Chicken Lymphocytes Research Articles

Identification of broad-spectrum B-cell and T-cell epitopes of H9 subtype avian influenza virus HA protein using polypeptide scanning1

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

Exploring the immunomodulatory and antioxidant capacities of Berberis aristata in avian lymphocytes

India is one of the leading producers for eggs and broilers globally and thus has witnessed significant growth in the poultry sector. Different poultry stressors can negatively impact the growth, gut health, immune function and production reproductive performances of poultry. Antibiotic growth promoters (AGPs) have been used in the poultry industry as they also combat stress in poultry along with growth promotion. But the use of antimicrobials as growth promoter raises the concern of antibiotic resistance and its potential hazardous effect on human health as well as the environment. Several other alternatives are available for AGPs such as acidifiers, probiotics, prebiotics, antibacterial peptides and phytobiotics. Phytobiotics are known to possess several benefits for poultry as well as livestock such as immunomodulation, anti-inflammatory effect, antioxidant effect, maintain intestinal health etc. One such important plant is Berberis aristata belonging to the family Berberidaceae which is used in traditional system of medicine for the management of various inflammatory disorders. In the present study aqueous root extract of Berberis aristata (BAE) was evaluated for its immunomodulatory and antioxidant activity. The extraction yield of the roots of Berberis aristata in water was about 9.78%. The BAE showed presence of phenolics, flavonoids, tannins, terpenoids and contained about 104.97 mg/gm of BAE and 83.14 mg/gm of BAE of phenolics and flavonoids, respectively. BAE showed significant antioxidant activity in DPPH free radical scavenging assay. Maximum non-cytotoxic dose (MNCD) of BAE for lymphocyte culture was determined to be 600 µg/ ml. Lymphocytes proliferation assay (LPA) showed that BAE enhanced lymphocyte proliferation of B-cells stimulated by lipopolysaccharide (LPS) and T-cells stimulated by Concanavalin A and Phytohaemagglutinin M (PHA). Treatment with BAE significantly reduced lipid peroxidation (LPO) and increased the level of reduced glutathione, superoxide dismutase, and catalase in chicken lymphocytes, thus indicating antioxidant potential of BAE. The study reveals significant immunomodulatory and antioxidant properties of BAE, thus suggesting that it could be a potential alternative to AGPs in poultry production. But further in vivo studies are required to explore its efficacy and safety for its application in commercial poultry farming.

Impact of apitherapy on canine, equine, and chicken lymphocytes, in vitro

Apitherapy is a form of alternative medicine that utilizes products from the western honeybee (Apis mellifera), including honey, propolis, and honeybee venom, to improve the health status of human patients by altering host immunity. An added benefit of these products is that they are nutraceuticals and relatively inexpensive to aquire. Currently, little is known about the use of honeybee products in veterinary species, as well as their impact on host immunity. In the present in vitro study, honey, propolis, and honeybee venom were co-cultured with enriched canine, equine, and chicken peripheral blood lymphocytes (PBLs) with cell proliferation, cell viability/apoptosis, and cellular morphology evaluated. Concanavalin A (Con A) and dexamethasone were used as stimulatory and suppressive controls, respectively. Honeybee products’ effects on the three veterinary species varied by product and the species. Honey stimulated the PBLs proliferation in all three species but also displayed some increased cytotoxicity. Propolis stimulated proliferation in canine and equine PBLs, however, it suppressed proliferation in the chicken PBLs. Honeybee venom was the strongest PBL stimulant for all three species and in the equine, surpassed the stimulant response of Con A and yet, enhanced PBL cell viability post culture. In summary, the results of this preliminary in vitro study show that these three honeybee products do impact lymphocyte proliferation and viability in dogs, horses, and chickens, and that more research both in vitro and in vivo will be necessary to draw conclusions regarding their future use as immune stimulants or inhibitors.

