Equine Dendritic Cells Research Articles

The effect of cortisol on equine monocyte–derived dendritic cell phenotype and cytokine production

AbstractBackgroundIncreased risk of infection in neonates, including foals, is associated with the naivety of the immune system and the immaturity of the endocrine system. Foal dendritic cells (DCs) are phenotypically and functionally less mature than adult horse DCs. Exposure of foal DCs to foal plasma alters DC phenotypic maturation, cytokine secretion and bacterial endocytosis. Specific plasma factors that impact equine DC activity are unknown. Endocrine dysfunction, resulting in altered cortisol availability, is common in foals. Cortisol impacts DC maturation and function in other species, but the effect of cortisol on equine DCs is not described. We hypothesized that exposure to cortisol would impact both the phenotype and function of equine monocyte–derived DCs (MoDCs), perpetuating an immature phenotype and altered cytokine profile.MethodsMoDCs were generated from foals (n = 8) on 1, 7 and 30 days of age and adult horses (n = 9). MoDCs were exposed to killed bacteria in the presence or absence of cortisol. The expression of surface markers (MHC class‐II, CD86 and CD14) was measured by flow cytometry. Supernatant cytokine concentrations (IL‐4, IL‐17, IFN‐γ and IL‐10) were quantified using a validated bead‐based immunoassay.ResultsCortisol exposure reduced the percentage of equine MoDCs expressing MHC class‐II and CD86 (p ≤ 0.03). Adult horse MoDCs exposed to bacteria and cortisol produced less IL‐4, IL‐17, IFN‐γ and IL‐10 than cells not treated with cortisol, and foal MoDCs secreted less IL‐10 and IL‐4 (p ≤ 0.009).ConclusionsIn sum, cortisol exposure perpetuates an immature MoDC phenotype and alters cytokine responses, which might impact the foal's susceptibility to infection.

The Effect of Varying Thawing Temperatures and Preserving Conditions on the Recovery Rate of Cryopreserved Equine Dendritic Cells

The Effect of Varying Thawing Temperatures and Preserving Conditions on the Recovery Rate of Cryopreserved Equine Dendritic Cells

The effect of foal or adult horse plasma on equine monocyte-derived dendritic cell phenotype and function

Immunological and endocrine immaturity in foals increases foal morbidity and mortality from bacterial sepsis. Dendritic cells (DC) are critical in activating the adaptive immune response, but foal DC are phenotypically and functionally different than those of adult horses. Age-related variations in availability of some soluble plasma factors, such as hormones, might govern some age-related differences in DC function. Effects of exposure to plasma factors on equine DC phenotype and function have not been described. We hypothesized that exposure to plasma from foals or adult horses would differentially impact monocyte-derived DC (MoDC) phenotype and function. Eight healthy adult horses and 8 healthy foals were divided into pairs of one adult horse and one foal. Blood was collected from each pair for MoDC generation when foals were 1 and 30 days of age. MoDC from horses and foals were then exposed to killed whole-cell bacteria in the presence of their own age-matched plasma, plasma from the opposite-aged animal in the pair, and serum-free medium alone (control). Expression of DC-relevant surface markers (MHC class-II, CD86, and CD14) and endocytosis capability were measured by flow cytometry. Supernatant cytokine concentrations (IL-4, IL-17, IFN-γ, and IL-10) were quantified with a validated bead-based immunoassay. Data were analyzed using linear mixed-effects and Tobit regression models (P < 0.05). The percentage of MoDC expressing surface markers MHC class-II and CD86 was reduced in MoDC derived from 1-day-old foals in comparison to adult horse MoDC when cultured in medium alone or with either source of plasma (P = 0.0001). Foal and adult horse MoDC cultured in either source of plasma expressed more CD86 and less CD14 than cells cultured in serum-free medium alone (P ≤ 0.02). Adult horse and foal MoDC exposed to bacterial antigen in the presence of 1-day-old foal plasma secreted less IL-10 (P ≤ 0.0008) compared to those cultured in adult horse plasma. Endogenous production of IL-17 by MoDC from foals at day 1 of age cultured in adult plasma was increased compared to foal MoDC cultured in serum-free medium (P = 0.004). Phagocytosis of killed, labeled Staphylococcus aureus was reduced when MoDC generated from foals or adult horses were exposed to plasma from foals at day 1 or 30 of age (P ≤ 0.03). Age-related variation in soluble plasma factors appear to regulate equine MoDC function, but specific plasma factors capable of regulating MoDC phenotype or function were not defined in this study.

Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

BackgroundDendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5+ T lymphocytes to assess their T cell stimulatory capacity.ResultsThe microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum were found to be superior in their functional T lymphocyte priming capacity and to elicit significantly less non-specific proliferation.ConclusionsEqMoDC generated with horse serum-supplemented medium showed improved morphological characteristics, higher cell viability and exhibited a more robust performance in the functional T cell assays. Therefore, horse serum was found to be superior to FBS for generating equine monocyte-derived dendritic cells.

