracellular calcium signal was recorded over time (216 s This suggests that PAF seems to act in a specific receptor (PAFR)-mediated fashion in chicken macrophages as in mammalian cells these data revealed that PAFR is expressed at the protein level and can be downregulated and partially degraded by PAF in chicken macrophages Platelet-activating factor receptor (PAFR) protein expression in chicken primary macrophages can be modulated by exogenous platelet-activating factor (PAF) or lipopolysaccharide (LPS) (A) Immunofluorescence (IF) analysis of PAFR protein expression using an anti-PAFR polyclonal antibody in B13/B13 histocompatible chicken bone marrow-derived macrophages (BMDM) BMDM were stimulated with PAF (10 µM) Escherichia coli LPS (10 ng/ml) or PCA 4248 (10 µM) for 2 h before IF analysis PAFR expression is shown in green (Alexa-Fluor 488-conjugated secondary antibody) and the nucleus in blue (DAPI staining) (B) Representative flow cytometry dot plots showing gating strategy for PAFR expression analysis in BMDM and histogram showing PAFR expression upon BMDM stimulation for 2 h with PAF and/or PCA 4248 as compared to isotype control (C) Western blot analysis of PAFR expression in BMDM whole cell lysates following different treatments for 2 h Target protein size is indicated with black arrows (D) PAFR/PTAFR and LPCAT2/LPCAT2 gene expression analysis in BMDM stimulated with PAF or dead bacteria (10 multiplicity of infection) for 6 h Data are expressed as relative normalized expression (as compared to vehicle control group) IF images were captured using an Axiovert 200 M inverted epi-fluorescence microscope equipped with an EC Plan-Neofluar 40 × /1.3 oil/Dic objective (ApoTome system **P < 0.01 when compared to negative (vehicle) control group #P < 0.05 when compared to positive control groups In addition to this physiological phenomenon, we observed that PAFR gene expression is increased upon E. coli LPS or DB stimulation, but not upon exogenous PAF treatment, for 6 h as compared to vehicle-treated group (Figure 2D), as was assessed by IF analysis (Figure 2A LPCAT2 gene expression was also upregulated by these pro-inflammatory bacterial stimuli which suggests that bacteria-associated molecular patterns are positive signals for the upregulation of PAFR and LPCAT2 genes the latter being important for the metabolism of PAF during inflammation To further address the functional role of PAF in chicken primary macrophages, we first assessed whether exogenous PAF were cytotoxic to these cells. We showed that PAF (1 and 10 µM) was not cytotoxic to chicken BMDM after 6 h stimulation as evaluated through annexin V/PI double-staining strategy (Figure 3A) and caspase 3–7 activation assay (Figure 3B) both canonical markers for early and late cell apoptosis coli LPS and DB were cytotoxic to chicken BMDM within the same experimental settings Exogenous platelet-activating factor (PAF) treatment does not impact cell viability and induces reactive oxygen species (ROS) release by chicken primary macrophages (A) Representative flow cytometry dot plot and bar graph showing gating strategy for annexin V and propidium iodide (PI) expression analysis in bone marrow-derived macrophages (BMDM) from B13/B13 histocompatible chicken and percentage of double-positive stained cells following treatment with PCA 4248 (10 µM) Escherichia coli lipopolysaccharide (LPS) (10 ng/ml) or dead bacteria (10 multiplicity of infection) for 6 h (B) Caspase 3–7 activity in BMDM following exposure to different stimuli for 6 h (C) Delta percentage of BMDM containing zymosan-fluorescein isothiocyanate particles following different stimuli for 1 h as an index of phagocytosis (delta calculated after incubation at 4°C and 40°C) (D) Representative flow cytometry dot plot and histograms showing ROS release by BMDM as determined by intracellular DCFDA fluorescence intensity (E) Bar graph showing the mean fluorescence intensity (MFI) of DCFDA-positive BMDM following different stimuli for 2 h **P < 0.01 when compared to negative (vehicle or medium) control groups We next asked whether PAF could enhance BMDM phagocytosis of fluorescent zymosan beads. We observed that PAF (10 µM) treatment for 1 h significantly enhanced chicken macrophage phagocytosis and that PAFR antagonist PCA 4248 abrogated this phenomenon (Figure 3C) In another biologically relevant assay, we assessed whether PAF induces ROS release by chicken macrophages. Using a fluorescent probe, we showed that both PAF (10 µM) and E. coli LPS stimulation for 2 h promoted enhanced ROS release by chicken BMDM. However, PAFR antagonist PCA 4248 reverted ROS release only in PAF-treated BMDM, revealing that ROS release is induced by PAF in a PAFR-dependent manner (Figures 3D,E) Altogether these data suggest that PAF promotes two acute macrophage-related inflammatory events in a PAFR-dependent fashion and independent of any cytotoxic effect in chicken macrophages These data suggest that PAF produced by chicken macrophages upon LPS stimulation contributes to the overall pro-inflammatory effects of LPS via PAFR Endogenous platelet-activating factor (PAF) contributes to the pro-inflammatory response elicited by lipopolysaccharide (LPS) and exogenous PAF potentiates LPS-induced inflammation in chicken primary macrophages