Chicken Peripheral Blood Mononuclear Cells Research Articles

Citrus pectin modulates chicken peripheral blood mononuclear cell proteome in vitro

Citrus pectin (CP) is a dietary fiber used in animal nutrition with anti-inflammatory properties. CP downregulates chicken immunoregulatory monocytes' functions, like chemotaxis and phagocytosis, in vitro. The molecular underlying background is still unknown. This study investigated the activity of CP on chicken peripheral blood mononuclear cells (PBMC) proteome. An overall number of 1503 proteins were identified and quantified. The supervised sparse variant partial least squares-discriminant analysis (sPLS-DA) for paired data highlighted 373 discriminant proteins between CP-treated and the control group, of which 50 proteins with the highest abundance in CP and 137 in the control group were selected for Gene Ontology (GO) analyses using ProteINSIDE. Discriminant Protein highly abundant in CP-treated cells were involved in actin cytoskeleton organization and negative regulation of cell migration. Interestingly, MARCKSL1, a chemotaxis inhibitor, was upregulated in CP-treated cells. On the contrary, CP incubation downregulated MARCKS, LGALS3, and LGALS8, which are involved in cytoskeleton rearrangements, cell migration, and phagocytosis. In conclusion, these results provide a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro downregulation of phagocytosis and chemotaxis is related to changes in proteins related to the cytoskeleton.

Flagellin Enhances the Immunogenicity of Pasteurella multocida Lipoprotein E Subunit Vaccine.

Fowl cholera, caused by Pasteurella multocida infection, poses challenges for prevention because of its many serotypes. Bacterins are currently widely used for vaccination against fowl cholera, but protection is limited to homologous strains. Live attenuated vaccines of P. multocida provide some heterologous protection, but side effects are considerable. More recently, protein-based antigens are promising subunit vaccines when their low immunogenicity has been addressed with effective adjuvants. Bacterial flagellin has been widely considered a promising adjuvant for vaccines. In this study, we tested the adjutancy of flagellin in a subunit vaccine against P. multocida in a mice and chicken models. For vaccine formulation, the antigen fPlpE (P. multocida liporotein E) was combined with fFliC (Salmonella Typhimurium flagellin). The recombinant proteins of fPlpE and fFliC were successfully expressed using the Escherichia coli system as the expected sizes of 55 kDa and 70 kDa, respectively. The fFliC elicited strong expression levels of proinflammatory cytokine (IL-1β, IL-8, and IL-6) when stimulated in native chicken peripheral blood mononuclear cells. Immunization of mice and chickens with the subunit vaccines containing fFliC accelerated the antibody response. In the challenge tests, fFliC increased vaccine protective efficacy against the heterologous strain P. multocida A1 and highly virulent strain Chu01 in mice and chickens, respectively. These data indicated potential possibilities of using fFliC as an immunostimulant adjuvant in developing a subunit vaccine against fowl cholera.

Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant

BackgroundIn recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed.ResultsThe in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group.ConclusionThese results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Inhibitory effect of Eimeria maxima IFN-γ inhibitory molecules on the immune function of T cell subsets in chickens