Equine herpesvirus type 1 (EHV1) induces alterations in the immunophenotypic profile of equine monocyte-derived dendritic cells

Equine herpesvirus 1 (EHV1) is an α-herpesvirus that can infect a variety of different cells in vitro and in vivo, including dendritic cells (DC) which are essential in the immune response against EHV1. Infection of equine monocyte-derived DC (MDDC) with EHV1 induced down-regulation of major histocompatibility complex I (MHCI), CD83, CD86, CD206, CD29 and CD172a, but not of CD11a/CD18 and MHCII. This down-regulation was not mediated by the virion host-shutoff (VHS) protein or pUL49.5. Interestingly, down-regulation of CD83 and CD86 was in part mediated by pUL56. Taken together, these data indicate that EHV1 employs different and still unresolved mechanisms to induce down-regulation of several functionally important cell surface proteins on equine DC.

Differentiation and culture of dendritic cells from equine peripheral blood mononuclear cells

Dendritic cells(DCs) derived from myeloid cells are the most potent antigen-presenting cells. In the mouse, it is possible to differentiate a number of DCs from bone marrow. However, the horse has trouble in sacrificing with economic problems. To our knowledge, the culture of equine DCs has not been performed yet in our country. Furthermore, the cytokine set for culture of equine DCs has not been established in this area. Thus, we tried some sets of cytokines and milogen for this purpose. Mononuclear cells were isolated from blood by Ficoll histopaque, versene solution and an adhesion process and then cultured for 4 days. Specific morphology and viability of DC were determined by visual observation using an optical microscope. Human Granulocyte Macrophage-Colony Stimulating Factor(GM-CSF) and equine interleukin(lL)-4 were certainly effective for culturing equine DCs. Antigen-stimulatory ability of equine DCs was confirmed by Mixed Leukocyte Reaction using &lt;sup&gt;3&lt;/sup&gt;H-thymidine incorporation assay. To verify antigen-uptake capability of DC, we usedfluorescein isothiocyanate-dextran and analyzed by using FACSCalibur&lt;sup&gt;Ⓡ&lt;/sup&gt; flow cytometer(BD Biosciences). It was evident from our results that human GM-CSF could be used as an alternative cytokine for equine GM-CSF due to its genetic homogeneity. Taken together, this study demonstrated that human GM-CSF and equine IL-4 were effective to diflerentiate and proliferate DCs from peripheral blood mononuclear cells and enhanced its viability. The information from this study may provide the study of horse immunity with new insights and basic technology.

Ex vivo generation of mature equine monocyte-derived dendritic cells

Dendritic cells (DCs) are innate immune cells specialized in antigen detection and presentation. They perform an essential role in initiating and guiding the immune response, the direction of which largely depends upon the activation state of the DCs. The objective of this study was to generate mature equine monocyte-derived DCs and, in doing so, to develop a method for measuring the activation state of these cells. Equine DCs were stimulated with UV-inactivated Escherichia coli ( E. coli), and the activation status was measured by analyzing cell surface marker expression, cytokine production, and endocytic capacity. Comparisons for each parameter measured were performed between macrophages, non-stimulated DCs and stimulated DCs. Equine monocyte-derived DCs may be distinguished from macrophages based on cell surface expression of MHC class II ( p < 0.0001) and CD206 ( p < 0.0001), their capacity for endocytosis of FITC-dextran ( p < 0.05), and production of TNF-α upon stimulation ( p < 0.001). Furthermore, stimulated DCs can be distinguished from non-stimulated DCs based on increased cell surface expression of MHC class II ( p < 0.0001) and upregulation of pro-inflammatory cytokine mRNA, particularly IL-12/IL-23p40 ( p < 0.05) and IL-23p19 ( p < 0.05). The ability to measure DC activation state will facilitate future investigations of equine DC function.

Generation and characterisation of equine dendritic cells from highly enriched peripheral blood monocytes

Generation and characterisation of equine dendritic cells from highly enriched peripheral blood monocytes

An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: Divergent maturation programs by IL-4 and LPS

Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V ® medium on dishes coated with extracellular matrix components (Primaria ®) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed into three subsets of (i) MHC II neg cells, (ii) MHC II low immature, endocytic cells and (iii) MHC II high spontaneously mature, non-endocytic DC. The immature DC fraction of the GM-CSF cultures matured, as detected by MHC II up-regulation, upon LPS exposure overnight. DC cultures in GM-CSF plus IL-4 resulted in higher cell yields, a loss of the immature MHC II low population but increased mature MHC II high DC, suggesting maturation. However, the MHC II high DC fraction was still endocytically active and did not lose their endocytic function after LPS treatment. They marginally up-regulated MHC II expression but this did not result in an enhanced stimulation of an allogeneic mixed lymphocyte reaction. However, LPS treatment clearly induced mRNA for IL-12p35 and p40, which was not observed by addition of IL-4 alone. Together our data indicate that IL-4 and LPS induce two different maturation programs. IL-4 induces a semi-maturation where the cells are still endocytic, which can be further matured to secrete cytokines in a second step by LPS.

Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes.