Review on Virulence and Diagnostic Methods of Mareks Disease Virus in Chickens

Marek’s Disease is a highly contagious oncogenic and neuropathic disease of chickens caused by Marek’s Disease Virus which is a highly cell-associated Alpha herpes virus that replicates in chicken lymphocytes and establishes a latent infection within T cells. Evolution of Marek’s Disease Virus towards higher virulence have been a concern over the years from mild to virulent, very virulent and very virulent plus. Mareks Disease is diagnosed by clinical examination of infected chickens accompanied with demonstration of postmortem lesions. The technique for diagnosis of infection with Marek’s Disease Virus involves isolation and identification of Marek’s Disease Virus from infected tissues. Virus isolation is usually by virus propagation in cell culture and identification/ quantification by cytopathic changes. Further confirmatory diagnosis of Marek’s Disease Virus can be done by serological test such as Agar Gel Immuno Diffusion Test and Enzyme Linked Immuno Sorbent Assay and molecular based technique such as Polymerase Chain Reaction. High-throughput sequencing technology which is a second-generation sequencing, provide the possibility to rapidly obtain the full sequence of pathogen genome at once for studying virus evolution and development of novel vaccines. Mareks Disease can be controlled by live-attenuated vaccines. In Ethiopia, reports coming from various regions of the country indicated that Marek’s Disease possesses a great threat to the flourishing young poultry industry.

Resveratrol alleviates imidacloprid-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Imidacloprid (IMI) is a neonicotinoid insecticide with the highest global market share, and IMI exposure in the environment can negatively affect many nontarget organisms (a general term for organisms affected by drugs other than target organisms). Resveratrol (RSV), a non-flavonoid polyphenolic organic compound derived from peanuts, grapes, and other plants, has anti-inflammatory and antioxidant effects. It is currently unclear how RSV protects against cell damage caused by IMI. Therefore, we established an experimental model of chicken lymphocyte lines exposed to 110 μg/mL IMI and/or 0.5 μM RSV for 24 h. According to the experimental results, IMI markedly raised intracellular reactive oxygen species levels and diminished the activity of the cellular antioxidant enzymes (CAT, SOD, and GPx), leading to MDA accumulation and decreased T-AOC. JNK, ERK, and P38, the essential components of the mitogen-activated protein kinase (MAPK) signaling pathway, were also expressed more when IMI was present. Additionally, IMI resulted in upregulation of mitochondrial apoptosis (Caspase 3, Caspase 9, Bax, and Cyt-c) and necroptosis (Caspase 8, RIPK1, RIPK3, and MLKL) related factors expression, downregulation of Bcl-2 expression, induction of upregulation of cytokine IL-6 and TNF-α expression, and downregulation of IFN-γ expression. The combined treatment of RSV and IMI significantly reduced cellular oxidative stress levels, inhibited the MAPK signaling pathway, and alleviated IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction. To summarize, RSV antagonized IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Immunomodulatory effect of Atractylodis macrocephala Koidz. polysaccharides in vitro

Vaccination is still the main method of preventing most infectious diseases, but there are inefficiencies and inaccuracies in immunization. Studies have reported that Atractylodis macrocephalae Koidz. polysaccharides (RAMP) have immunomodulatory effects, but the mechanisms involved in whether they can modulate the immune response in chickens are not yet clear. The aim of this study was to investigate the effect of RAMP on lymphocytes functions by analyzing cell proliferation, cell cycle, mRNA expression of cytokines and CD4 +/CD8+ratio. To identify potential molecules involved in immune regulation, we performed a comprehensive transcriptome profiling of chicken lymphocytes. In addition, the adjuvant effect of RAMP was evaluated by detecting indicators of hemagglutination inhibition. When lymphocytes were cultured with RAMP in vitro, the proliferation rate of lymphocytes was increased (P < 0.01), more cells in S phase and G2/M phase (P < 0.01) and the mRNA expression of IFN-γ was upregulated (P < 0.05), while the mRNA expression of TGF-β (P < 0.01) and IL-4 (P < 0.05) was downregulated and the CD4 +/CD8+ratio was increased (P < 0.05). Transcriptomic results showed that RAMP increased the expression of HIST1H46 (P < 0.05) and CENPP (P < 0.05). Validation of qPCR showed that RAMP may play an important role in regulating cellular immunity by downregulating the Notch pathway. The results also showed that RAMP could increase the serum Newcastle disease virus antibody levels in chickens. These data suggest that RAMP could enhance immune function of lymphocytes and was a candidate vaccine adjuvant in chickens.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

LACpG10-HL Functions Effectively in Antibiotic-Free and Healthy Husbandry by Improving the Innate Immunity.

