Equine Peripheral Blood Mononuclear Cells Research Articles

Monoclonal antibodies for equine IL-1β enable the quantification of mature IL-1β in horses

Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.

Effects of tamoxifen on the immune response phenotype in equine peripheral blood mononuclear cells.

Tamoxifen (TAM) is widely utilized in the prevention and treatment of human breast cancer and has demonstrated the potential to modulate the immune response. It has been proposed as a therapeutic tool for immune-mediated diseases. TAM has been investigated as a possible treatment for asthma-like conditions in horses, revealing specific impacts on the innate immune system. While the effects of TAM on equine neutrophils are well-documented, its influence on lymphocytes and the modulation of the immune response polarization remains unclear. This in vitro study employed peripheral blood mononuclear cells (PBMC) from healthy horses, exposing them to varying concentrations of the TAM and assessing the expression of genes involved in the polarization of the immune response (TBX21, IFNG, GATA3, IL4, IL10, FOXP3, and CTLA4) in PBMC stimulated or not with PMA/ionomycin. Additionally, the effect of TAM over the proportion of regulatory T cells (Treg) was also assessed. TAM did not significantly affect the expression of these genes and Treg at low concentrations. However, at the highest concentration, there was an impact on the expression of GATA3, IL4, IL10, and CTLA4 genes. These alterations in genes associated with a Th2 and regulatory response coincided with a noteworthy increase in drug-associated cytotoxicity but only at concentrations far beyond those achieved in pharmacological therapy. These findings suggest that the effects of TAM, as described in preclinical studies on asthmatic horses, may not be attributed to the modification of the adaptive response.

Therapeutic Potential in Wound Healing of Allogeneic Use of Equine Umbilical Cord Mesenchymal Stem Cells.

Wound healing after skin injury is a complex process, particularly in equines where leg wounds are prevalent and their repair is complicated due to the anatomical characteristics. Conventional treatments are not effective enough. The umbilical cord offers an unlimited source of adult mesenchymal stem cells (ucMSCs) from Wharton's jelly tissue. The present study aims to demonstrate the safety and therapeutic potential of the allogeneic use of equine ucMSCs (e-ucMSCs) in the healing of severe equine leg wounds. The methods employed were the isolation, culture and expansion of e-ucMSCs. Flow cytometry and a PCR assay were used for cell characterization. This study included an immunomodulation assay, a murine pre-clinical trial and the first phase of an equine clinical trial. Our results showed that e-ucMSCs express a functional HLA-G homolog, EQMHCB2. In the immunomodulation assay, the e-ucMSCs inhibited the proliferation of activated equine peripheral blood mononuclear cells (e-PBMCs). In the murine pre-clinical trial, e-ucMSCs reduced healing time by 50%. In the equine clinical trial, the injection of e-ucMSCs into severe leg lesions improved the closure time and quality of the tissues involved, regenerating them without fibrous tissue scar formation. In conclusion, the results of this study suggest that e-ucMSCs can be used allogeneically for wound healing by creating a tolerogenic environment.

Immunogenicity analysis of BPV-1 positive equine sarcoid-derived cultured fibroblasts

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)− 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/− 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies.

In vitro effects of monophosphoryl lipid A and Poly I:C combination on equine cells.

Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

Effect of different adjuvant formulations on the antibody response of horses to porcine zona pellucida proteins

Immunization with porcine zona pellucida (PZP) proteins is being used successfully to induce infertility in wildlife including horses. However, widespread adoption of this method to control the growth of horse populations requires further refinement in order to induce long-term infertility, reduce the frequency and severity of injection site reactions, and make the vaccines easier to administer. The next generation of PZP-based vaccines will likely be a controlled-release formulation with different adjuvants from the Freund’s adjuvants used in existing vaccines. We evaluated the response of equine peripheral blood mononuclear cells to a cationic nanoparticle adjuvant, Nano-11, alone and with the TLR agonists poly(I:C) and CpG ODN as a screen to develop an adjuvant system suitable for immunization of horses. The secretion of IL-1β, TNF and CXCL10 were used as readouts. The combination of poly(I:C) with Nano-11 significantly increased the secretion of IL-1β and TNF in comparison with Nano-11 only, with little effect of further addition of CpG ODN. The efficacy of the Nano-11/poly(I:C) adjuvant to enhance the immune response to native PZP proteins was determined in horses. Horses were immunized twice with the licensed Zonastat-H vaccine or PZP with Nano-11/poly(I:C) emulsified in silicone oil. A third group received PZP with the saponin adjuvant QA-21 emulsified in silicone oil. The horse sera collected monthly after the injections had increased anti-PZP IgG antibodies with the strongest response observed with Zonastat-H. We conclude that Nano-11/poly(I:C) is a potential candidate for the development of a controlled release formulation of a next generation PZP-based immunocontraception.