It has been reported that infection of chicken coccidian could inhibit the production of Th1 cytokine IFN-γ, thereby evading clearance by the host immune system. The present study aimed to have a further investigation into the effects of Eimeria maxima IFN-γ inhibitory molecules (EmHPSP-2 and EmHPSP-3) on the immune function of chicken peripheral blood mononuclear cells (PBMC) and various T cell subsets. First, separated PBMC or sorted T cell subsets were used for incubation with recombinant proteins of EmHPSP-2 (rEmHPSP-2) and EmHPSP-3 (rEmHPSP-3). Subsequently, the effects of rEmHPSP-2 and rEmHPSP-3 on proliferative capacity, nitric oxide (NO) release and mRNA levels of cytokines of the above cells were detected. The sorting purity of CD8+, CD4+ CD25-, CD4+, and CD4+ CD25+ T cells was 93.01, 88.88, 87.04, and 81.26%, respectively. The NO release of PBMC was significantly inhibited by rEmHPSP-2 and rEmHPSP-3. The proliferation of PBMC and CD4+ T cells was significantly inhibited by rEmHPSP-2 and rEmHPSP-3, whereas CD8+, CD4+ CD25-, and CD4+ CD25+ T cells was significantly promoted by the 2 proteins. The 2 proteins significantly downregulated interferon-gamma (IFN-γ) mRNA level, upregulated the transcriptional levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) in PBMC. IFN-γ and IL-2 transcriptional levels were markedly inhibited in CD8+ T cells. IFN-γ transcriptional level was significantly inhibited, but IL-4 was promoted by rEmHPSP-2 and rEmHPSP-3 in CD4+ CD25- T cells. Meanwhile, the inhibitory effects of rEmHPSP-2 and rEmHPSP-3 on the transcriptional levels of IFN-γ and IL-2 were more obvious in CD4+ T cells containing CD25+ cells compared with the CD25+ cells depletion group. It was found that IL-10, TGF-β1, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) mRNA levels were significantly upregulated upon stimulation of chicken CD4+ CD25+ T cells by proteins. This study is not only of great significance to clarify the immune evasion mechanism of chicken coccidia, but also provides candidate antigen molecules for development of a novel vaccine against chicken coccidiosis.

Role of Macrophages in the Pathogenesis of Genotype VII Newcastle Disease Virus in Chickens

Long-term evolution of Newcastle disease virus (NDV) results in substantial alteration in viral pathogenesis. NDVs of genotype VII, a late genotype, show marked tropism to lymphoid tissues, especially to macrophages in chickens. However, the role of macrophages in the pathogenesis of genotype VII NDV is still unclear. Herein, NDV infectivity in macrophages and the role of macrophages in the pathogenesis of genotype VII NDV in chickens were investigated. We reported that NDV strains of genotype VII (JS5/05) and IV (Herts/33) can replicate in the adherent (predominantly macrophages) and non-adherent cells (predominantly lymphocytes) derived from chicken peripheral blood mononuclear cells (PBMCs), and significantly higher virus gene copy was detected in the adherent cells. In addition, JS5/05 had significantly higher infectivity in PBMC-derived adherent cells than Herts/33, correlating with its enhanced tropism to macrophages in the spleen of chickens. Interestingly, the depletion of 68% of macrophages exerted no significant impact on clinical signs, mortality and the systematic replication of JS5/05 in chickens, which may be associated with the contribution of non-depleted macrophages and other virus-supportive cells to virus replication. Macrophage depletion resulted in a marked exacerbation of tissue damage and apoptosis in the spleen caused by JS5/05. These findings indicated that macrophages play a critical role in alleviating tissue damage caused by genotype VII NDV in chickens. Our results unveiled new roles of macrophages in NDV pathogenesis in chickens.

Expression of toll-like receptor 4 and its associated cytokines from peripheral blood mononuclear cells in Leghorn chickens.

Immune cells require toll-like receptor 4 (TLR4) to respond to lipopolysaccharides (LPS) by releasing pro-inflammatory cytokines. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or pathogens. This study aimed to assess TLR4 gene expression in PBMCs from Leghorn chicken and the release of related cytokines. Peripheral blood mononuclear cells were isolated from blood samples obtained from Leghorn chicks. The PBMC cultures were stimulated with various concentrations of LPS (0.01-1 μg/ml). Polymerase chain reaction was used to detect TLR4 expression. The production of tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1β and IL-6) was quantified using an enzyme-linked immunosorbent assay. We found that TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs. In addition, the release of TNF-α, IL-1β, and IL-6 levels in Leghorn chicken PBMCs increased significantly with an increase in LPS concentration (0.01-1 μg/mL) (p < 0.05). Although TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs, its expression was significantly higher in LPS-stimulated PBMCs Therefore, the chicken's endotoxin response can be assessed by evaluating the pro-inflammatory cytokine production from PBMCs.