coli LPS (10 ng/ml) in the presence or absence of PCA 4248 (10 µM) or human PAF-acetylhydrolase (PAF-AH) (10 µg/ml) for 6 h B13/B13 histocompatible chicken-derived bone marrow-derived macrophage (BMDM) supernatants were assessed for the presence of (A) nitric oxide (NO) and cell lysates were used to analyze gene expression of (B) iNOS/NOS2 and (D) IL-1β/IL1B by quantitative real-time PCR (E) PAF (0.1–10 µM) alone or together with LPS was added to BMDM and NO production in the supernatants was assessed after 6 h (F) NO production by BMDM derived from the bone marrow of the outbred PA12 chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h (G) NO production by peripheral blood mononuclear cells (PBMC) from B13/B13 histocompatible chickens following exposure to PAF (10 µM) and/or LPS in the presence or absence of PCA 4248 (10 µM) for 6 h and ***P < 0.001 when compared to negative (vehicle) control groups #P < 0.05 and ##P < 0.01 when compared to positive control groups Exogenous platelet-activating factor (PAF) potentiates the pro-inflammatory response elicited by lipopolysaccharide (LPS) in chicken primary macrophages in a PAF receptor (PAFR)-dependent manner After 6 h stimulation with PAF (10 µM) and/or E coli LPS (10 ng/ml) in the presence or absence of PAFR antagonist PCA 4248 (10 µM) B13/B13 histocompatible chicken-derived bone marrow-derived macrophage (BMDM) supernatants were assessed for the presence of (A) nitric oxide and (D) COX-2/PTGS2 by quantitative real-time PCR (E) Prostaglandin E2 dosage by mass spectrometry in BMDM supernatants following the same aforementioned stimuli for 6 h **P < 0.01 and ***P < 0.001 when compared to negative (vehicle) control groups in a magnitude that does not appear to interfere with their inhibitory action on PAFR signaling and its potentiation effects as suggested We did not observe any statistically significant effects in the aforementioned parameters when the p38 MAPK-mediated signaling pathway was blocked using the specific inhibitor SB 203580 (data not shown) The potentiation effects of platelet-activating factor (PAF) on lipopolysaccharide (LPS)-induced pro-inflammatory response in chicken primary macrophages are partially dependent on CaM KII- and phosphatidylinositol 3-kinase (PI3K)/Akt-mediated signaling pathways After 6 h stimulation with PAF (10 µM) and/or Escherichia coli LPS (10 ng/ml) in the presence or absence of PI3K antagonist wortmannin (WMN (E) Delta percentage of BMDM containing zymosan-fluorescein isothiocyanate particles following different stimuli for 1 h as an index of phagocytosis (delta calculated after incubation at 4°C and 40°C) (F) Western blot analysis of CaM KII and Akt expression in BMDM whole cell lysates following different treatments for 6 h **P < 0.01 and ***P < 0.001 when compared to negative (vehicle or medium) control groups We also confirmed that Akt (which is phosphorylated by PI3K) and CaMK II are expressed in chicken BMDM at protein levels (Figure 6F) although we could not determine the presence of PI3K due to the unavailability of validated commercial antibodies known to recognize this enzyme in chickens We therefore presume that PAF exerts its pro-phagocytic and potentiating effects over LPS-induced pro-inflammatory responses through PI3K/Akt- and/or CaMK II-mediated intracellular signaling pathways we showed that PAF is able to promote increased intracellular bacteria killing together with an increased pro-inflammatory response we suggest that APEC infection is likely to promote increased PAF signaling and production by chicken macrophages Exogenous platelet-activating factor (PAF) treatment favors intracellular bacteria killing and amplifies the inflammatory response in infected chicken macrophages (A) HD11 macrophage-like cells were infected with 10 multiplicity of infection (MOI) of avian pathogenic Escherichia coli (APEC) strains BEN2908 or BEN3421 (both O2 serotype strains) followed by treatment with PAF (1 or 10 µM) for 6 or 24 h Intracellular bacterial load was evaluated through colony-forming unit counts (B) Cell viability was evaluated through a colorimetric assay based on methylthiazoletetrazolium bromide cellular metabolism following the same in vitro infection protocol Data are expressed in optical density at 550 nm (C) Nitric oxide production in the supernatants of HD11 cells following infection with 10 MOI of APEC BEN2908 in the presence or absence of PAF (D) Immunofluorescence (IF) analysis of PAF receptor (PAFR) protein expression using an anti-PAFR polyclonal antibody PAFR expression is shown in green (Alexa-Fluor 488-conjugated secondary antibody) and the nucleus in blue (DAPI immunostaining) (E) PAFR/PTAFR and (F) LPCAT2/LPCAT2 gene expression analysis in HD11 cells following infection with 10 MOI of the APEC BEN2908 strain Quantitative real-time PCR data are expressed as relative normalized expression (as compared to vehicle control group) and ***P < 0.001 when compared to negative (vehicle or medium) control groups the gene expressions of PAFR/PTAFR and LPCAT2/LPCAT2 are upregulated together with pro-inflammatory gene expression such as COX-2/PTGS2 and IL-1β/IL1B as compared to vehicle-instilled animals These data suggest that during pulmonary colibacillosis and metabolism are overexpressed concomitantly