Mechanisms of type I interferon production by chicken TLR21.

The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-κB and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-κB and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1ß, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFß1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Evolutionary diversification of defensins and cathelicidins in birds and primates.

Divergent evolution for more than 310 million years has resulted in an avian immune system that is complex and more compact than that of primates, sharing much of its structure and functions. Not surprisingly, well conserved ancient host defense molecules, such as defensins and cathelicidins, have diversified over time. In this review, we describe how evolution influenced the host defense peptides repertoire, its distribution, and the relationship between structure and biological functions. Marked features of primate and avian HDPs are linked to species-specific characteristics, biological requirements, and environmental challenge.

Uncovering the core principles of the gut-lung axis to enhance innate immunity in the chicken.

Research in mammals has evidenced that proper colonization of the gut by a complex commensal microbial community, the gut microbiota (GM), is critical for animal health and wellbeing. It greatly contributes to the control of infectious processes through competition in the microbial environment while supporting proper immune system development and modulating defence mechanisms at distant organ sites such as the lung: a concept named 'gut-lung axis'. While recent studies point to a role of the GM in boosting immunity and pathogen resilience also in poultry, the mechanisms underlying this role are largely unknown. In spite of this knowledge gap, GM modulation approaches are today considered as one of the most promising strategies to improve animal health and welfare in commercial poultry production, while coping with the societal demand for responsible, sustainable and profitable farming systems. The majority of pathogens causing economically important infectious diseases in poultry are targeting the respiratory and/or gastrointestinal tract. Therefore, a better understanding of the role of the GM in the development and function of the mucosal immune system is crucial for implementing measures to promote animal robustness in commercial poultry production. The importance of early gut colonization in the chicken has been overlooked or neglected in industrial poultry production systems, where chicks are hampered from acquiring a complex GM from the hen. Here we discuss the concept of strengthening mucosal immunity in the chicken through GM modulation approaches favouring immune system development and functioning along the gut-lung axis, which could be put into practice through improved farming systems, early-life GM transfer, feeding strategies and pre-/probiotics. We also provide original data from experiments with germ-free and conventional chickens demonstrating that the gut-lung axis appears to be functional in chickens. These key principles of mucosal immunity are likely to be relevant for a variety of avian diseases and are thus of far-reaching importance for the poultry sector worldwide.

Chronic dietary exposure to a glyphosate-based herbicide results in total or partial reversibility of plasma oxidative stress, cecal microbiota abundance and short-chain fatty acid composition in broiler hens.

Glyphosate-based herbicides (GBHs) are massively used in agriculture. However, few studies have investigated the effects of glyphosate-based herbicides on avian species although they are largely exposed via their food. Here, we investigated the potential reversibility of the effects of chronic dietary exposure to glyphosate-based herbicides in broiler hens. For 42 days, we exposed 32-week-old hens to glyphosate-based herbicides via their food (47 mg/kg/day glyphosate equivalent, glyphosate-based herbicides, n = 75) corresponding to half glyphosate's no-observed-adverse-effect-level in birds. We compared their performance to that of 75 control animals (CT). Both groups (glyphosate-based herbicides and control animals) were then fed for 28 additional days without glyphosate-based herbicides exposure (Ex-glyphosate-based herbicides and Ex-control animals). Glyphosate-based herbicides temporarily increased the plasma glyphosate and AMPA (aminomethylphosphonic acid) concentrations. Glyphosate and aminomethylphosphonic acid mostly accumulated in the liver and to a lesser extent in the leg muscle and abdominal adipose tissue. Glyphosate-based herbicides also temporarily increased the gizzard weight and plasma oxidative stress monitored by TBARS (thiobarbituric acid reactive substances). Glyphosate-based herbicides temporarily decreased the cecal concentrations of propionate, isobutyrate and propionate but acetate and valerate were durably reduced. The cecal microbiome was also durably affected since glyphosate-based herbicides inhibited Barnesiella and favored Alloprevotella. Body weight, fattening, food intake and feeding behavior as well as plasma lipid and uric acid were unaffected by glyphosate-based herbicides. Taken together, our results show possible disturbances of the cecal microbiota associated with plasma oxidative stress and accumulation of glyphosate in metabolic tissues in response to dietary glyphosate-based herbicides exposure in broiler hens. Luckily, glyphosate-based herbicides at this concentration does not hamper growth and most of the effects on the phenotypes are reversible.

New Insights in the Interplay Between African Swine Fever Virus and Innate Immunity and Its Impact on Viral Pathogenicity.