Abstract 6791: Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma

Abstract BACKGROUND: Colorectal cancer (CRC) is the second-most deadly cancer. Therapeutic resistance to immuno-oncology drives the need for new treatments. Stimulated tumor cell (STC) vaccine (Brenus Pharma) is composed of selected tumor cell lines, stimulated to overexpress tumor-associated or tumor-specific antigens and neoantigens including resistance factors. Haptenization of these proteins forms an immunogenic complex which stimulate the immune system to recognize and target the patient’s tumor cells expressing same resistance factors. We report in vivo results of STC-1010 vaccine, on human CRC adenocarcinoma from HT29 cell using chorioallantoic membrane (CAM) assay developed by Inovotion in immune reactive model. METHODS: This study was carried out in 2 steps: Firstly, three batches of naïve chicken embryos were stimulated by injections of STC1010 at Embryo Development Day (EDD) 11 and EDD13. At EDD18 of each batch, chicken peripheral blood mononuclear cells (PBMCs) were collected and used as anti-tumor reagent to treat respectively, at EDD 11, EDD 13 and EDD16 chicken embryos xenografted with HT29 cells. Activation of PBMCs was evaluated by IL2 and IL12 secretion quantified by ELISA. At EDD18, i.e., 9 days post-graft, in ovo anti-tumor efficacy was evaluated by tumor weight, metastatic invasion (qPCR analysis of human Alu sequence in lower CAM) and quantification of tumor-infiltrating by CD8, CD4, IFN-gamma, Perforin and TNF-alpha. RESULTS: Compared to negative control, STC-1010 vaccine induced: 1) significant increase of IL-12 and IL-2 secretion in peripheric blood during the generation of all three batches of PBMCs, confirming previous results (IL-12: +52%, p=0.0003 ; IL-2: +482%, p=0.0033); 2) a significant expression of IFN-gamma in tumor (+130,83%, p=0.0185); 3) a tendency to increase infiltrating cells: CD4+: +79,2%, CD8+: +29,4% , Perforin: +105,5%, TNFα : +78,63% confirmed by immunohistochemistry and translated into 4) a significant increase of tumor necrosis (p = 0.0267); and 5) a tendency of metastasis regression (-49%); with 6) no embryonic toxicity/mortality (daily evaluation of embryonic viability) induced by STC-1010. CONCLUSION: This in ovo study confirms efficacy of the STC-1010 observed in previous CRC syngeneic models and gives more insight about STC mechanism of action with the activation and maturation of dendritic cells, induction of CD8+ and LTh1 against tumor as the main driver of the response, all without toxicity. Inovotion’s CAM model could be used for indication screening and as a pre-proof-of-concept before syngeneic model study. Citation Format: Yan Wang, Arnaud Peyronnier, Benoit Pinteur, Lionel Chalus, Corinne Tortorelli, Paul Bravetti, Jean Viallet, François Ghiringhelli, Philippe Fornies. Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6791.

PD-1/PD-L1 Checkpoint Inhibitors Are Active in the Chicken Embryo Model and Show Antitumor Efficacy In Ovo.

Simple SummaryCancer immunotherapy, also known as immuno-oncology (IO), has made impressive progress in recent decades and is becoming an essential approach for cancer treatments. For IO drug development, a pertinent preclinical model is indispensable for the rapid and efficient transition from preclinical evaluation through to clinical progress. To date, rodents represent the most-often used models for preclinical evaluation. However, their use presents several drawbacks, including ethical constraints, and time-consuming and costly experiments, which could slow down IO drug development. The aim of our study was to assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for evaluating IO drugs. We confirmed in ovo the anti-tumor efficacy of programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) checkpoint inhibitors based on the Chicken Chorioallantoic Membrane (CAM) assay, revealing the pertinence of the chicken embryo model in its use for IO research.(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3+, CD4+, CD8+ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell’s cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery.

Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Epigenetic Regulation of Host Defense Peptide Synthesis: Synergy Between Histone Deacetylase Inhibitors and DNA/Histone Methyltransferase Inhibitors.