to genes related to the acute inflammatory response which is in agreement with the data found in APEC-infected macrophages Overexpression of platelet-activating factor receptor (PAFR) and LPCAT2 is correlated with increased pro-inflammatory gene expression in chicken organs during pulmonary colibacillosis (A) Bacterial load in the lungs of B13/B13 histocompatible white leghorn chickens at 6 h following intratracheal infection with 109 colony forming units of APEC BEN2908 (O2 serotype) strain (B) Lung or (C) liver samples from APEC BEN2908-infected chickens were analyzed for PAFR/PTAFR and IL-1β/IL1B gene expression by quantitative real-time PCR ***P < 0.001 when compared to negative (vehicle) control group these data reveal that PAF is active in chicken endothelial cells and promotes increased cell permeability potentiates bacterial-derived inflammatory responses and may indirectly contribute to leukocyte adhesion to the chicken endothelium Exogenous platelet-activating factor (PAF) contributes to chicken endothelial cell dysfunction and increased pro-inflammatory response to bacteria-associated molecular patterns (A) PAF receptor (PAFR)/PTAFR gene expression as evaluated by quantitative real-time PCR (qRT-PCR) analysis in chicken aortic endothelial cells (chAEC) from PA12 outbred chickens following treatment with PAF (10 µM) (B) Fluorescein isothiocyanate (FITC)-dextran relative fluorescence units (RFU) as an index of chAEC monolayer permeability in a transwell system following treatment with PAF (1 or 10 µM) in the presence or absence of PCA 4248 (10 µM) (C) FITC-dextran RFU in the same transwell system at 30 min post chAEC treatment with PAF in the presence or absence of PCA 4248 Data are expressed as fold increase as compared to vehicle control group as evaluated through a colorimetric assay based on methylthiazoletetrazolium bromide cellular metabolism (E) IL-1β/IL1B and (F) CXCLi2/IL8L2 gene expression in chAEC following treatment with PAF and DB for 6 h in the presence or absence of PCA 4248 qRT-PCR data are expressed as relative normalized expression (as compared to vehicle control group) and ###P < 0.001 when compared to positive control groups Exogenous platelet-activating factor receptor (PAF) directly promotes upregulation of adhesion molecules gene expression in chicken endothelial cells (A) E-selectin/SELE and (B) VCAM-1/VCAM1 gene expression in chicken aortic endothelial cells (chAEC) following treatment with PAF (10 µM) *P < 0.05 and **P < 0.01 when compared to negative (vehicle) control groups The physiological and pathological roles of lipid mediators in livestock animals are often neglected and little is known on their presence and mechanisms of action we provide novel insights into the function of PAF as a pro-inflammatory mediator in chickens and the mechanism through which PAF contributes to chicken macrophage activation and responses to bacterial LPS PAF contributes to intracellular bacterial killing and EC dysfunction two phenomena that may be correlated with the overexpression of PAFR and LPCAT2 in organs of chickens presenting colibacillosis we provided evidence that PAF may be an early orchestrator of the inflammatory response to pathogenic bacteria in chickens paracrine- or autocrine-produced PAF is likely to play a direct role in the inflammatory response to Gram-negative bacteria-associated molecular patterns in chickens notably at the toll-like receptor 4 (TLR4) level The biological relevance of these findings merits to be further investigated in vivo as suggested by the data in the present manuscript the contribution of PAF to heterophil (the avian ortholog of neutrophils) arrest and recruitment via PI3K/Akt signaling pathway merits to be investigated this is the first study highlighting the role of a lipid mediator in promoting EC dysfunction in chickens PAF treatment led to the upregulation of genes coding for the leukocyte adhesion molecules VCAM-1 and E-selectin produced within the endothelium and adjacent tissues could favor not only leukocyte activation but also leukocyte arrest and recruitment and ECs during host-response to Gram-negative bacteria could contribute to local and sustained inflammatory responses in chickens we demonstrated that PAF is able to promote increased bacterial clearance by primary chicken macrophages in vitro together with a strong amplification of the inflammatory response Our gene expression data showed that PAFR and LPCAT2 genes are overexpressed in lungs and liver of APEC-infected chickens at early time points postinfection While these data reveal that key components of PAF recognition and biosynthesis are locally expressed in parallel to a strong inflammatory response to APEC we are still unable to define which cell populations are expressing these molecules it is reasonable to assume that PAF is indeed produced and acts within infected sites thereby amplifying inflammation and/or contributing to bacterial clearance as observed in vitro Both PAFR and LPCAT2 genes are overexpressed in chicken macrophages and chAEC upon LPS stimulation in vitro which suggests that at least these cell types may be involved in the response to colibacillosis in vivo LPCAT2 is believed to be the main pro-inflammatory LPCAT in the remodeling pathway for the