Beta defensins as biomarkers: detectable in LPS-stimulated equine peripheral blood mononuclear cells and normal, aseptic, and probable septic equine synovial fluid.

Diagnosis of equine septic arthritis is not straightforward, and increasing time between onset, diagnosis, and treatment can have serious consequences for quality of life. Defensins are used in diagnosis of human joint infection. The presence of beta defensins (BDs) in equine synovial fluid and their utility as a biomarker of sepsis has not been investigated; therefore, our objectives were to (1) compare in vitro gene expression of lipopolysaccharide (LPS)-stimulated equine neutrophils to unstimulated neutrophils and (2) compare BD protein expression from normal, aseptically inflamed, and septic equine joints. 5 horses for isolated neutrophil BD expression and 21 synovial fluid samples from 14 horses. RT-qPCR analysis was performed for BD gene expression of stimulated and unstimulated equine peripheral neutrophils. BD protein expression was evaluated from equine joints with no disease, aseptic inflammation, and septic inflammation using a commercial ELISA designed for horses and analyzed with a Kruskal-Wallis test (significant at P < .05). A significant increase was noted in expression of BD-3 in LPS stimulated as compared to unstimulated neutrophils. There were no significant differences in BD expression noted between joints with no disease, aseptic inflammation, and septic inflammation. Low case numbers and different types of cases in the aseptic inflammation group were main limitations. BD expression patterns in samples from stimulated equine peripheral neutrophils and synovial fluid were identified. BDs are detectable in equine synovial fluid and can be stimulated from peripheral neutrophils. Further examination is needed to define their role as biomarkers of joint disease.

In vitro anti-trypanosomal effect of ivermectin on Trypanosoma evansi by targeting multiple metabolic pathways.

High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. evansi, mRNA expression profile of 13 important drug target genes has been studied at 12, 24, and 48 h interval. In the in vitro growth inhibition assay, ivermectin inhibited T. evansi growth and multiplication significantly (p < 0.001) with IC50 values of 13.82 μM, indicating potent trypanocidal activity. Cytotoxicity assays on equine peripheral blood mononuclear cells (PBMCs) and Vero cell line showed that ivermectin affected the viability of cells with a half-maximal cytotoxic concentration (CC50) at 17.48 and 22.05 μM, respectively. Data generated showed there was significant down-regulation of hexokinase (p < 0.001), ESAG8 (p < 0.001), aurora kinase (p < 0.001), casein kinase 1 (p < 0.001), topoisomerase II (p < 0.001), calcium ATPase 1 (p < 0.001), ribonucleotide reductase I (p < 0.05), and ornithine decarboxylase (p < 0.01). The mRNA expression of oligopeptidase B remains refractory to the exposure of the ivermectin. The arginine kinase 1 and ribonucleotide reductase II showed up-regulation on treatment with ivermectin. The ivermectin was found to affect glycolytic pathways, ATP-dependent calcium ATPase, cellular kinases, and other pathway involved in proliferation and maintenance of internal homeostasis of T. evansi. These data imply that intervention with alternate strategies like nano-formulation, nano-carriers, and nano-delivery or identification of ivermectin homologs with low cytotoxicity and high bioavailability can be explored in the future as an alternate treatment for surra in animals.Graphical abstract

Evaluation of equine xenogeneic mixed lymphocyte reactions using 5-ethynyl-2’-deoxyuridine (EdU)