Host defense peptides (HDPs) are an integral part of the innate immune system acting as the first line of defense. Modulation of HDP synthesis has emerged as a promising host-directed approach to fight against infections. Inhibition of histone deacetylation or DNA methylation is known to enhance HDP gene expression. In this study, we explored a possible synergy in HDP gene induction between histone deacetylase inhibitors (HDACi) and DNA/histone methyltransferase inhibitors (DNMTi/HMTi). Two chicken macrophage cell lines were treated with structurally distinct HDACi, HMTi, or DNMTi individually or in combinations, followed by HDP gene expression analysis. Each epigenetic compound was found to be capable of inducing HDP expression. To our surprise, a combination of HDACi and HMTi or HDACi and DNMTi showed a strong synergy to induce the expressions of most HDP genes. The HDP-inducing synergy between butyrate, an HDACi, and BIX01294, an HMTi, were further verified in chicken peripheral blood mononuclear cells. Furthermore, tight junction proteins such as claudin 1 were also synergistically induced by HDACi and HMTi. Overall, we conclude that HDP genes are regulated by epigenetic modifications. Strategies to increase histone acetylation while reducing DNA or histone methylation exert a synergistic effect on HDP induction and, therefore, have potential for the control and prevention of infectious diseases.

Pro-inflammatory cytokine release from chicken peripheral blood mononuclear cells stimulated with lipopolysaccharide.

Background and Aim:The principal cytokines released by the host on infection include pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF). These cytokines were regarded as regulators of the host’s response to infection. This study aimed to determine the release of pro-inflammatory cytokines from chicken peripheral blood mononuclear cells (PBMCs) following lipopolysaccharide (LPS) stimulation.Materials and Methods:Blood samples were collected from six Betong chickens. To isolate PBMCs, density gradient centrifugation was utilized. PBMC culture in RPMI1640 with 10% fetal bovine serum was stimulated with various concentrations of LPS (0, 0.01, 0.1, and 1.0 μg/mL). The production of TNF-α, IL-1β, and IL-6 was determined using an enzyme-linked immunosorbent assay.Results:When the PBMCs were cultured for 24 h with varying doses of LPS, there was no significant variation in cell viability. TNF-α, IL-1β, and IL-6 levels were measured in Betong chicken PBMC. The release of these cytokines increased considerably as LPS concentration (0.01-1 μg/mL) increased (p<0.05).Conclusion:In vitro studies of the chicken immune response, notably the release of pro-inflammatory cytokines, can be conducted using PBMCs obtained from chicken blood.

Chicken Peripheral Blood Mononuclear Cells Response to Avian Leukosis Virus Subgroup J Infection Assessed by Single-Cell RNA Sequencing.

Chicken peripheral blood mononuclear cells (PBMCs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Here we performed single-cell RNA sequencing (scRNA-seq) of PBMCs in Avian leukosis virus subgroup J (ALV-J) infected and control chickens at 21 days post infection (DPI) to determine chicken PBMCs subsets and their specific molecular and cellular characteristics. Eight cell populations and their potential marker genes were identified in PBMCs. T cell populations had the strongest response to (ALV-J) infection, based on the detection of the largest number of differentially expressed genes (DEGs), and could be further grouped into four subsets: activated CD4+ T cells, Th1-like cells, Th2-like cells, and cytotoxic CD8+ T cells. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection activated CD4+ T cell was probably inclined to differentiate into Th1-like cells. Compared to the control PBMCs, ALV-J infection also had an obvious impact on PBMCs composition. B cells showed inconspicuous response and their numbers decreased in PBMCs from ALV-J infected chicken. Proportions of cytotoxic Th1-like cells and CD8+ T cells increased in the T cell population of PBMCs from ALV-J infected chicken, which were potentially key mitigating effectors against ALV-J infection. More importantly, our results provide a rich resource of gene expression profiles of chicken PBMCs subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

Natural Cyclooxygenase-2 Inhibitors Synergize With Butyrate to Augment Chicken Host Defense Peptide Gene Expression.