biosynthesis of PAF in mammals Our protein homology data suggest that chicken LPCAT2 is closely related to human and mouse counterparts upon contact with bacteria or bacteria-associated molecular patterns and other unidentified tissue parenchyma cells PAF would in turn amplify local inflammation through eicosanoid and chemokine production to facilitate initial bacterial clearance and trigger the inflammatory response parenchymal) and endothelial PAF production would directly or indirectly (e.g. via chemokine production) promote increased leukocyte adhesion and recruitment (e.g. contributing to the orchestration of classical cellular and molecular events that take place at the onset of the inflammatory response We therefore suggest that PAF might be an important mediator of inflammation in the host response to Gram-negative bacteria causing economically relevant diseases in poultry farms worldwide and a proof of concept on the use of PAFR antagonists to limit exacerbated inflammation associated with experimental colibacillosis are currently being performed Although the feasibility of such therapeutic strategy in the poultry industry is still difficult to envisage these experiments will bring biologically relevant information on how lipid mediators and their receptors could be pharmacologically manipulated in chickens so as to pave the way to alternative therapies in face of the widespread antibiotic resistance in poultry farms and the lack of efficacious vaccines for certain avian diseases we believe that the present study sheds light on the role and function of PAF in bridging endothelium function and macrophage-associated innate immunity in chickens in response to bacteria-derived stimuli these activity profiles may be shared by other yet to be identified lipid mediators of inflammation a line of research that should be of interest to the scientific community in the fields of avian immunology All animals used in the avian pathogenic Escherichia coli (APEC) infection protocol were treated according to EU recommendations for animal welfare and the protocol was approved by the French regional ethics committee number 19 (Comité d’Ethique en Expérimentation Animale Val de Loire) under the reference CL2007-44 and AL performed the experiments and analyzed data The reviewer TK and handling editor declared their shared affiliation We thank Nathalie Lallier for her technical assistance on the experimental infection model and the personnel from the experimental unit PFIE (Plateforme d’Infectiologie Expérimentale) at the Centre INRA Val de Loire (Nouzilly Brazil) for valuable discussions on the experimental design setup This work was supported by the French National Institute for Agricultural Research (INRA) through a young researcher grant (to RG) from the Animal Health division (Département Santé Animale The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fvets.2017.00226/full#supplementary-material CrossRef Full Text | Google Scholar Lipid mediator networks in cell signaling: update and impact of cytokines Prostaglandins Other Lipid Mediat (2005) 77(1–4):197–209 Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators Nat Rev Immunol (2008) 8(5):349–61 J Biol Chem (1990) 265(29):17381–4 Google Scholar Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis Crit Care Med (2002) 30(5 Suppl):S294–301 Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation Platelet-activating factor and its analogs: metabolic pathways and related intracellular processes Biochim Biophys Acta (1995) 1254(3):231–49 CrossRef Full Text | Google Scholar Dual phase regulation of experimental allergic encephalomyelitis by platelet-activating factor A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransferase Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2 Paf acetylhydrolase as a novel calcium-independent phospholipase-A2 Google Scholar Roles of cytosolic phospholipase A(2) and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF Biochem Biophys Res Commun (2000) 271(3):812–7 Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent Increased biosynthesis of platelet-activating factor in activated human eosinophils Stress-induced platelet-activating factor synthesis in human neutrophils Biochim Biophys Acta (2005) 1733(2–3):120–9 Platelet-activating factor augments tumor necrosis factor and procoagulant activity The macrophage response to endotoxin requires platelet activating factor Hypotensive activity of PAF-acether in rats Agents Actions (1982) 12(5–6):725–30 CrossRef Full Text | Google Scholar Acute circulatory collapse caused by platelet-activating factor (PAF-acether) in dogs Eur J Pharmacol (1983) 86(3–4):403–13 Recombinant tumour necrosis factor-alpha and platelet-activating factor synergistically increase intercellular adhesion molecule-1 and E-selectin-dependent neutrophil adherence to endothelium in vitro Ether lipids and platelet-activating factor: evolution and cellular function Biochemistry (Mosc) (1997) 62(10):1103–8 Platelet-activating factor production occurs through stimulation of cholinergic and dopaminergic receptors in the chick retina Activation of washed chicken thrombocytes by 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (platelet-activating factor) Comp Biochem Physiol A Comp Physiol (1985) 82(1):145–51 