Allogeneic solid organ transplantation is currently the only treatment option for end stage organ disease. The shortage of available donor organs has driven efforts to utilize xenogeneic organs for transplantation. In vitro methods for evaluating immune-compatibility are a quick and low cost means of screening novel tissue products prior to more involved, expensive, and invasive live animal studies. Recently, a new analog of the DNA base thymidine, 5-ethynyl-2’-deoxyuridine (EdU), was developed. It may be used in a fast, efficient and specific means of evaluating cell proliferation via flow cytometry. This study was designed to test and optimize this platform for assessing equine xenogeneic one-way mixed lymphocyte reaction (MLR) to porcine stimulator cells. Furthermore, it was hypothesized that an enriched T-lymphocyte (T-cell) population would generate a stronger proliferative response to stimulation, and higher levels of cytokine production when compared to unfractionated peripheral blood mononuclear cells (PBMCs). PBMCs and T-cells were isolated from 3 horses and 4 pigs. Equine xenogeneic MLRs were set up using porcine allogeneic MLRs as a reference for clinically acceptable levels of cell proliferation. Equine T-cells showed significantly greater EdU incorporation in one-way xenogeneic MLRs than equine PBMCs. However, there was no significant difference in cell proliferation between porcine T-cell and PBMC as responders in allogenic one-way MLRs. Given the results of this study, we consider that enriched equine T-cells should be used in preference to unfractionated PBMCs when attempting to evaluate the equine xenogeneic response using the EdU assay as an indicator of suitability for transplant in vivo.

Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet+ B cells

BackgroundTraditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers.ResultsHere, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1− lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse.ConclusionsTaken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.

Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro.

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-α and C–C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/mL and three with <10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-α and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Inflammatory Responses in Equine Peripheral Blood Mononuclear Cells, an In Vitro Study.

Simple SummaryNatural compounds are often an important source of biologically active molecules, which can find important applications in the treatment or pharmacological prevention of several pathologies. Silibinin is a natural polyphenolic flavonoid that is extracted from plant milk thistle, Silybum marianum. Silibinin has been reported to have antioxidant and immunomodulatory and anti-inflammatory activities. In horses, in particular, inflammation secondary to bacterial infection or translocation is one of the most frequent causes of morbidity and mortality. The aim of this study was to test the effect of silibinin on lipopolysaccharide (LPS)-induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). Taken together, our results showed an interesting prospective in therapeutic use of silibinin in equine inflammatory disease. Furthermore, the results from this study support the evidence of use equine PBMCs as an in vitro model to study inflammatory and immune response and for drug screening into the target specie.Although inflammation is an important physiological response, it plays a prominent role in several diseases across the mammalian species. In horses, in particular, inflammation secondary to bacterial infection or translocation is one of the most frequent causes of morbidity and mortality. Research in new molecules with anti-inflammatory and immunomodulatory proprieties and safe use profile is constantly an active field; natural compounds are an important source of molecules with peculiar properties such as antioxidants, anti-inflammatory and immune modulating. Silibinin, a natural polyphenolic flavonoid, extracted from plant milk thistle, Silybum marianum, has been reported to have actions such as antioxidant immunomodulatory and anti-inflammatory. The aim of this study was to test the effect of silibinin on lipopolysaccharide (LPS)-induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). Our results showed the protective effect of silibinin 10 μM and 50 μM in equine PBMCs stimulated with LPS. Silibilinin was able to prevent the LPS induced increased levels of TNF-α, IL-1β, IL-6 and IL-8. The results from this study on LPS-stimulated equine PBMCs showed that silibinin could be a useful pharmacological approach in treatment or prevention of several inflammatory conditions in horse.

Recombinant horse interleukin-4 and interleukin-10 induced a mixed inflammatory cytokine response in horse peripheral blood mononuclear cells.

Background and Aim:Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in Escherichia coli. Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs).Materials and Methods:Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction.Results:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis displayed a molecular weight of 15 kDa for IL-4 and 19 kDa for IL-10. Recombinant IL-4 and IL-10 significantly induced cell proliferation at 250 ng/ml. The results demonstrated that IL-4 enhanced expression of interferon-gamma (IFN-γ), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10, while recombinant horse IL-10 induced expression of IL-6, IFN-γ, and TNF-α.Conclusion:The present study demonstrated that biologically active horse IL-4 and IL-10 could be produced in E. coli.

Can levamisole upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) T-helper 1 (CD4 Th1) T-cytotoxic (CD8) immune response in vitro?

