Analysis of chicken intestinal natural killer cells, a major IEL subset during embryonic and early life

Restrictions on antimicrobials demand alternative strategies to improve broiler health, such as supplying feed additives which stimulate innate immune cells like natural killer (NK) cells. The main objective of this study was to characterize intestinal NK cells in broiler chickens during embryonic and early life and compare these to NK cells in spleen, blood and bone marrow. Also T-cell subsets were determined. The majority of intestinal NK cells expressed IL-2Rα rather than 20E5 and 5C7, and showed low level of activation. Within intestinal NK cells the activation marker CD107 was mostly expressed on IL-2Rα+ cells while in spleen and blood 20E5+ NK cells primarily expressed CD107. High percentages of intestinal CD8αα+, CD8αβ+ and from 2 weeks onward also gamma delta T cells were found. Taken together, we observed several intestinal NK subsets in broiler chickens. Differences in NK subsets were mostly observed between organs, rather than differences over time. Targeting these intestinal NK subsets may be a strategy to improve immune-mediated resistance in broiler chickens.SUPPLEMENTARY MATERIAL : Table S1. Characterization of immune cells generated in broiler chickens in the present study compared to data known in layer chickens.The Dutch Research Council (NWO) and by Cargill Animal Nutrition and Health.http://www.elsevier.com/locate/desalhj2022Veterinary Tropical Disease

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

Monitoring COVID‐19 vaccine effectiveness against COVID‐19 hospitalisation and death using electronic health registries in ≥65 years old population in six European countries, October 2021 to November 2022

Background: Within the ECDC-VEBIS project, we prospectively monitored vaccine effectiveness (VE) against COVID-19 hospitalisation and COVID-19-related death using electronic health registries (EHR), between October 2021 and November 2022, in community-dwelling residents aged 65-79 and ≥80 years in six European countries. Methods: EHR linkage was used to construct population cohorts in Belgium, Denmark, Luxembourg, Navarre (Spain), Norway and Portugal. Using a common protocol, for each outcome, VE was estimated monthly over 8-week follow-up periods, allowing 1 month-lag for data consolidation. Cox proportional-hazards models were used to estimate adjusted hazard ratios (aHR) and VE = (1 - aHR) × 100%. Site-specific estimates were pooled using random-effects meta-analysis. Results: For ≥80 years, considering unvaccinated as the reference, VE against COVID-19 hospitalisation decreased from 66.9% (95% CI: 60.1; 72.6) to 36.1% (95% CI: -27.3; 67.9) for the primary vaccination and from 95.6% (95% CI: 88.0; 98.4) to 67.7% (95% CI: 45.9; 80.8) for the first booster. Similar trends were observed for 65-79 years. The second booster VE against hospitalisation ranged between 82.0% (95% CI: 75.9; 87.0) and 83.9% (95% CI: 77.7; 88.4) for the ≥80 years and between 39.3% (95% CI: -3.9; 64.5) and 80.6% (95% CI: 67.2; 88.5) for 65-79 years. The first booster VE against COVID-19-related death declined over time for both age groups, while the second booster VE against death remained above 80% for the ≥80 years. Conclusions: Successive vaccine boosters played a relevant role in maintaining protection against COVID-19 hospitalisation and death, in the context of decreasing VE over time. Multicountry data from EHR facilitate robust near-real-time VE monitoring in the EU/EEA and support public health decision-making.European Centre for Disease Prevention and Control, Grant/Award Numbers ECDC/2021/018, RS/2022/DTS/24104.info:eu-repo/semantics/publishedVersio

Glucose oligosaccharide and long-chain glucomannan feed additives induce enhanced activation of intraepithelial NK cells and relative abundance of commensal lactic acid bacteria in broiler chickens

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and longchain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.The Dutch Research Council (NWO) in conjunction with Cargill Animal Nutrition and Health in the context of stimulating Public–Private research collaboration and part of the research program of NWO Earth and Life Sciences (ALW).http://www.mdpi.com/journal/vetscipm2022Veterinary Tropical Disease

Relative vaccine effectiveness against COVID-19 hospitalisation in persons aged ≥ 65 years: results from a VEBIS network, Europe, October 2021 to July 2023