Antibiotics are broadly restricted in modern husbandry farming, necessitating the need for efficient and low-cost immunomodulatory preparations in antibiotic-free and healthful farming. As is known to all, CpG oligonucleotides (CpG-ODNs, an effective innate immunostimulatory agent) recognized by TLR9 in mammals (while TLR21 in avians) could collaborate with some united agent to induce stronger immune responses, but the cost is prohibitively expensive for farmers. Here, considering the coordination between TLR2 and TLR9/TLR21, we firstly proposed the idea that the well-fermented Lactococcus lactis could be utilized as a CpG-plasmid carrier (LACpG10) to enhance the host’s innate immunity against pathogenic invasion. In the present study, after obtaining LACpG10-HL from homogenized and lyophilized recombinant strain LACpG10, we treated primary chicken lymphocytes, two cell lines (HD11 and IPEC-J2), and chickens with LACpG10-HL, CpG plasmids (pNZ8148-CpG10), and other stimulants, and respectively confirmed the effects by conducting qRT-PCR, bacterial infection assays, and a zoological experiment. Our data showed that LACpG10-HL could induce excellent innate immunity by regulating autophagy reactions, cytokine expression, and motivating PRRs. Interestingly, despite having no direct antiseptic effect, LACpG10-HL improved the antibacterial capacities of lymphocytes and enterocytes at the first line of defense. Most importantly, water-supplied LACpG10-HL treatment reduced the average adverse event rates, demonstrating that LACpG10-HL maintained its excellent immunostimulatory and protective properties under farming conditions. Our research not only contributes to revealing the satisfactory effects of LACpG10-HL but also sheds new light on a cost-effective solution with optimal immune effects in green, antibiotic-free, and healthful husbandry farming.

Immunomodulatory and antioxidant effects of Glycyrrhiza uralensis polysaccharide in Lohmann Brown chickens.

Glycyrrhiza polysaccharide extract 1 (GPS-1) is a bioactive component isolated from Glycyrrhiza uralensis, also known as Chinese licorice. It appears to be pharmacologically active as an antibacterial, antiviral, and anti-tumor agent. GPS-1 has also been shown to buffer liver health and regulate the immune system. Moreover, GPS-1 is low cost and easy to extract. More study was needed to elucidate the biochemical pathways underlying the immunomodulatory and antioxidant benefits observed in Glycyrrhiza polysaccharide extract 1 (GPS-1). in vitro experiments on chicken lymphocytes and dendritic cells (DCs) show that GPS-1 significantly promotes the proliferation of immune cells and is linked to lymphocytes' secretion of IL-12, IFN-γ, and TNF-α by. DC secretion of NO, IL-2, IL-1β, IFN-γ, TNF-α, and IL-12p70 was also increased significantly. Additionally, GPS-1 also displayed a significant antioxidant effect in vitro, able to scavenge DPPH, hydrogen peroxide, ABTS, and other free radicals like superoxide anions. Separately, GPS-1 was tested in vivo in combination with the Newcastle disease virus (NDV) – attenuated vaccine. 120 Lohmann Brown chickens were vaccinated, while another 30 became the unvaccinated blank control (BC) group. For three consecutive days 1 mL of GPS-1 was administered at doses of 19.53 μg/mL, 9.77 μg/mL, or 4.88 μg/mL to the ND-vaccinated birds, except for the vaccine control (VC), where n = 30 per group. In vivo results show that GPS-1 combined with Newcastle disease (ND) vaccine had the best efficacy at significantly increasing chickens' body weight and ND serum antibody titer, enhancing their secretion of IL-2 and IFN- γ, and promoting the development of immune organs. The results also indicate that GPS-1 was able increase the proliferation of in vitro immune cells and elevate their cytokine secretion, which enhances the body's immune response. GPS-1 also clearly has the potential to be used as an immune adjuvant alongside ND vaccination.