BackgroundEquine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM‐affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses.ObjectiveLevamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T‐helper 1 (CD4 Th1), and T‐cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro as compared to mitogen alone.AnimalsTen neurologically normal adult horses serologically negative for Sarcocystis neurona.MethodsProspective study. Optimal conditions for levamisole were determined based on cellular proliferation. Peripheral blood mononuclear cells were then cultured using optimal conditions of mitogen and levamisole to identify the immune phenotype, based on subset‐specific activation markers, intracellular cytokine production, and cytokine concentrations in cell supernatants. Subset‐specific proliferation was determined using a vital stain.ResultsConcanavalin A (conA) with levamisole, but not levamisole alone, resulted in a significant decrease (P < .05) in PBMC proliferation compared to conA alone. Levamisole alone did not elicit a specific immune phenotype different than that induced by conA.Conclusion and Clinical ImportanceLevamisole co‐cultured with conA significantly attenuated the PBMC proliferative response as compared with conA. If the mechanisms by which levamisole modulates the immune phenotype can be further defined, levamisole may have potential use in the treatment of inflammatory diseases.

C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine C-C motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology.Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stained with the anti-CCL mAbs. Flow cytometric analysis confirmed the specificity of the different mAbs for the respective chemokine. In addition, equine PBMC were stained after isolation, culture in medium, or stimulation with LPS, or PMA and ionomycin.CCL2 was detected in few cluster of differentiation (CD)14+ monocytes in PBMC stimulated with PMA and ionomycin for 2 h. CCL3 was produced by CD14+ monocytes after 4–6 h culture in medium. After stimulation with PMA and ionomycin for 12–24 h, CCL3 was also expressed in lymphocytes, mainly in CD4+ T cells. Stimulation with LPS reduced the percentage of CCL3+ monocytes in PBMC. CCL5 was detected in PBMC ex vivo in CD4+ and CD8+ T cells. Culture of PBMC for longer than 6 h or stimulation with PMA and ionomycin reduced the percentage of CCL5+ cells. CCL11 was produced by CD4+ T cells in PBMC after stimulation with PMA and ionomycin for 4–24 h. After LPS stimulation of PBMC, CCL2, CCL5, and CCL11 production were comparable to culture in medium alone.ELISAs for each of the four chemokines were developed using pairs of anti-equine CCL mAbs. Supernatants from PMA and ionomycin stimulated PBMC contained detectable amounts of CCL2, CCL3 and CCL5, while CCL11 secretion could be stimulated from equine tracheal epithelial cells in response to IL-4.The newly generated mAbs for equine CCL chemokines facilitate the quantitative analysis of intracellular chemokine production by flow cytometry and soluble chemokines by ELISA. The CCL mAbs are valuable tools to improve the evaluation of innate immune responses in horses.

Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood

Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3− cells in addition to CD8+ T cells. Further analysis using co-labeling with CD3 and multiple B cell antibodies revealed that most of these CVS21+CD3− cells were B cells. Clone CVS8 and anti-equine CD4 clone CVS4 only labeled CD3+ cells and were mutually exclusive. To identify equine B-cell subpopulations, anti-equine Pan-Ig (clone CVS36), IgM (clone 1–22), and IgG1 (clone CVS45), as well as anti-human CD21 (clone B-ly4) were tested. Anti-equine Pan-Ig antibody labeled 88 ± 7.6% of CD3− lymphocytes. Anti-equine IgM often produced a continuum of stain intensity of PBMC from several adult horses. Anti- IgG1 and −CD21 labeled overlapping subsets of CD3− cells. Based on these results, combined with conjugate availability, a final panel of anti-CD4 (CVS4), −CD8 (CVS8), −IgG1 (CVS45), and −CD21 (B-ly4) antibodies was used to sort CD4+ T cells, CD8+ T cells, and CD21+ and/or IgG1+ B cells simultaneously. The identity of the sorted populations was confirmed by qRT-PCR for cell lineage markers. The described analysis emphasizes the need for increased availability of reagents and use of multispectral flow for equine immunophenotypic analysis. This new multispectral flow cytometry protocol will facilitate studies on equine lymphocyte responses and will have broad application across studies of infectious and immune-mediated disease.

Equine PBMC Cytokines Profile after In Vitro α- and γ-EHV Infection: Efficacy of a Parapoxvirus Ovis Based-Immunomodulator Treatment.

