A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were signifcantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was signifcantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were signifcantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specifc antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specifc T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.ADDITIONAL FILE 1. Gating strategy of IELs and splenic lymphocytes in broiler chickens. Gating strategy included consecutive selection for lymphocytes (FSC-A vs SSC-A), singlets (FSC-A vs FSC-H) and viable cells (Live/Dead marker-negative) followed by selection of NK and T cell subsets in ileum and spleen. Furthermore, activation of NK and T cells was analyzed by surface expression of CD107 and intracellular expression of IFNγ. Conjugate controls are shown for IELs and splenic lymphocytes.ADDITIONAL FILE 2. Effect of SE infection on numbers of splenic NK cells in broiler chickens. A Numbers (cells/mg) of splenic IL-2Rα+ and B 20E5+ NK cells per mg spleen in uninfected (uninf) and SE-infected (SE-inf) chickens in the course of time. C Gene expression levels of NK cell lineage marker (NFIL3), IL-7Rα and perforin 1 (PRF1) by RT-qPCR in sorted IL-2Rα+ and 20E5+ NK cell subsets. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 7 dpi n = 4 and for gene expression levels n = 1.ADDITIONAL FILE 3. Staining and sorting controls associated with Figure 4. A The staining controls for the gating strategy are shown. The left panel depicts splenocytes without the viability dye. The middle and right panels show splenocytes that are gated according to Figure 4A, but without the primary antibodies that bind MRC1LB and CD11, respectively. B The graphs show the gating strategy and purity of a representative sample of splenocytes that was sorted into CD11+ MRC1LB+, CD11+ MRC1LB− FSClow and CD11+ MRC1LB− FSChigh subpopulations. The splenocytes that are gated as CD11+ MRC1LB− in the upper panels are shown in the lower panels to visualize their FSC-A vs SSC-A pattern. C The absolute numbers of sorted APC subpopulations are shown.ADDITIONAL FILE 4. Phenotypic characterization of splenic APCs upon SE infection. A-B The presence (%) and C-D numbers (cells/mg spleen) of FSClow DCs and and FSChigh DCs in uninfected (uninf) and SE-infected (SE-inf) chickens were assessed over time. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 0 dpi n = 3 and at 7 dpi n = 4. Statistical significance is indicated as ** p < 0.01.ADDITIONAL FILE 5. The gating strategy used to determine the activation status of the APC subsets as depicted in Figure 5. The three identified splenic APC subsets A macrophages, B FSClow DCs and C FSChigh DCs were assessed for CHIR-AB1, CD40, CD80 and MHC-II. For CHIR-AB1, CD40 and CD80, the cells expressing the respective markers were selected and expressed as a percentage. The expression of MHC-II by each subset was expressed as the geometric mean fluorescent intensity (gMFI).ADDITIONAL FILE 6. Numbers of intraepithelial and splenic γδ T cells and cytotoxic T cells expressing either CD8αα or CD8αβ in broiler chickens upon SE infection. A Numbers (cells/mg) of intraepithelial CD8αα+ γδ T cells, B CD8αβ+ γδ T cells, C cytotoxic CD8αα+ T cells and D CD8αβ+ T cells per mg ileum in uninfected (uninf) and SE-infected (SE-inf) chickens in the course of time. E Numbers (cells/mg) of splenic CD8αα+ γδ T cells, F CD8αβ+ γδ T cells, G cytotoxic CD8αα+ T cells and H CD8αβ+ T cells per mg spleen in uninfected and SE-infected chickens. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 1 dpi in the IELs and spleen n = 4 due to numbers of events acquired in the gate of interest were < 100, and at 7 dpi in spleen n = 4. Statistical significance is indicated as * p < 0.05, ** p < 0.01. *** p < 0.001.ADDITIONAL FILE 7. Numbers of CD4 + T cells in the spleen of broiler chickens upon SE infection. Numbers (cells/mg) of splenic CD4+ αβ T cells per mg spleen in uninfected (uninf) and SE-infected (SE-inf) chickens in the course of time. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 7 dpi n = 4.ADDITIONAL FILE 8. T cell activation in the IEL population and spleen of broiler chickens upon SE infection. A Percentages of intraepithelial CD8+ T cells expressing CD107 (including both γδ and αβ T cells) in uninfected (uninf) and SE-infected (SE-inf) chickens in the course of time. B Percentages of splenic CD8+ T cells expressing CD107 (including both γδ and αβ T cells), C CD8+ γδ T cells expressing IFNγ, D CD4+ αβ T cells expressing IFNγ and E CD8+ αβ T cells expressing IFNγ in uninfected (uninf) and SE-infected (SE-inf) chickens over time. Mean + SEM per treatment and time point is shown (n = 5), for uninfected chickens at 7 dpi in spleen n = 4 and at 1 and 3 dpi in the IELs percentages were not determined (n.d.) due to numbers of events acquired in the gate of interest were < 100.The Dutch Research Council (NWO) and by Cargill Animal Nutrition and Health.http://www.veterinaryresearch.orgpm2022Veterinary Tropical Disease