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.

Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-β2-ε-β1 Toxoids to Induce Protective Immunity.

Clostridium perfringens (C. perfringens) is a bacterium that commonly causes zoonotic disease. The pathogenicity of C. perfringens is a result of the combined action of α, β, and ε exotoxins. In this study, Lactobacillus crispatus (pPG-T7g10/L. crispatus) expressing the main toxoids of C. perfringens, α, ε, β1, and β2, with EGFP-labeling, was constructed, and the protective effect was estimated in chickens. The α-β2-ε-β1 toxoid was constitutively expressed for confirmation by laser confocal microscopy and western blotting, and its immunogenicity was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical assays. After booster immunization, the probiotic vaccine group showed significantly higher levels (p < 0.05) of specific secretory IgA (sIgA) and IgY antibodies in the serum and intestinal mucus. Furthermore, the levels of cytokines, including interferon (IFN)-γ, interleukin (lL)-2, IL-4, IL-10, IL-12, and IL-17, and the proliferation of spleen lymphocytes in chickens orally immunized with pPG-E-α-β2-ε-β1/L. crispatus increased significantly. Histopathological observations showed that the intestinal pathological changes in chickens immunized with pPG-E-α-β2ε-β1/L. crispatus were significantly alleviated. These data reveal that the probiotic vaccine could stimulate mucosal, cellular, and humoral immunity and provide an active defense against the toxins of C. perfringens, suggesting a promising candidate for oral vaccines against C. perfringens.

Reversal of Phorate Induced Immunotoxicity and Oxidative Stress due to in vitro Exposure of Indigenous Hill Cattle Cow Urine in Chicken Lymphocytes

Cow urine has been described in ‘Sushrut Samhita’ and ‘Ashtanga Sangraha’ to be the most effective secretion of animal origin with innumerable therapeutic values. Cow urine or gau-mutra has a unique place in Ayurveda and is suggested for improving general health. Cow urine has many beneficial properties particularly in the areas of agriculture and therapeutics. Phorate is a broad spectrum, non-biocumulative organophosphorus insecticide and acaricide. It is extremely toxic to mammals and other non-target organisms. Present paper reports ameliorating effects of Badri cow urine distillate (CUD) against phorate induced immunotoxicity and oxidative stress in chicken lymphocytes culture employing lymphocyte proliferation assay and nitric oxide (NO) estimation. In vitro exposure of thousand times dilution of No Observable Effect Level (NOEL/103) dose of phorate caused immunotoxic effects as displayed by decrease in B and T cells proliferation. In case of combination treatments of phorate and CUD, there was increase in B and T cells proliferation as compared to only pesticide treated cells. There was an increase in oxidative stress on measuring nitric oxide level in phorate treated cells as compared to control cells and combination treated groups. Thus, it could be inferred that cow urine ameliorated the phorate induced immunotoxicity and oxidative stress in chicken lymphocytes.

Development of lymphocyte subpopulations in local breed chickens.

Background and Aim:Local breeds of chicken are known to have relatively higher disease resistance to many endemic diseases and diseases that are highly virulent in commercial chickens. This study aimed to address the lymphocyte subpopulations in three constitutive immune system organs (thymus, bursa of Fabricius, and spleen) in 30, 8-week-old, male local breed chickens.Materials and Methods:The T (CD3+) and B lymphocytes (Bu-1+) were identified through one-color, direct immunofluorescent staining of the thymus, bursa, and spleen lymphocytes. Likewise, two-color, direct immunofluorescent staining was performed to identify the CD4- and/or CD8-defined T lymphocytes. The proportions of T and B lymphocytes and CD4- and/or CD8 defined chicken lymphocyte subsets in lymphoid suspensions prepared from the thymus, bursa, and spleen were determined by flow cytometry.Results:CD3+ cells, particularly those positive for CD4+CD8–, were dominant in the thymus, whereas cells expressing the Bu-1 marker were predominant in the bursa of Fabricius. The proportion of T and B cells was almost equal in the spleen, with more cells expressing the CD4–CD8+ marker in the red pulp.Conclusion:These findings indicate that local breeds of chicken could serve as a reliable model for studying the immune system of commercial light chicken breeds, due to the similarity in the presence and the distribution of the immune cells.