Enhancing the synthesis of microbicidal and immunomodulatory host defense peptides (HDP) is a promising host-directed antimicrobial strategy to combat a growing threat of antimicrobial resistance. Here we investigated the effect of several natural cyclooxygenase-2 (COX-2) inhibitors on chicken HDP gene regulation. Our results indicated that phenolic COX-2 inhibitors such as quercetin, resveratrol, epigallocatechin gallate, anacardic acid, and garcinol enhanced HDP gene expression in chicken HTC macrophage cell line and peripheral blood mononuclear cells (PBMCs). Moreover, these natural COX-2 inhibitors showed a strong synergy with butyrate in augmenting the expressions of multiple HDP genes in HTC cells and PBMCs. Additionally, quercetin and butyrate synergistically promoted the expressions of mucin-2 and claudin-1, two major genes involved in barrier function, while suppressing lipopolysaccharide-triggered interleukin-1β expression in HTC macrophages. Mechanistically, we revealed that NF-κB, p38 mitogen-activated protein kinase, and cyclic adenosine monophosphate signaling pathways were all involved in the avian β-defensin 9 gene induction, but histone H4 was not hyperacetylated in response to a combination of butyrate and quercetin. Because of their HDP-inducing, barrier-protective, and antiinflammatory activities, these natural COX-2 inhibitors, when combined with butyrate, may be developed as novel host-directed antimicrobial therapeutics.

Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment.

Cellular metabolic preference is a culmination of environment, nutrition, genetics, and individual variation in poultry. The Seahorse XFe24 analyzer was used to generate foundational immune cellular metabolic data in layer, broiler, and legacy genetic strains using fresh chicken peripheral blood mononuclear cells (PBMCs). Baseline mitochondrial respiration [oxygen consumption rate (OCR)] and glycolytic activity [extracellular acidification rate (ECAR)] were determined in modern commercial laying hen (Bovans White) and broiler (Ross 308) lines, as well as the highly inbred lines of Iowa State University (L8, Fayoumi M-15.2, Spanish, Ghs-6), partially inbred broiler line, and advanced intercrosses of broiler by Fayoumi M-15.2 and broiler by Leghorn lines. Commercial broiler vs. Bovans layer and unvaccinated vs. vaccinated Bovans layer immune cell metabolic potential were compared following an in-assay pathway inhibitor challenge. Titrations consistently showed that optimal PBMC density in laying hens and broilers was 3 million cells per well monolayer. Assay media substrate titrations identified 25 mM glucose, 1 mM glutamine, and 1 mM sodium pyruvate as the optimal concentration for layer PBMCs. Pathway inhibitor injection titrations in Bovans layers and broilers showed that 0.5 μM carbonyl cyanide-4 phenylhydrazone (FCCP) and 1 μM oligomycin were optimal. Baseline OCR and ECAR were significantly affected by genetic line of bird (p < 0.05), with the dual-purpose, L8 inbred line showing the highest OCR (mean 680 pmol/min) and the partially inbred broiler line showing the greatest ECAR (mean 74 mpH/min). ECAR metabolic potential tended to be greater in modern layers than broilers (p < 0.10), indicating increased ability to utilize the glycolytic pathway to produce energy. OCR was significantly higher in vaccinated than unvaccinated hens (p < 0.05), while baseline ECAR values were significantly lower in vaccinated Bovans laying hens, showing increased oxidative capacity in activated immune cells. These baseline data indicate that different genetic strains of birds utilized the mitochondrial respiration pathway differently and that modern commercial lines may have reduced immune cell metabolic capacity compared with legacy lines due to intense selection for production traits. Furthermore, the Seahorse assay demonstrated the ability to detect differences in cellular metabolism between genetic lines and immune status of chickens.

Immunostimulatory potential of Nigella sativa seeds extract on chicken macrophage function

In present study, the effect of Nigella sativa seeds extract on the chicken peripheral blood mononuclear cells (PBMCs) was investigated. The chicken PBMCs were stimulated with two different doses (125?g/ml; 250?g/ml) of Nigella sativa seeds extract and the cells were harvested at different time points till 48h post-stimulation for analysis of iNOS gene expression by quantitative PCR and nitric oxide (NO) estimation at 24 and 48h post-stimulation. High dose (250?g/ml) of Nigella sativa seed extract showed remarkable induction of iNOS transcripts expression and NO production at 48h post-stimulation, which were 12.8040±1.03347 folds and 5.7089±.64535µM respectively. The results indicated the immunostimulatory potential of the Nigella sativa seed extract on the chicken PBMCs.

Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro.

Simple SummaryKnowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and promote the health of animals. To investigate the mechanisms of action of immunomodulating compounds, controlled in vitro systems using freshly isolated immune cells from blood represent a promising alternative to animal experiments. Immune cell isolation from the blood of chickens is a complex and difficult process since the immune cell fractions are significantly contaminated with red blood cells and platelets. To our knowledge, a robust protocol for immune cell isolation from chicken blood and the subsequent cultivation of immune cells is not available. Here, we established a protocol for blood sampling and immune cell isolation and cultivation from chicken blood, which could be applied for the investigation of direct effects of immunomodulating compounds. This protocol, combining different techniques of immune cell isolation, cultivation, and differentiation of distinct immune cell populations, will serve as a potential alternative to animal testing in vivo. By gaining knowledge about the mechanisms of action of immunomodulating compounds, this in vitro model will contribute to promote health and welfare in chicken farming.Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

The Transcriptional Differences of Avian CD4+CD8+ Double-Positive T Cells and CD8+ T Cells From Peripheral Blood of ALV-J Infected Chickens Revealed by Smart-Seq2.

It is well known that chicken CD8+ T cell response is vital to clearing viral infections. However, the differences between T cell subsets expressing CD8 receptors in chicken peripheral blood mononuclear cells (PBMCs) have not been compared. Herein, we used Smart-Seq2 scRNA-seq technology to characterize the difference of chicken CD8high+, CD8high αα+, CD8high αβ+, CD8medium+, and CD4+CD8low+ T cell subsets from PBMCs of avian leukosis virus subgroup J (ALV-J)-infected chickens. Weighted gene co-expression network analysis (WGCNA) and Trend analysis revealed that genes enriched in the “Cytokine–cytokine receptor interaction” pathway were most highly expressed in the CD8high αα+ T cell population, especially T cell activation or response-related genes including CD40LG, IL2RA, IL2RB, IL17A, IL1R1, TNFRSF25, and TNFRSF11, suggesting that CD8high αα+ T cells rather than other CD8 subpopulations were more responsive to ALV-J infections. On the other hand, genes involved in the “FoxO signaling pathway” and “TGF-beta signaling pathway” were most highly expressed in the CD4+CD8low+ (CD8low+) T cell population and the function of CD4+CD8low+ T cells may play roles in negatively regulating the functions of T cells based on the high expression of CCND1, ROCK1, FOXO1, FOXO3, TNFRSF18, and TNFRSF21. The selected gene expressions in CD8+ T cells and CD4+CD8low+ double-positive T cells confirmed by qRT-PCR matched the Smart-Seq2 data, indicating the reliability of the smart-seq results. The high expressions of Granzyme K, Granzyme A, and CCL5 indicated the positive response of CD8+ T cells. Conversely, CD4+CD8+ T cells may have the suppressor activity based on the low expression of activation molecules but high expression of T cell activity suppressor genes. These findings verified the heterogeneity and transcriptional differences of T cells expressing CD8 receptors in chicken PBMCs.

Assessment of chicken peripheral blood mononuclear cells isolated from freshly drawn blood versus 24 h refrigerated blood.

Background and Aim:The peripheral blood mononuclear cell (PBMC) is an excellent cell source for in vitro studies, particularly those involving immunology. The aim of this study was to determine the quality and quantity of chicken PBMCs isolated from freshly drawn blood as well as blood that had been chilled for 24 h. In addition, the survival of PBMCs cultured in medium was investigated.Materials and Methods:Blood samples were collected from 12 Betong and 12 Leghorn chickens. Hemograms were analyzed. Density gradient centrifugation was used to isolate PBMCs. PBMCs (2×106 cells/mL) were cultured in a culture medium and incubated in a CO2 incubator for 5 consecutive days. The number of viable cells was determined using the trypan blue dye exclusion method.Results:Blood samples were obtained from healthy chickens. There was no statistically significant difference in the total amount of PBMC between fresh and refrigerated blood samples from both chicken breeds. The viability of PBMCs isolated from fresh blood (95%) was significantly greater than blood refrigerated for 24 h (90-92%) in both breeds. Furthermore, the viability of PBMCs isolated from both blood samples decreased significantly over time, from 90-95% to 60-65%.Conclusion:The total number of PBMC in fresh and refrigerated blood was not significantly different. Fresh blood-derived PBMCs had significantly higher viability than 24 h refrigerated blood PBMCs. Furthermore, the viability of PBMCs decreased significantly over time.