The continuous spread of African swine fever virus (ASFV) in Europe and Asia represents a major threat to livestock health, with billions of dollars of income losses and major perturbations of the global pig industry. One striking feature of African swine fever (ASF) is the existence of different forms of the disease, ranging from acute with mortality rates approaching 100% to chronic, with mild clinical manifestations. These differences in pathogenicity have been linked to genomic alterations present in attenuated ASFV strains (and absent in virulent ones) and differences in the immune response of infected animals. In this mini-review, we summarized current knowledge on the connection between ASFV pathogenicity and the innate immune response induced in infected hosts, with a particular focus on the pathways involved in ASFV detection. Indeed, recent studies have highlighted the key role of the DNA sensor cGAS in ASFV sensing. We discussed what other pathways may be involved in ASFV sensing and inflammasome activation and summarized recent findings on the viral ASFV genes involved in the modulation of the interferon (IFN) and nuclear factor kappa B (NF-κB) pathways.

Methodologies to Assess the Bioactivity of an Herbal Extract on Immunity, Health, Welfare and Production Performance in the Chicken: The Case of Melissa officinalis L. Extract.

The potential of herbal extracts containing bioactive compounds to strengthen immunity could contribute to reducing antimicrobial use in poultry. This study aimed at developing a reliable and robust methodological pipeline to assess the ability of herbal extracts to strengthen chicken innate defenses, especially concerning inflammation and oxidative stress. This methodology was applied to Melissa officinalis L. (MEL) extract, recognized for its biological activities including antioxidant and anti-inflammatory properties. Different methods were used to (1). guarantee the quality of MEL extract and its capacity to stimulate the innate immune system; (2). evaluate the relevance of an ex vivo model to mimic inflammatory and oxidative stress challenges to replace LPS injection in chickens; (3). analyse the effects of feed supplemented with MEL extract on inflammation and oxidative stress induced ex vivo; (4). assess the effects of MEL extract on the redox balance, health, welfare and performance in broilers exposed to suboptimal starting conditions through a large-scale approach. The quality of MEL extract preparations, through phytochemical quantification of rosmarinic acid (RA), revealed varying concentrations of RA in the different MEL extracts. RA concentrations remained stable for at least 9 months and in feed three months after incorporating MEL extract. When incubated with chicken cell lines MEL extract showed potential metabolic activation and ability to stimulate immune functions but induced cytotoxicity at high concentrations. The original ex vivo model of inflammation developed on chicken blood cells enabled inflammation and oxidative stress biomarkers to be expressed and revealed antioxidative and anti-inflammatory properties of blood cells from chickens fed MEL extract. The experimental model of chicken suboptimal starting conditions validated beneficial effects of MEL extract on the redox balance and also evidenced improved performance during the growth phase, a tendency for fewer muscle defects but a higher severity of pododermatitis lesions without affecting other welfare indicators. This study grouped methods and tools that could be combined according to the plant extract, the needs of professionals working in poultry production systems and staff responsible for animal health, welfare and feeding.

Upregulation of gut cathepsin L during Eimeria tenella infection.

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial ß-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian ß-defensins that are key molecules of innate immunity.

Cellular and molecular insights on the regulation of innate immune responses to experimental aspergillosis in chicken and turkey poults.

Across the world, many commercial poultry flocks and captive birds are threatened by infection with Aspergillus fumigatus. Susceptibility to aspergillosis varies among birds; among galliform birds specifically, morbidity and mortality rates seem to be greater in turkeys than in chickens. Little is known regarding the features of avian immune responses after inhalation of Aspergillus conidia, and to date, scarce information on inflammatory responses during aspergillosis exists. Thus, in the present study, we aimed to improve our understanding of the interactions between A. fumigatus and economically relevant galliform birds in terms of local innate immune responses. Intra-tracheal aerosolization of A. fumigatus conidia in turkey and chicken poults led to more severe clinical signs and lung lesions in turkeys, but leukocyte recovery from lung lavages was higher in chickens at 1dpi only. Interestingly, only chicken CD8+ T lymphocyte proportions increased after infection. Furthermore, the lungs of infected chickens showed an early upregulation of pro-inflammatory cytokines, including IL-1ß, IFN-γ and IL-6, whereas in turkeys, most of these cytokines showed a downregulation or a delayed upregulation. These results confirmed the importance of an early pro-inflammatory response to ensure the development of an appropriate anti-fungal immunity to avoid Aspergillus dissemination in the respiratory tract. In conclusion, we show for the first time that differences in local innate immune responses between chickens and turkeys during aspergillosis may determine the outcome of the disease.

Aspergillus fumigatus infection may cause mortality in poultry, depending on species sensitivity. This study confirms the earlier activation of chickens' pro-inflammatory effectors to control Aspergillus dissemination, whereas turkeys' immune response enables the exacerbation of lung lesions.

Microbiota Changes Due to Grape Seed Extract Diet Improved Intestinal Homeostasis and Decreased Fatness in Parental Broiler Hens.