Expression analysis of genes related to lipid metabolism in peripheral blood lymphocytes of chickens challenged with reticuloendotheliosis virus

The underlying molecular mechanism of lipid metabolism in peripheral blood lymphocytes from chicken infected with reticuloendotheliosis virus (REV) remains poorly understood. Therefore, this scientific question was explored in vitro and in vivo. The results indicated that triglyceride content was significantly reduced, but the free fatty acid content and carnitine palmitoyltransferase-1 activity were significantly increased in blood lymphocytes after REV infection. By RNA sequencing, 97 known differentially expressed genes (DEG) related to lipid metabolism or glycometabolism were screened via Gene Ontology term analysis. On the basis of these 97 DEG, enriched pathways, including the peroxisome proliferators-activated receptor (PPAR) signaling pathway, were identified. Among these 97 DEG, some representative genes were related to lipolysis and fatty acid utilization (PPARG, LPL, PLIN2, ACOX1, ACSL1, FABP3, and FABP4). However, other genes related to lipid biosynthesis (ACSL3, ACSL6, DGAT2, LPIN1, and LPIN2) were downregulated. The quantitative polymerase chain reaction results confirmed the accuracy of the RNA sequencing data, and the in vivo outcome supports theses in vitro results. Our findings revealed that REV regulates fatty acid and lipid metabolism in peripheral blood lymphocytes from chicken. After the lymphocytes were infected with REV, the exogenous fatty acids were preferentially used; genes involved in fatty acid utilization were upregulated via the PPAR pathway, whereas genes involved in lipid and fatty acid biosynthesis were downregulated.

Lactobacillus reuteri-derived extracellular vesicles maintain intestinal immune homeostasis against lipopolysaccharide-induced inflammatory responses\xa0in\xa0broilers

BackgroundLactobacillus reuteri strains are widely used as probiotics to prevent and treat inflammatory bowel disease by modulating the host’s immune system. However, the underlying mechanisms by which they communicate with the host have not been clearly understood. Bacterial extracellular vesicles (EVs) have been considered as important mediators of host-pathogen interactions, but their potential role in commensals-host crosstalk has not been widely studied. Here, we investigated the regulatory actions of EVs produced by L. reuteri BBC3, a gut-associated commensal bacterium of Black-Bone chicken, in the development of lipopolysaccharide (LPS)-induced intestinal inflammation in a chicken model using both in vivo and in vitro experiments.ResultsL. reuteri BBC3 produced nano-scale membrane vesicles with the size range of 60–250 nm. Biochemical and proteomic analyses showed that L. reuteri BBC3-derived EVs (LrEVs) carried DNA, RNA and several bioactive proteins previously described as mediators of other probiotics’ beneficial effects such as glucosyltransferase, serine protease and elongation factor Tu. In vivo broiler experiments showed that administration of LrEVs exerted similar effects as L. reuteri BBC3 in attenuating LPS-induced inflammation by improving growth performance, reducing mortality and decreasing intestinal injury. LrEVs suppressed the LPS-induced expression of pro-inflammatory genes (TNF-α, IL-1β, IL-6, IL-17 and IL-8), and improved the expression of anti-inflammatory genes (IL-10 and TGF-β) in the jejunum. LrEVs could be internalized by chicken macrophages. In vitro pretreatment with LrEVs reduced the gene expression of TNF-α, IL-1β and IL-6 by suppressing the NF-κB activity, and enhanced the gene expression of IL-10 and TGF-β in LPS-activated chicken macrophages. Additionally, LrEVs could inhibit Th1- and Th17-mediated inflammatory responses and enhance the immunoregulatory cells-mediated immunosuppression in splenic lymphocytes of LPS-challenged chickens through the activation of macrophages. Finally, we revealed that the reduced content of both vesicular proteins and nucleic acids attenuated the suppression of LrEVs on LPS-induced inflammatory responses in ex vivo experiments, suggesting that they are essential for the LrEVs-mediated immunoregulation.ConclusionsWe revealed that LrEVs participated in maintaining intestinal immune homeostasis against LPS-induced inflammatory responses in a chicken model. Our findings provide mechanistic insight into how commensal and probiotic Lactobacillus species modulate the host’s immune system in pathogens-induced inflammation.