Vertebrate class distribution of 1-alkyl-2-acetyl-Sn-glycero-3-phosphocholine acetylhydrolase in serum Comp Biochem Physiol B (1984) 78(1):37–40 Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad J Biol Chem (1995) 270(43):25481–7 Characterization of leukotriene production in vivo and in vitro in resident and elicited peritoneal macrophages in chickens and mice Prostaglandins Leukot Essent Fatty Acids (1997) 56(1):41–9 Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4) Gen Comp Endocrinol (2008) 157(2):99–106 Bacterial toll-like receptor agonists induce sequential NF-kappaB-mediated leukotriene B4 and prostaglandin E2 production in chicken heterophils Vet Immunol Immunopathol (2012) 145(1–2):159–70 Avian colibacillosis: still many black holes Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E Vet Immunol Immunopathol (2009) 127(1–2):77–84 Infections with avian pathogenic and fecal Escherichia coli strains display similar lung histopathology and macrophage apoptosis Pathobiology of the intravenous infusion of acetyl glyceryl ether phosphorylcholine (AGEPC) a synthetic platelet-activating factor (PAF) Google Scholar Role of the platelet-activating factor (PAF) receptor during pulmonary infection with gram negative bacteria Br J Pharmacol (2002) 137(5):621–8 Platelet-activating factor receptor plays a role in lung injury and death caused by influenza A in mice Pivotal advance: avian colony-stimulating factor 1 (CSF-1) and CSF-1 receptor genes and gene products Characterization of the pattern of inflammatory cell influx in chicks following the intraperitoneal administration of live Salmonella enteritidis and Salmonella enteritidis-immune lymphokines Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites J Chromatogr B Analyt Technol Biomed Life Sci (2013) 932:123–33 Escherichia coli colonization of the trachea in poultry: comparison of virulent and avirulent strains in gnotoxenic chickens Modelling the P2Y purinoceptor using rhodopsin as template Drug Des Discov (1995) 13(2):133–54 Molecular tinkering of G protein-coupled receptors: an evolutionary success Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors Biochem Biophys Res Commun (2002) 293(4):1233–41 Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptor Br J Pharmacol (2003) 140(5):932–8 Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction Biochemistry (1999) 38(18):5764–71 Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals Proc Natl Acad Sci U S A (2003) 100(10):5783–8 and down-regulation of the human platelet-activating factor receptor J Biol Chem (2003) 278(48):48228–35 Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K-ERK1/2 pathway Br J Pharmacol (2004) 141(2):311–21 Platelet-activating factor induces matrix metalloproteinase-9 expression through Ca(2+)- or PI3K-dependent signaling pathway in a human vascular endothelial cell line Platelet-activating factor may act as a second messenger in the release of icosanoids and superoxide anions from leukocytes and endothelial cells Proc Natl Acad Sci U S A (1990) 87(8):3215–9 The development of anti-inflammatory drugs for infectious diseases Google Scholar Platelet-activating factor: a role in preterm delivery and an essential interaction with toll-like receptor signaling in mice Calcium/calmodulin-dependent kinase II is required for platelet-activating factor priming Platelet-activating factor receptor-mediated PI3K/AKT activation contributes to the malignant development of esophageal squamous cell carcinoma Unique properties of the chicken TLR4/MD-2 complex: selective lipopolysaccharide activation of the MyD88-dependent pathway Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo Wortmannin inhibits mitogen-activated protein kinase activation induced by platelet-activating factor in guinea pig neutrophils J Biol Chem (1994) 269(48):30485–8 Morphologic basis of increased vascular permeability induced by acetyl glyceryl ether phosphorylcholine The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC) Microb Pathog (2008) 45(5–6):361–9 Uptake of particulate antigens in a nonmammalian lung: phenotypic and functional characterization of avian respiratory phagocytes using bacterial or viral antigens Identification of a novel noninflammatory biosynthetic pathway of platelet-activating factor J Biol Chem (2008) 283(17):11097–106 Phosphorylation of lysophosphatidylcholine acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-stimulated macrophages J Biol Chem (2010) 285(39):29857–62 Effect of a novel potent platelet-activating factor antagonist Am J Respir Crit Care Med (1995) 151(5):1331–5 Keywords: avian pathogenic Escherichia coli Schouler C and Guabiraba R (2017) Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens Received: 19 October 2017; Accepted: 06 December 2017; Published: 18 December 2017 Copyright: © 2017 Garrido, Chanteloup, Trotereau, Lion, Bailleul, Esnault, Trapp, Quéré, Schouler and Guabiraba. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Rodrigo Guabiraba, cm9kcmlnby5ndWFiaXJhYmEtYnJpdG9AaW5yYS5mcg== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish we have chosen five municipalities as pilot municipalities of the Healthy Municipalities Project They are located in "Central Agreste" region (the Agreste region lies between the coastal rainforest region and instate semiarid lands of Northeast We took into consideration the economic situation the existence or lack of similar projects and the willingness of the municipality to collaborate These 5 municipalities are between 110 to 130km from the state capital of Recife All have agriculture and livestock farming as the major industry The total population of these 5 municipalities is 97,295 (in the year 2000) and their Human Development Index is 0.588 (in 2000) Their HDI is practically at the level considered for low-income countries (0.554 in the 2000 Human Development Report) which has previously have worked with Healthy Municipalities aided by the Ministry of Health is also integrated as one of the pilot municipalities of the project We then selected pilot communities in each municipality we chose one life cycle as a theme for each municipality through interviews and existing information we selected one community from each pilot municipality - the community with an accumulation of social capital relevant to the selected life cycle we selected the youth life cycle theme in Bonito We selected one pilot community where there were a large number of youth groups Figure5 : Localization of Pilot Municipalities In the concept of Healthy Municipalities, health would be improved not only through improvements to health care services, but also by the effects of various factors in everyday life (q.v. Figure 2) We call this the "Idea of Comprehensive Health" we should not treat health and life within only a few sectors such as health care education or industries; we should treat them more widely and cross-sectionally the framework of the life cycle comes to the fore Thinking in terms of life cycle phases such as children we can treat health and life in a more comprehensive fashion Our Healthy Municipalities Project proposes the life cycle framework and performs trial runs of specific policy-making and intersectoral cooperation This is just a gateway of activities and it is possible for each pilot municipality to alter or amplify the life cycle themes later on The life cycle themes of each municipality are cited below Barra de Guabiraba The life cycle theme : adult men Camocim de São Felix The life cycle theme : adult women and children Sairé The life cycle theme : elderly São Joaquim do Monte The life cycle theme : adult women and children Itambé The life cycle theme : infants municipality and community development takes a long time All precedent cases of municipality and community building generally show that there are no concrete outcomes at first but they become gradually clear in the process of 3 to 5 years Particularly when involving the development of human resources and social capital (capacity development) it is important to support development with patience as well as considerable time and effort in supporting "municipality and community development" through a technical cooperation project that calls for certain results within limited time the Healthy Municipalities Project has chosen as its objective the creation and implementation of "mechanisms" that may be developed and concretized during the project period "Mechanisms" are social devices that permit continuity in the collaborated activities of residents and local government There are three different levels on which where these activities are implemented Figure6 : Mechanism of Healthy Municipalities residents freely discuss and take an active part in the undertakings Facilitators have the role of encouraging community gatherings and leading meetings "Facilitators" work in cooperation with "supervisors" (mentioned later) to promote community activities always seeking to have new members participate in them The participation of "collaborators of healthy municipalities" who help facilitators and supervisors is also indispensable there is EAPPPS (Center for Healthy Public Policy Articulation and Promotion) for the integration and coordination of public policies giving priority to health and life Supervisors coordinate and link activities from the micro and mid levels here these include the intersectoral adjustment of public policies that reflect the concerns of the residents the training of the human resources necessary for Healthy Municipalities implementation and evaluation of community activities the coordination of federal and state government programs or projects to meet the community's needs participation in the development planning of municipalities On the macro level (group of adjacent municipalities) there arise discussions on broad-based common issues that can be more effectively resolved together than by an individual municipality as the referral center for "Healthy Municipalities" the UFPE trains the necessary human resources for the "mechanisms" and lend technical support to the activities The UFPE also hosts the network of healthy municipalities and diffuses the "mechanisms" to other regions the state government has introduced "Healthy Municipalities" in its development strategy and offers information on various public programs or projects to the EAPPPS of each municipality to support the planning of healthy public policies In the course of the discussions on the intervention method of the Healthy Municipalities Project in communities the