VEBIS-Lot 4 working group: James Humphreys, Alexis Sentís, Joris Van Loenhout, Pierre Hubin, Katrine Finderup Nielsen, Chiara Sacco, Daniele Petrone, Patrizio Pezzotti, Itziar Casado, Aitziber Echeverria, Camino Trobajo-Sanmartín, Stijn Andeweg, Anja Bråthen Kristoffersen, Irina Kislaya, Patricia Soares, Carlos Dias, Ausenda Machado.Since 2021, the Vaccine Effectiveness, Burden and Impact Studies of coronavirus disease 2019 (COVID-19) and influenza (VEBIS) project monitors vaccine effectiveness (VE) in real-world conditions to inform vaccination programmes in the European Union/European Economic Area (EU/EEA) countries [1]. One project aims to monitor real-time COVID-19 VE using electronic health registries (EHR) in multiple countries, with initial findings previously published [2-4]. We report pooled VE results against hospitalisation due to COVID-19 by number of doses received and time since vaccination in a community-dwelling resident population aged ≥ 65 years between October 2021 and July 2023.info:eu-repo/semantics/publishedVersio

Estimated number of deaths directly averted in people 60 years and older as a result of COVID-19 vaccination in the WHO European Region, December 2020 to November 2021

Since December 2019, over 1.5 million SARS-CoV-2-related fatalities have been recorded in the World Health Organization European Region - 90.2% in people ≥ 60 years. We calculated lives saved in this age group by COVID-19 vaccination in 33 countries from December 2020 to November 2021, using weekly reported deaths and vaccination coverage. We estimated that vaccination averted 469,186 deaths (51% of 911,302 expected deaths; sensitivity range: 129,851-733,744; 23-62%). Impact by country ranged 6-93%, largest when implementation was early.S

Improving health of broiler chickens by early life modulation of immune responsiveness and intestinal microbiota : Recruiting Natural Killer cells for increased resistance

The poultry sector provides a major contribution to the world food supplies and its meat and egg production is expected to increase sharply in the next ten years, leading to poultry becoming the most widely consumed meat. Hence, the health of broiler chickens is of great importance, however, the current ban on preventive treatment of antimicrobials has led to an increase in intestinal health problems in poultry. This urges for alternative strategies to improve health of broiler chickens. Young chickens are more susceptible to infectious diseases than adult ones since the adaptive immune system is not fully developed yet. Therefore, strategies should aim at increase of resistance to pathogens early in the life of chickens. At that stage, the immune responsiveness of chickens to invading pathogens relies on the innate immune system and natural killer (NK) cells, abundantly present within the intestinal intraepithelial lymphocyte (IEL) population, are among the first to respond. The aim of this thesis was to investigate stimulation of immune responsiveness, modulation of intestinal microbiota and the interaction between these by nutritional supplementation in order to increase the resistance and consequently improve the health of young broiler chickens. For this purpose, number and functions of natural killer (NK) and T cell subsets in the intestine and other tissues of broiler chickens were assessed and acquired insights used for subsequent studies pertaining modulation of these cells. Furthermore, the interaction between the immune system and intestinal microbiota in young broiler chickens was investigated by early exposure to adult-derived microbiota. This showed to accelerate the maturation of the intestinal microbiota paralleled by increased NK cell responsiveness. Then, the potential of selected nutritional strategies to modulate immune responsiveness and microbiota composition was investigated. Nutritional compounds were consecutively screened for enhanced activity of NK cells in vitro, and evaluated for safety during embryonic development in ovo and impact on performance traits in vivo. Based on these results, two nutritional strategies were selected for further investigation that showed potential to stimulate immune responsiveness directly and indirectly through modulation of the intestinal microbiota. Furthermore, a significant role for NK cells in the immune response to Salmonella enterica serotype Enteritidis (SE), an important intestinal pathogen in chickens, was recognized. Finally, nutritional supplementation resulted in increased resistance to SE infection in young broiler chickens by stimulation of NK cell responsiveness, modulation of intestinal microbiota as well as the interaction between these. In conclusion, the studies described in this dissertation contribute to the development of alternative strategies to increase the resistance of young chickens and consequently, to improve the health of chickens

Improving health of broiler chickens by early life modulation of immune responsiveness and intestinal microbiota : Recruiting Natural Killer cells for increased resistance