In poultry, the selection of broilers for growth performance has induced a deterioration in the health of the parental hens associated with poor reproductive efficiency. To improve these parameters, we administered to laying parental broiler hens a regular diet supplemented or not (Control) with a moderate (1%) or a high level (2%) of grape seed extract (GSE). The 1% GSE diet was administered from a young age (from 4 to 40 weeks of age) and the high level of 2% GSE was administered only during a 2-week period (from 38 to 40 weeks of age) in the laying period. The analysis of 40-week-old hens showed that 2% GSE displayed a reduction in the fat tissue and an improvement in fertility with heavier and more resistant eggs. Seven monomer phenolic metabolites of GSE were significantly measured in the plasma of the 2% GSE hens. GSE supplementation increased the relative abundance of the following bacteria populations: Bifidobacteriaceae, Lactobacilliaceae and Lachnospiraceae. In conclusion, a supplementation period of only 2 weeks with 2% GSE is sufficient to improve the metabolic and laying parameters of breeder hens through a modification in the microbiota.

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines. The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry. Eggs were quickly collected after laying at a broiler breeder farm. They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator. The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Precision cut lung slices: a novel versatile tool to examine host-pathogen interaction in the chicken lung.

The avian respiratory tract is a common entry route for many pathogens and an important delivery route for vaccination in the poultry industry. Immune responses in the avian lung have mostly been studied in vivo due to the lack of robust, relevant in vitro and ex vivo models mimicking the microenvironment. Precision-cut lung slices (PCLS) have the major advantages of maintaining the 3-dimensional architecture of the lung and includes heterogeneous cell populations. PCLS have been obtained from a number of mammalian species and from chicken embryos. However, as the embryonic lung is physiologically undifferentiated and immunologically immature, it is less suitable to examine complex host-pathogen interactions including antimicrobial responses. Here we prepared PCLS from immunologically mature chicken lungs, tested different culture conditions, and found that serum supplementation has a detrimental effect on the quality of PCLS. Viable cells in PCLS remained present for ≥ 40 days, as determined by viability assays and sustained motility of fluorescent mononuclear phagocytic cells. The PCLS were responsive to lipopolysaccharide stimulation, which induced the release of nitric oxide, IL-1ß, type I interferons and IL-10. Mononuclear phagocytes within the tissue maintained phagocytic activity, with live cell imaging capturing interactions with latex beads and an avian pathogenic Escherichia coli strain. Finally, the PCLS were also shown to be permissive to infection with low pathogenic avian influenza viruses. Taken together, immunologically mature chicken PCLS provide a suitable model to simulate live organ responsiveness and cell dynamics, which can be readily exploited to examine host-pathogen interactions and inflammatory responses.

The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella.

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Chicken cGAS Senses Fowlpox Virus Infection and Regulates Macrophage Effector Functions.

The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, 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). Here we show that in chicken macrophages, 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. In the context of fowlpox, an avian DNA virus infection, 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.

Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice.

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens.

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC. RESEARCH HIGHLIGHTS Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host-pathogen interactions. pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons. pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

Interleukin-33 contributes to disease severity in Dengue virus infection in mice.

The excessive inflammation often present in patients with severe dengue infection is considered both a hallmark of disease and a target for potential treatments. Interleukin-33 (IL-33) is a pleiotropic cytokine with pro-inflammatory effects whose role in dengue has not been fully elucidated. We demonstrate that IL-33 plays a disease-exacerbating role during experimental dengue infection in immunocompetent mice. Mice infected with dengue virus serotype 2 (DENV2) produced high levels of IL-33. DENV2-infected mice treated with recombinant IL-33 developed markedly more severe disease compared with untreated mice as assessed by mortality, granulocytosis, liver damage and pro-inflammatory cytokine production. Conversely, ST2-/- mice (deficient in IL-33 receptor) infected with DENV2 developed significantly less severe disease compared with wild-type mice. Furthermore, the increased disease severity and the accompanying pathology induced by IL-33 during dengue infection were reversed by the simultaneous treatment with a CXCR2 receptor antagonist (DF2156A). Together, these results indicate that IL-33 plays a disease-exacerbating role in experimental dengue infection, probably driven by CXCR2-expressing cells, leading to elevated pro-inflammatory response-mediated pathology. Our results also indicate that IL-33 is a potential therapeutic target for dengue infection.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge.

Mammalian type I interferons (IFNα/ß) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNß act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1ß and notably IFNB/IFNß. Neutralization of IFNß during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape.

Endotheliotropism is a hallmark of gallinaceous poultry infections with highly pathogenic avian influenza (HPAI) viruses and a feature that distinguishes HPAI from low pathogenic avian influenza (LPAI) viruses. Here, we used chicken aortic endothelial cells (chAEC) as a novel in vitro infection model to assess the susceptibility, permissiveness, and host response of chicken endothelial cells (EC) to infections with avian influenza (AI) viruses. Our data show that productive replication of AI viruses in chAEC is critically determined by hemagglutinin cleavability, and is thus an exclusive trait of HPAI viruses. However, we provide evidence for a link between limited (i.e. trypsin-dependent) replication of certain LPAI viruses, and the viruses' ability to dampen the antiviral innate immune response in infected chAEC. Strikingly, this cell response pattern was also detected in HPAI virus-infected chAEC, suggesting that viral innate immune escape might be a prerequisite for robust AI virus replication in chicken EC.