central issue was how a community becomes empowered (how is it possible to improve the autonomy of the community effectively) There are two fundamental problems in this region people who live in Northeast region of Brazil have historically leaned heavily upon the hope of assistance from patrons the notion of autonomy within the community is fragile it is desirable for the community to solve their problems themselves through the empowerment process but there is difficulty in achieving this with community efforts alone due to the serious problems stemming from poverty Dealing with these two problems simultaneously is daunting we need to take a different path from conventional ways we have developed a method that seeks to make the best use of Existing Potentials (utilizing the abilities of one person who is good at making things etc.) in the community to set up goals that can be accomplished by the community itself We believe that their autonomy can be enhanced by achieving these goals (The traditional approach has been a "cause-seeking approach" that analyzes the cause of problems to be solved.) The development of project method is based on the SOJO (System-Oriented Joyful Operation) model developed by Japanese researcher Toshihiro IWANAGA and others people feel a sense of accomplishment through the implementation of small actions through joint efforts boosting their self-respect and lending continuity for further actions with confidence This name derives from the image of bamboo: Flexibility - flexible application of the method is one aspect; the firm roots of bamboo - a sound base in reality and also symbolizes the collaboration of Brazil with the Orient JICA website uses cookies to provide you with a better service.By closing this message or just continuing to browse the site, you are agreeing to our use of cookies in our Privacy Policy RSS FeedsRSS Feeds Volume 7 - 2016 | https://doi.org/10.3389/fimmu.2016.00125 Immunology developed under the notion of the immune system exists to fight pathogens the discovery of interactions with commensal microbiota that are essential to human health initiated a change in this old paradigm we argue that the immune system has major physiological roles extending far beyond defending the host Immune and inflammatory responses share the core property of sensing defining the immune system also as a sensory system The inference with the immune system collects places it in close relationship to the nervous system which suggests that these systems may have a profound evolutionary connection indicating its theoretical basis should be updated There is a consensus among scientists that the ability of microorganisms or microbial products to trigger inflammation and immune responses are important for immune function We would like to suggest that immune functions extend far beyond interaction with pathogens targeting the host immune response instead of the causative agent itself may be an effective option in the context of disease inflammation is prone to imbalance as many physiological machineries in the organism and loss of balance relates to pathogenic states we argue that inflammation and innate immunity are biological-related processes which must operate under similar premises and toward the common goal of reaching homeostasis Figure 1. Incorporation of inflammation into immunology. Over the last decades, inflammation and immunology were progressively merged as biomedical research evolves. (A) Scientific papers, in numbers, retrieved from queries for “immunology,” “inflammation AND immunology,” and “inflammation” at PubMed (http://www.ncbi.nlm.nih.gov/pubmed) Results span 1 year (forth to fifth year) over four consecutive decades (80’ to 2010’) (B) Scientific papers retrieved from “immunology” for the given year/decade were queried for “inflammation,” and vice versa Exposure to microbiota or to microbial products promptly restores the ability of germ-free mice to respond as conventional mice indicating that the immune tissues and cells were present and functional in the absence of microbes This concept of responsiveness to host leads to broader considerations that the immune system does not depend on pathogens to function or exist Biomedical research is biased toward disease although disease does not represent homeostasis in the vertebrate host thus the immune system must operate in such spectrum The pyramid estimates the proportion of immune responses that go unnoticed by current experimental techniques in cold colors in comparison to less frequent immune responses in the context of disease immune responses are categorized by abundance and intensity the categories of inflammation are depicted If the immune system is part of a living functioning organism, constantly reaching for homeostasis, interacting with microbiota, remodeling tissue, dealing with injury, and so forth; interacting with pathogens is a small fraction of the activities of the immune system (27). Contact with a pathogen may result in infection, and infection could lead to disease (28) Based on these possibilities and the fact that the immune system is fully active for a lifetime given the impact of disease in human health it comes as no surprise that biomedical research is focused on disease though disease does not