Equine herpesvirus-1 (EHV-1) causes serious disease in horses throughout the world, despite the frequent use of vaccines. CTLs are thought to be critical for protection from primary and reactivating latent EHV-1 infections. However, the antigen-specificity of EHV-1-specific CTLs is unknown. The aim of this study was to identify EHV-1 genes that encode proteins containing CTL epitopes and to determine their MHC I (or ELA-A in the horse) restriction. Equine dendritic cells, transfected with a series of EHV-1 genes, were used to stimulate autologous CTL precursor populations derived from previously infected horses. Cytotoxicity was subsequently measured against EHV-1-infected PWM lymphoblast targets. Dendritic cells were infected with EHV-1 (positive control) or transfected with plasmids encoding the gB, gC, gD, gE, gH, gI, gL, immediate-early (IE) or early protein of EHV-1 using the PowderJect XR-1 research device. Dendritic cells transfected with the IE gene induced CTL responses in four of six ponies. All four of these ponies shared a common ELA-A3.1 haplotype. Dendritic cells transfected with gC, gD, gI and gL glycoproteins induced CTLs in individual ponies. The cytotoxic activity was ELA-A-restricted, as heterologous targets from ELA-A mismatched ponies were not killed and an MHC I blocking antibody reduced EHV-1-specific killing. This is the first identification of an EHV-1 protein containing ELA-A-restricted CTL epitopes. This assay can now be used to study CTL specificity for EHV-1 proteins in horses with a broad range of ELA-A haplotypes, with the goal of developing a multi-epitope EHV-1 vaccine.

Modulating immune responses with dendritic cells: an attainable goal in veterinary medicine?

Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8− “myeloid” DCs, CD4−/CD8+ “lymphoid” DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells. We describe the influence of cytokines on the phenotype of DCs generated in the rat. Using bone marrow cells as the source of precursors we generated “myeloid-type” DCs from the adherent population using granulocyte–macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or “lymphoid-type” DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFα. In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors. We report on the structure and function of this molecule.

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10–100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.

Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses. As with other herpesviruses, cell-mediated immunity is considered important for both recovery and protection. Although virus-specific T-cell proliferation and cytotoxicity can be detected following in vivo infection, little is known about the role of antigen presenting cells such as dendritic cells (DCs) in these processes. Peripheral blood DCs were shown to express the viral glycoprotein gB perinuclearly following exposure to EHV-1 in vitro, demonstrating EHV-1 replication within them. Co-culture of infected DCs or their supernatants with a susceptible cell line (RK13) demonstrated that EHV-1 infection was productive. In vitro-infected DCs showed cytopathic effects, including loss of viability and syncytial formation. However, they were superior to other antigen presenting cells in stimulating both peripheral blood T-cell proliferation and cytotoxicity. Although ponies which had been intranasally infected with EHV-1 exhibited T-cell proliferation to live virus presented on DCs, the responses began to decline as early as 15 weeks and cease at 22 weeks post-in vivo infection. Cytotoxic responses were not detected 35 weeks after the first intranasal infection but were seen again 7 weeks following a second infection. These findings show that equine DCs, which are infected with EHV-1 in vitro, can stimulate memory T-cell responses but appear unable to circumvent the short-lived memory response found following this infection in vivo.

Isolation and characterisation of equine dendritic cells

Despite their important role in initiating T-cell responses in other species, dendritic cells have not been studied in the horse. A method for isolating blood dendritic cells by adherence and metrizamide gradients was adapted to equine cells. A number of monoclonal antibodies (mAbs), including some which label dendritic cells in other species, were tested for immunochemical reactivity with the isolated blood dendritic cells, and sections of lymph node and spleen. 62 ± 6% of the isolated blood cells were MHC Class II positive and had typical dendritic cell morphology and only 4 ± 2% contained non-specific esterase, a marker of mature macrophages. These dendritic cells also expressed MHC Class I, LFA-1, EqWC1 and EqWC2. Amongst the potentially cross-reactive antibodies a mAb against bovine CD1b was the most interesting by staining lymph node, but not blood, dendritic cells. Monoclonal antibodies against equine CD5 (T-cells), surface immunoglobulin (B-cells) and macrophages (CZ2.2) were used to enumerate the contaminating cells in preparations from blood by flow cytometry. 39 ± 7% of the cells did not express T and B cell markers or CZ2.2 but were large and MHC Class II positive. Comparison of immunochemistry and flow data, together with examination of alveolar macrophages and adhered blood cells, all support the view that CZ2.2 detects a myeloid marker not seen on mature macrophages and possibly shared with dendritic cell precursors. The functional capacity of the isolates was assessed in terms of their stimulating ability in the mixed leukocyte reaction (MLR). Dendritic cell enriched isolates were more potent stimulators of MLRs than peripheral blood mononuclear cells or adherent cells. Thus equine dendritic cells isolated from blood express high levels of MHC Class I and II and LFA-1 and stimulate a vigorous MLR. They do not express markers characterising T and B cells but, by virtue of expression of the equine macrophage marker CZ2.2, appear closely related to mononuclear phagocytes.