The poultry sector provides a major contribution to the world food supplies and its meat and egg production is expected to increase sharply in the next ten years, leading to poultry becoming the most widely consumed meat. Hence, the health of broiler chickens is of great importance, however, the current ban on preventive treatment of antimicrobials has led to an increase in intestinal health problems in poultry. This urges for alternative strategies to improve health of broiler chickens. Young chickens are more susceptible to infectious diseases than adult ones since the adaptive immune system is not fully developed yet. Therefore, strategies should aim at increase of resistance to pathogens early in the life of chickens. At that stage, the immune responsiveness of chickens to invading pathogens relies on the innate immune system and natural killer (NK) cells, abundantly present within the intestinal intraepithelial lymphocyte (IEL) population, are among the first to respond. The aim of this thesis was to investigate stimulation of immune responsiveness, modulation of intestinal microbiota and the interaction between these by nutritional supplementation in order to increase the resistance and consequently improve the health of young broiler chickens. For this purpose, number and functions of natural killer (NK) and T cell subsets in the intestine and other tissues of broiler chickens were assessed and acquired insights used for subsequent studies pertaining modulation of these cells. Furthermore, the interaction between the immune system and intestinal microbiota in young broiler chickens was investigated by early exposure to adult-derived microbiota. This showed to accelerate the maturation of the intestinal microbiota paralleled by increased NK cell responsiveness. Then, the potential of selected nutritional strategies to modulate immune responsiveness and microbiota composition was investigated. Nutritional compounds were consecutively screened for enhanced activity of NK cells in vitro, and evaluated for safety during embryonic development in ovo and impact on performance traits in vivo. Based on these results, two nutritional strategies were selected for further investigation that showed potential to stimulate immune responsiveness directly and indirectly through modulation of the intestinal microbiota. Furthermore, a significant role for NK cells in the immune response to Salmonella enterica serotype Enteritidis (SE), an important intestinal pathogen in chickens, was recognized. Finally, nutritional supplementation resulted in increased resistance to SE infection in young broiler chickens by stimulation of NK cell responsiveness, modulation of intestinal microbiota as well as the interaction between these. In conclusion, the studies described in this dissertation contribute to the development of alternative strategies to increase the resistance of young chickens and consequently, to improve the health of chickens

Variation of the EuII emission wavelength by substitution of fluoride by hydride in fluorite-type compounds EuHxF2-x (0.20 ≤ x ≤ 0.67)

Mixed-hydride fluorides EuHxF2-x were prepared by the solid-state reaction of EuF2 and EuH2 under hydrogen gas pressure in an autoclave. EuII luminescence is observed for 0.20 > x > 0.67, while pure EuF2 does not show any emission. The energy of the emission depends strongly on the degree of substitution x. For low hydride contents, yellow emission is observed, whereas higher hydride contents lead to red emission. The red shift is attributed to the nephelauxetic effect of the hydride anion. Remarkably, limited concentration quenching is observed in EuHxF2-x (0.20 > x > 0.67). This observation is explained by suppression of long-range energy migration due to disorder in the local environment of Eu2+ in the mixed H/F crystals. The strong x dependence of the luminescence maxima proves hydride-fluoride substitution to be a valuable tool to tune the emission wavelength of EuII-containing phosphors

Variation of the Eu^II Emission Wavelength by Substitution of Fluoride by Hydride in Fluorite-Type Compounds EuH_<i>x</i>F_2–<i>x</i> (0.20 ≤ <i>x</i> ≤ 0.67)

Mixed-hydride fluorides EuH_<i>x</i>F_2–<i>x</i> were prepared by the solid-state reaction of EuF₂ and EuH₂ under hydrogen gas pressure in an autoclave. Eu^II luminescence is observed for 0.20 ≤ <i>x</i> ≤ 0.67, while pure EuF₂ does not show any emission. The energy of the emission depends strongly on the degree of substitution <i>x</i>. For low hydride contents, yellow emission is observed, whereas higher hydride contents lead to red emission. The red shift is attributed to the nephelauxetic effect of the hydride anion. Remarkably, limited concentration quenching is observed in EuH_<i>x</i>F_2–<i>x</i> (0.20 ≤ <i>x</i> ≤ 0.67). This observation is explained by suppression of long-range energy migration due to disorder in the local environment of Eu²⁺ in the mixed H/F crystals. The strong <i>x</i> dependence of the luminescence maxima proves hydride–fluoride substitution to be a valuable tool to tune the emission wavelength of Eu^II-containing phosphors