represent or explain homeostasis between systems in a host Although extensive knowledge on disease allows us to explore it and propose treatments the challenge relies on studying processes that did not lead to disease The greatest challenge in reinterpreting the immune system is to define what the system is and what it does to maintain homeostasis. The basis for that may be the overlap of inflammation and immune responses. In terms of core mechanism, their common ground is sensing and recognition of molecules of variable compositions, forms, sources, and properties (14) the immune system is unable to respond or to sustain interaction inflammation is unable to start or to resolve the ability of the immune system to sense things is not conflicting with sustaining interactions with both microbes and host we propose that the immune system is a sensory system vascular tissues are in constant scrutiny by circulating leukocytes These interpretations can be exemplified by the combination of signals (cytokines and antibodies) that define leukocyte recruitment which stands for information interpretation and reply the bulk of information that the immune system manages is what allows the system to define and pursue homeostasis Information management by the immune system The nervous and immune systems handle large volumes of information the immune system contributes to host homeostasis This process start with information sensing obtained through interaction with molecules of different origins and composition causing specific leukocyte activation and recruitment information is communicated and replied to other components of the immune system such as in antigen-presenting cells and T CD4+ lymphocytes The immune system is able to store information for which adaptive memory is the most studied process Stored information can be promptly accessed exemplified by quick adaptive immune responses upon secondary challenges adding to the ongoing flow of information through the immune system those examples corroborate our hypothesis by showing how specialized the immune system is to sense and manage alterations in the host homeostasis (e.g. over other body systems not directly involved in sensing in case of intrinsic failure of the immune system a simple stimulus could lead to disastrous consequences Inferring that the immune system plays an important role in sustaining intrahost interactions would also explain why immune responses and inflammation have great influence over other host systems immunity can be described as a host system of information management which allows all possible interactions the host may have to sustain The nervous system is traditionally associated with sensing and management of information in the vertebrate host The suggestion that the primary function of the immune system is also sensing and management of information may be conflicting since these are already entrusted to another evolutionary-conserved body system sensing is a general property of multicellular organisms and essential for host fitness Although it may appear controversial to have two complex systems in charge of the same function the nervous and immune systems are not redundant These systems mediate interactions between host and environment that exceed interactions made by any other body systems in number and complexity The nervous and immune systems share remarkable similarities such as full coverage of the host and interactivity The nervous system is responsible for sensing physical stimuli This panel is complemented by the immune system’s sensing of chemical stimuli both systems confer a notion of “self” to the host by defining what the host is from different perspectives The notion of self is a key aspect for interaction between the host and other organisms these systems could manage immeasurable amounts of information and interaction in vertebrate organisms regularly The understanding of the immune system has changed drastically in the last decade (62). A homeostatic paradigm of immunity is already accepted by a significant part of the scientific community, and new ideas, such as disease tolerance (9), add interesting perspectives to the field. Accordingly, recent findings suggest the immune system also maintain virus–host interactions (63) Although great advances have been made in interpreting the roles of immunity from a homeostatic perspective unbiased by pathogens thorough research and thinking are still needed The complexity of the immune system is only matched by its ability to sustain greater complexity As the immune system role in supporting host–microbiota interactions was consolidated scientists now turn to the role of the immune system in other biological processes or systems The field of neuroimmunology shows promise as the immune and nervous systems seem intimately related in function Pain is a connecting point between neurology and immunology where sensing and interactive properties of nervous and immune systems converge pain translates into a great opportunity for research and MT contributed with both intellectual and written information All authors agreed on the final version of the manuscript and are accountable for its contents The authors would like to thank Caio Tavares Fagundes and Nadia Neto for comments on the manuscript