TLR-Mediated Cytokine Gene Expression in Chicken Peripheral Blood Mononuclear Cells as a Measure to Characterize Immunobiotics.

Immunobiotics are probiotics that promote intestinal health by modulating immune responses. Immunobiotics are recognized by Toll-like receptors (TLRs) and activate cytokine gene expression. This study aimed to characterize cytokine gene expression in the chicken peripheral blood mononuclear cells (PBMC) stimulated with purified TLR ligands and live probiotics. PBMC were isolated from the whole blood. PBMC were stimulated with: lipopolysaccharide (LPS), CpG ODN, Pam3CSK4, Zymosan, galactooligosaccharides (GOS), Lactococcus lactis subsp. cremoris (L. lactis), and Saccharomyces cerevisiae at 42.5 °C and 5% CO2 for 3 h, 6 h, and 9 h. After each time-point, PBMC were harvested for RNA isolation. Relative gene expression was analyzed with RT-qPCR for cytokine genes (IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12p40, and IFN-ɣ) and reference genes (ACTB and G6PDH). Genes were clustered into pro-inflammatory genes, Th1/Th2 genes, and Th1-regulators. The gene expression differed between treatments in IL1-β, IL-6, IL-8, IL-10, and IL-12p40 (p < 0.001). The genes IL-1β, IL-6, and IL-8 had the highest fold change of mRNA expression at 3 h in response to TLR ligands. L. lactis up-regulated the pro-inflammatory genes at the 6 h time-point. L. lactis did not activate the anti-inflammatory IL-10 gene, but activated IL-12p40 at 6 h. Hereby, L. lactis was proven to exert immunostimulatory properties in PBMC.

Assay of extracellular matrix degradation and transmigration of chicken peripheral blood mononuclear cells after infection with genotype VII Newcastle disease virus in vitro

Previous studies showed that, compared to genotype IV Newcastle disease virus (NDV), genotype VII NDV induced extensive extracellular matrix (ECM) degradation by up-regulating the protein expression of matrix metalloproteinase (MMP)-14 in chicken spleens. To investigate potential relationship between MMP-14 function and the ECM degradation, an in vitro peripheral blood mononuclear cells (PBMCs) infection model was established to study the effect of genotype VII NDV (JS5/05) infection on MMP-14 expression, ECM degradation and cell transmigration. The gene and protein expression levels of MMP-14 in NDV-infected chicken PBMCs were measured by quantitative real-time PCR (qRT-PCR) and Western blot, and the subcellular location of MMP-14 was analyzed using immunofluorescence microscopy. A fluorescence-based collagen degradation assay was optimized to measure ECM degradation in PBMCs. Additionally, parameters of a transwell-based transmigration assay were also optimized to determine chemotaxis and transmigration of virus-infected PBMCs. The results showed that JS5/05 up-regulated significantly the expression of MMP-14 in PBMCs at the mRNA and protein levels compared to genotype IV NDV (Herts/33). MMP-14 was transported towards the membrane and accumulated on the cell surface of the JS5/05-infected cells, whereas it remained mainly in the cytoplasm of the Herts/33-infected cells. Collagen degradation assay showed that JS5/05-infected cells exhibited significant collagen degradation compared to the Herts/33-infected cells, and the areas of collagen degradation co-localized with cell surface MMP-14 in the JS5/05-infected cells. The transwell-based transmigration system showed that the transmigration of the JS5/05-infected PBMCs was enhanced significantly compared to the Herts/33-infected cells. These results demonstrated that genotype VII NDV induced up-regulation and surface accumulation of MMP-14 in PBMCs, leading to enhanced ECM degradation and cell migration, and the assays optimized for this study were useful for investigating the regulation of cell behaviour by NDV.