Equine herpesviruses (EHV) infect horses early during life and the persistence of these viruses through establishment of latency represents a real risk. A better understanding of the immune response to EHV infection is necessary to improve our methods of prevention and decrease the risk of transmission. The objectives of this study were to characterise the cytokine gene expression profile of peripheral blood mononuclear cells (PBMC) after in vitro EHV-1, EHV-4, and EHV-2 infection and to determine the efficacy of inactivated Parapoxvirus ovis (iPPVO) against these 3 viruses. PBMC were isolated from 3 horses and infected in vitro with EHV-1, EHV-4, or EHV-2 in the presence or absence of iPPVO. In vitro culture of PBMC with EHV-1, EHV-4, and iPPVO induced a significant increase of IFN-α, IFN-β, and IFN-γ gene expression. EHV-4 also triggered a significant increase of IL-6 and TNF-α mRNA. EHV-2 triggered a significant increase of IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, and TNF-α mRNA. The presence of iPPVO induced an earlier and stronger expression of IFN-α, IFN-β, and IFN-γ mRNA during EHV infection and reduced the inflammatory response induced by EHV-2. In conclusion, this study suggests that the presence of iPPVO potentiates the development of the immune response to in vitro EHV infection.

A monoclonal antibody for detection of intracellular and secreted interleukin-2 in horses

Interleukin-2 (IL-2) is a T cell growth factor and major modulator of T helper (Th) cell differentiation. Here, we have developed and characterized a monoclonal antibody to equine IL-2 (anti-IL-2 mAb, clone 158-1). The IL-2 mAb detected rIL-2 by ELISA, intracellular staining and flow cytometry analysis and Western blotting. The IL-2 mAb was also paired with a polyclonal IL-2 detection antibody in both ELISA and a fluorescent bead-based assay. When these two assays were compared using identical reagents there was an improved analytical sensitivity (46pg/ml) and wider linear quantification range (46–100,000pg/ml) of IL-2 quantification using the fluorescent bead assay. Equine rIL-2 standards were expressed in both yeast and mammalian cells but the mammalian cell-expressed rIL-2 standard was found to be most accurate for native IL-2 quantification. Using this system we found that stimulation of equine peripheral blood mononuclear cells (PBMC) with phorbol 12-myristate 13-acetate (PMA) and ionomycin induced IL-2 secretion most potently. Pokeweed mitogen (PWM) consistently resulted in low amounts of IL-2 from PBMC, while concanavalin A (ConA), phytohemagglutinin-L (PHA-L) and lipopolysaccharide (LPS) either marginally stimulated or failed to stimulate IL-2 secretion from equine PBMC. After stimulation of equine PBMC with PMA and ionomycin, IL-2 production was detected in 13.0% (range 7.5-16.8%) of the lymphocytes by flow cytometric analysis. IL-2 expression was mainly stimulated in CD4+ cells, in a sub-population of CD8+ cells, and also in CD4-/CD8- cell population. In addition, both IFN-γ+/IL-2+ and IL-4+/IL-2+ producing cells were observed. Testing of serum and colostrum samples from 15 healthy horses showed that IL-2 was not detectable in these samples (<46pg/ml). In summary, the equine IL-2 mAb provides a new tool for the characterization of IL-2 producing equine cells and the quantification of secreted equine IL-2 in sensitive assays.

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing.

The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.

LPS-induced modules of co-expressed genes in equine peripheral blood mononuclear cells

BackgroundLipopolysaccharide (endotoxin, LPS) is a strong inducer of the innate immune response. It is widespread in our environment, e.g. in house dust and contributes to asthma. Compared to humans, horses are even more sensitive to LPS. However, data on LPS effects on the equine transcriptome are very limited. Using RNA-seq we analysed LPS-induced differences in the gene expression in equine peripheral blood mononuclear cells at the gene and gene-network level in two half-sib families and one group of unrelated horses.Results24 h-LPS challenge of equine immune cells resulted in substantial changes in the transcriptomic profile (1,265 differentially expressed genes) showing partial overlap with human data. One of the half-sib families showed a specific response different from the other two groups of horses. We also identified co-expressed gene modules that clearly differentiated 24 h-LPS- from non-stimulated samples. These modules consisted of 934 highly interconnected genes and included genes involved in the immune response (e.g. IL6, CCL22, CXCL6, CXCL2), however, none of the top ten hub genes of the modules have been annotated as responsive to LPS in gene ontology.ConclusionsUsing weighted gene co-expression network analysis we identified ten co-expressed gene modules significantly regulated by in vitro stimulation with LPS. Apart from 47 genes (5%) all other genes highly interconnected within the most up- and down-regulated modules were also significantly differentially expressed (FDR < 0.05). The LPS-regulated module hub genes have not yet been described as having a role in the immune response to LPS (e.g. VAT1 and TTC25).