Augmented Blastogenic Response in B and T Cells due to in vitro Exposure of Bauhinia variegata (L.) Benth Leaf Extract in Chicken Lymphocytes

Bauhinia variegata (L.) Benth commonly known as kachnar belongs to the family Fabaceae. This plant is known for many therapeutic usages traditionally viz., anti-tumor, anti-inflammatory, anti-diabetic, antioxidant activity, etc. The present study was conducted to explore its immunomodulatory potential employing chicken lymphocytes culture system through lymphocytes proliferation assay (LPA). Leaves of Bauhinia variegata were used for preparation of fifty percent hydro-methanolic extract (BVE) with extraction yield of 5.44%. Various concentrations of BVE were used to give in vitro exposure to chicken lymphocytes and maximum non-cytotoxic dose of BVE was found to be 0.1mg/ml, which was further used in B and T cells blastogenesis. LPA revealed a significant immunopotentiating effect on B and T cells’ proliferation under mitogenic (LPS, ConA and PHA) stimulation. Thus, it could be concluded that BVE has immunopotentiating activity. However, there is need of further in vitro and in vivo analyses before it could be exploited as poultry feed additive.

Protective Effect of Epigallocatechin-3-Gallate in Hydrogen Peroxide-Induced Oxidative Damage in Chicken Lymphocytes.

Epigallocatechin-3-gallate (EGCG) is one of the fundamental compounds in green tea. The present study was to evaluate the protective effect of EGCG in oxidative damage and apoptosis induced by hydrogen peroxide (H2O2) in chicken lymphocytes. Results showed that preincubation of lymphocytes with EGCG significantly decreased H2O2-reduced cell viability and apoptotic cells with DNA damage, restored the H2O2-dependent reduction in total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), glutathione (GSH), and glutathione disulfide (GSSG), and suppressed the increase in intracellular reactive oxygen species (ROS), nitric oxide (NO), nitric oxide synthesis (NOS), malondialdehyde (MDA), lipid peroxide (LPO), and protein carbonyl (Carbonyl). In addition, preincubation of the cells with EGCG increased mitochondrial membrane potential (MMP) and reduced calcium ion ([Ca2+]i) load. The protective effect of EGCG in oxidative damage in lymphocytes was accompanied by mRNA expression of SOD, Heme oxygenase-1 (HO-1), Catalase (CAT), GSH-PX, nuclear factor erythroid 2-related factor 2 (Nrf2), and thioredoxin-1 (Trx-1). As EGCG had been removed before lymphocytes were challenged with H2O2, the activation of genes such as Nrf2 and Trx-1 by preincubation with EGCG could be the main reason for EGCG to protect the cells from oxidative damage by H2O2. Since oxidative stress is an important mechanism of biological damage and is regarded as the reasons of several pathologies, the present findings may be helpful for the use of tea products to prevent oxidative stress and maintain healthy in both humans and animals.