Overcoming scientific barriers in the transition from in vivo to non-animal batch testing of human and veterinary vaccines

INTRODUCTION : Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. AREAS COVERED : This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. EXPERT OPINION : For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.The Dutch Ministry of Agriculture, Nature and Food Quality, the National Institute of Public Health & the Environment and Intravacc.https://www.tandfonline.com/loi/ierv20am2022Veterinary Tropical Disease

Nitric oxide production and Fc receptor-mediated phagocytosis as functional readouts of macrophage activity upon stimulation with inactivated poultry vaccines in vitro

Vaccine batches must pass routine quality control to confirm that their ability to induce protection against disease is consistent with batches of proven efficacy from development studies. For poultry vaccines, these tests are often performed in laboratory chickens by vaccination-challenge trials or serological assays. The aim of this study was to investigate innate immune responses against inactivated poultry vaccines and identify candidate immune parameters for in vitro quality tests as alternatives for animal-based quality tests. For this purpose, we set up assays to measure nitric oxide production and phagocytosis by the macrophage-like cell line HD11, upon stimulation with inactivated poultry vaccines for infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and egg drop syndrome virus (EDSV). In both assays, macrophages became activated after stimulation with various toll-like receptor agonists. Inactivated poultry vaccines stimulated HD11 cells to produce nitric oxide due to the presence of mineral oil adjuvant. Moreover, inactivated poultry vaccines were found to enhance Fc receptor-mediated phagocytosis due to the presence of allantoic fluid in the vaccine antigen preparations. We showed that inactivated poultry vaccines stimulated nitric oxide production and Fc receptor-mediated phagocytosis by chicken macrophages. Similar to antigen quantification methods, the cell-based assays described here can be used for future assessment of vaccine batch-to-batch consistency. The ability of the assays to determine the immuno-potentiating properties of inactivated poultry vaccines provides an additional step in the replacement of current in vivo batch-release quality tests.The Innovative Medicines Initiative 2 Joint Undertakinghttp://www.mdpi.com/journal/vaccinesam2021Veterinary Tropical Disease

Macrophage activation assays to evaluate the immunostimulatory capacity of avibacterium paragallinarum in a multivalent poultry vaccine

High-quality vaccines are crucial to prevent infectious disease outbreaks in the poultry industry. In vivo vaccination tests are routinely used to test poultry vaccines for their potency, i.e., their capacity to induce protection against the targeted diseases. A better understanding of how poultry vaccines activate immune cells will facilitate the replacement of in vivo potency tests for in vitro assays. Using the chicken macrophage-like HD11 cell line as a model to evaluate innate immune responses, the current explorative study addresses the immunostimulatory capacity of an inactivated multivalent vaccine for infectious bronchitis, Newcastle disease, egg-drop syndrome, and infectious coryza. The vaccine stimulated HD11 cells to produce nitric oxide and to express pro-inflammatory cytokines IL-1 , TNF, and IL-12p40, chemokines CXCLi1 and CXCLi2, and the anti-inflammatory cytokine IL-10, but only when inactivated Avibacterium paragallinarum, the causative agent of infectious coryza, was present. Lipopolysaccharides from Avibacterium paragallinarum were crucial for the production of nitric oxide and expression of IL-1 and CXCLi1. The described immune parameters demonstrate the capacity of this multivalent vaccine to activate innate immune cells and may in the future, combined with antigen quantification methods, contribute to vaccine quality testing in vitro, hence the replacement of current in vivo vaccination tests.Figure S1: Cytotoxicity of the octavalent vaccine, extracted antigens and purified Av. paragallinarum bacteria, Figure S2: Gram staining of extracted antigens from the tri- and octavalent vaccines, Figure S3: The octavalent vaccine-induced gene expression of IL-10, whereas a trivalent vaccine without Av. paragallinarum antigens did not.The Innovative Medicines Initiative 2 Joint Undertakinghttp://www.mdpi.com/journal/vaccinesam2021Veterinary Tropical Disease