CD11d is a novel antigen on chicken leukocytes

In life sciences, antibodies are among the most commonly used tools for identifying, tracking, quantifying and isolating molecules, mainly proteins. However, it has recently become clear that antibodies often fall short with respect to specificity and selectivity and in many cases target proteins are not even known. When commercial availability of antibodies is scarce, e.g. for targeting proteins from farm animals, researchers face additional challenges: they often have to rely on cross-reactive antibodies, which are poorly characterized for their exact target, their actual cross-reactivity and the desired application.In this study, we aimed at identifying the true target of mouse monoclonal antibody 8F2, which was generated against chicken PBMC and used for decades in research, while it's actual target molecule remained unknown. We used 8F2 antibody for immunoprecipitation in chicken PBMC and subsequently identified its true target as CD11d, which was never described in chicken lymphocytes before, by quantitative LC-MSMS. The most abundant interactor of CD11d was identified as integrin beta 2.The existence of this alpha integrin was therefore clearly proven on protein level and provides a first basis to further assess the role of CD11d in chickens in future studies.Data are available via ProteomeXchange with identifier PXD017248. SignificanceOur studies determined CD11d as the true target of a previously uncharacterized mouse monoclonal antibody 8F2, generated against chicken peripheral blood derived mononuclear cells (PBMC). This is therefore now first member of alpha integrins in chickens, that existence was now clearly identified on protein level. The additional identification of CD11d interactors provides information on integrin-dependent regulation of signaling networks, allowing further functional studies.

A novel long noncoding RNA, ENSGALG00000021686, regulates the intracellular transport of fatty acids by targeting the FABP3 gene in chicken

Fatty acids (FAs) are essential for the vital movement of humans and animals. Their metabolism is, in part, regulated by FABP3. In our previous study, a novel lncRNA (ENSGALG00000021686, L21686) was identified, and FABP3 was predicted as its target gene. Here, using chicken myocytes, lymphocytes, and different tissues, L21686 target on the FABP3 gene, FABP3 mRNA expression, and their effect on FA metabolism are explored. The results show that the highest expression of L21686 is in muscle tissue, a significant energy-consuming tissue. L21686 expression is consistent with FABP3 mRNA expression. We also show that under the different treatments, the levels of FABP3 mRNA and protein in myocytes and lymphocytes change in tandem with L21686 expression. Moreover, the dual-luciferase reporter assay provided direct evidence that L21686 targets the FABP3 gene. Finally, it was found that the content of free FAs increases along with the up-regulation of L21686 and the FABP3 gene. Malonyl CoA content does not change under the different treatments, suggesting that L21686 regulates the intake of extracellular FAs in chicken. Further, the changes in lipoprotein lipase (LPL), sterol-regulatory element binding protein 1 (SREBP-1), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) mRNA levels support this view. In summary, our data show that the new lncRNA (L21686) regulates the intake of extracellular FAs in chicken cells in vitro by targeting the expression of the FABP3 gene. Our findings will help to establish the groundwork and provide a new clue for deciphering the regulation of FAs metabolism in chicken.

Selenium Prevents Lead-Induced Necroptosis by Restoring Antioxidant Functions and Blocking MAPK/NF-κB Pathway in Chicken Lymphocytes.

Recent studies have identified a new existence of a genetically programmed and regulated cell death characterized by necrotic cell death morphology, termed necroptosis. Lead (Pb) is a ubiquitously distributed environmental pollutant that is highly toxic to animals and human beings. However, no detailed report has been conducted on the necroptosis in lymphocytes caused by Pb. Selenium (Se), a trace element in the body, has been shown to exert cytoprotective effect in numerous pathological injury caused by heavy metals. Here, lymphocytes isolated from chicken spleen were divided into four groups, control group, Se group, Pb group, and Pb + Se co-treatment group to investigate the potential mechanism in the necroptosis triggered by Pb and in the antagonistic effect of Se on Pb toxicity. Flow cytometry analysis and AO/EB staining showed Pb caused typical necrosis characteristics in the lymphocytes. The expression of RIP1, RIP3, and MLKL was increased, whereas the level of caspase 8 was declined in Pb group, which proved the occurrence of necroptosis. Meanwhile, Pb exposure disrupted the antioxidant enzyme (SOD, GSH-Px, and CAT) balance, promoted the expression of MAPK/NF-κB pathway factors (ERK, JNK, p38, NF-κB, and TNF-α), and activated HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90). However, those Pb-induced changes were significantly alleviated in Se + Pb group. Our study revealed that Pb could trigger lymphocyte necroptosis through MAPK/NF-κB pathway activated by oxidative stress and that Se could antagonize Pb-induced necroptosis in chicken lymphocytes.