Training of primary chicken monocytes results in enhanced pro-inflammatory responses

Beta-glucan-stimulated mammalian myeloid cells, such as macrophages, show an increased responsiveness to secondary stimulation in a nonspecific manner. This phenomenon is known as trained innate immunity and is important to prevent reinfections. Trained innate immunity seems to be an evolutionary conserved phenomenon among plants, invertebrates and mammalian species. Our study aimed to explore the training of primary chicken monocytes. We hypothesized that primary chicken monocytes, similar to their mammalian counterparts, can be trained with β-glucan resulting in increased responses of these cells to a secondary stimulus. Primary blood monocytes of white leghorn chickens were primary stimulated with β-glucan microparticulates (M-βG), lipopolysaccharide (LPS), recombinant chicken interleukin-4 (IL-4) or combinations of these components for 48 h. On day 6, the primary stimulated cells were secondary stimulated with LPS. Nitric oxide (NO) production levels were measured as an indicator of pro-inflammatory activity. In addition, the cells were analyzed by flow cytometry to characterize the population of trained cells and to investigate the expression of surface markers associated with activation. After the secondary LPS stimulation, surface expression of colony stimulating factor 1 receptor (CSF1R) and the activation markers CD40 and major histocompatibility complex class II (MHC-II) was higher on macrophages that were trained with a combination of M-βG and IL-4 compared to unstimulated cells. This increased expression was paralleled by enhanced NO production. In conclusion, this study showed that trained innate immunity can be induced in primary chicken monocytes with β-glucan, which is in line with previous experiments in mammalian species. Innate immune training may have the potential to improve health and vaccination strategies within the poultry sector

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types

A method to differentiate chicken monocytes into macrophages with proinflammatory properties

Macrophages are part of the first line of defense against invading pathogens. In mammals, the in vitro culture of macrophages from blood monocytes or bone marrow cells is well established, including culturing conditions to differentiate them towards M1 or M2-like macrophages. In chicken, monocyte-derived macrophages have been used in several studies, but there is no uniform protocol or actual characterization of these cells. Therefore, to generate proinflammatory M1-like macrophages, in this study blood monocytes were differentiated using GM-CSF for 4 days and characterized based on cell morphology, surface marker expression and cytokine expression response to TLRs stimulation at each (daily) time point. Cell morphology showed that one-day-cultured cells contained a mixture of cell populations, while the homogenous population of cells on day 3 and day 4 were flat and had a ‘fried-egg’ like shape, similar to human M1 macrophages. In addition, cell surface marker staining showed that 3 and 4- days-cultured cells expressed a high level of MRC1L-B (KUL01) and MHC-II. Furthermore, LPS stimulation of the cultured cells induced gene expression of the proinflammatory cytokines IL-1β, IL-6 and IL-8 after 3 days of culture. Finally, it was shown that day 3 macrophages were able to phagocytose avian pathogenic E. coli (APEC) and respond by nitric oxide production. Overall, our systematic characterization of the monocyte derived cells from blood showed that a 3-days culture was optimal to obtain pro-inflammatory M1 like macrophages, increasing our knowledge about chicken macrophage polarization and providing useful information for studies on chicken macrophage phenotypes

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens