Equine Peripheral Blood Research Articles

Endogenous Adult Telomerase Positive Stem Cells Increase in Equine Peripheral Blood Following Exercise

Stout, et al., 2007 reported a base-line level of primitive endogenous adult Telomerase Positive Stem Cells (aTPSCs) circulating within adult porcine peripheral blood which increased dramatically, i.e., 23.5-fold, after just 90 minutes of trauma. Ongoing studies from our laboratory have shown the presence of similar primitive stem cells in adult equine blood and that the base-line levels of stem cells varied according to the particular breed of horse. The current study was undertaken to test the hypothesis that aTPSCs would increase in number in the peripheral blood following stress in the same horse. Stress in this study was defined as moderate exercise, i.e., 10 minutes of cantering. Blood withdrawal followed the guidelines of Fort Valley State University’s IACUC. Adult horses had their blood withdrawn immediately prior to and immediately after exercising. The blood was obtained by venipuncture. Isolated aTPSCs were assayed by trypan blue staining, CEA-CAM-1 for totipotent stem cells and SSEA-4 for pluripotent stem cells. This study demonstrates that moderate exercise will increase the level of these primitive pluripotent stem cells in the blood of adult equines. Studies are ongoing to address their functional significance during injury and repair.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15–1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15–1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping 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.

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.

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study.

The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Three adult horses with no evidence of ophthalmic or systemic disease. Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations. Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from "safe" to "retinotoxic" in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.

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.

Equine peripheral blood CD14+ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14+ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4 +IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4 +IL-10 priming increased CD86 immunopositivity by 2-fold (p = 0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1β compared to control (p = 0.012) and IL-4 +IL-10-primed (p = 0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p < 0.05). There were no differences in TGFβ-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p = 0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFβ1. Among the culture conditions evaluated, IL-4 +IL-10 priming for 24 h after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4 +IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

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

Moderate Differences in Plasma Leptin in Mares Have no Effect on Either the Amino Acid or the Fatty Acid Composition of the Uterine Fluid

Female mammalian reproductive functions are closely linked to body condition and metabolic status. Energy homeostasis is regulated by endocrine hormones such as insulin, IGF-I, leptin, and adiponectin via the hypothalamic-pituitary-adrenal axis. These metabolic hormones and their receptors are also expressed in reproductive tissues and the embryo. We investigated the relationship between circulating leptin and the fatty acid (FA) and amino acid (AA) composition of the equine uterine fluid (UF) and peripheral blood plasma (BP) by using a mass spectrometry-based approach. UF and BP were collected from ten broodmares on days 6 and 7 post ovulation, respectively. The mares were retrospectively assigned to two groups according to their BP leptin concentrations (high leptin [> 1.6 ng/mL] versus low leptin [<0.8 ng/mL]). Specific AA and FA compositions for BP and UF were found with different levels of respective metabolite abundances. The main FAs in BP were stearic, palmitic and linoleic acid. In UF, the three most abundant FAs were eicosapentaenoic, arachidonic and stearic acid. The AA profile of BP was dominated by glycine, glutamine, serine and alanine, which were likewise among the highly abundant AAs in UF. In UF, glutamic acid had by far the highest concentration. Therefore, BP leptin concentration within a physiological range does not seem to affect the specific FA nor the AA composition of the UF. The composition of the UF may therefore be mediated by local rather than by peripheral metabolic hormones.

The equine mesenchymal stromal cell secretome inhibits equid herpesvirus type 1 strain Ab4 in epithelial cells

Equid herpesvirus 1 (EHV-1) outbreaks occur when virus spreads from infected horses to in-contact horses, primarily via nasal shedding. This study evaluated the efficacy of factors secreted by equine peripheral blood derived mesenchymal stromal cells (PB-MSCs), collectively named the secretome, to inhibit the growth of EHV-1 in (i) 2D epithelial cell cultures (RK-13) in vitro, (ii) 3D equine nasal explants in vitro and (iii) an EHV-1 infection mouse model in vivo. The PB-MSC secretome was found to inhibit EHV-1 in RK-13 cells as well as in the epithelium of equine nasal explants. Although the PB-MSC secretome did not decrease overall severity of EHV-1 infection in mice, as determined by weight loss and viral titers in lungs, histological analyses indicated local reduction of EHV-1 infection in nasal epithelium. These results indicate that the PB-MSC secretome inhibits EHV-1 in epithelial cells in a context-dependent manner.

Engrafting Horse Immune Cells into Mouse Hosts for the Study of the Acute Equine Immune Responses.

Simple SummaryFor decades, studies using research mice as models for disease have been critical to our current understanding of disease processes and associated immune responses, highlighting the ways in which mouse physiology is different from human and other species. Recent work has been directed at creating mice that can host human immune cells, allowing the study and manipulation of the human immune response without harm to patients. The purpose of this study was to explore to use of mouse hosts for horse immune cells. Horses are difficult to study immunologically as they are expensive to keep, and keeping their environment free of immune triggers is very difficult. Using mice allows us to increase our study numbers and control the environment which improves study reproducibility. In this study, we transferred both horse blood lymphocytes as well as horse bone marrow into specially modified mouse hosts. We found that mice are able to host horse immune cells and that these transferred cells are active. Future work can now build on this study to understand the horse immune response to infectious agents using mice, helping to identify new therapeutic tools to help equine patients.Immunological studies in the horse are frequently hampered by lack of environmental control, complicated study design, and ethical concerns when performing high risk studies. The purpose of the current study was to investigate the utility of a xenograft model for studying acute equine immune responses. Immunocompromised non obese diabetic (NOD). sudden combined immunodeficiency (scid).gamma-/- (NSG) mice were engrafted with either equine peripheral blood lymphocytes (PBLs) or equine bone marrow to determine an optimal protocol for equine lymphocyte engraftment. We found that both PBL and bone marrow grafts populated the host mice successfully. Bone marrow transplants were technically more challenging and required further processing to retard graft versus host disease. Graft vs host disease was apparent at 28 days post-PBL transfer and 56 days post-bone marrow transfer. The results of these studies support the use of mouse hosts to study acute equine immune responses and that different engraftment techniques can be used depending on the experimental design.

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.

Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity.

Human IgE-binding monocytes are identified as allergic disease mediators, but it is unknown whether IgE-binding monocytes promote or prevent an allergic response. We identified IgE-binding monocytes in equine peripheral blood as IgE+/MHCIIhigh/CD14low cells that bind IgE through an FcεRI αɣ variant. IgE-binding monocytes were analyzed monthly in Culicoides hypersensitive horses and nonallergic horses living together with natural exposure to Culicoides midges. The phenotype and frequency of IgE-binding monocytes remained consistent in all horses regardless of Culicoides exposure. All horses upregulated IgE-binding monocyte CD16 expression following initial Culicoides exposure. Serum total IgE concentration and monocyte surface IgE densities were positively correlated in all horses. We also demonstrated that IgE-binding monocytes produce IL-10, but not IL-4, IL-17A, or IFN-γ, following IgE crosslinking. In conclusion, we have characterized horse IgE-binding monocytes for the first time and further studies of these cells may provide important connections between regulation and cellular mechanisms of IgE-mediated diseases.

Characterization of equine inflammasomes and their regulation.

Inflammasome, a cytosolic multi-protein complex, assembly is a response to sensing intracellular pathogenic and endogenic danger signals followed by caspase-1 activation, which maturates precursor cytokines such as interleukin (IL)-1β. Most inflammasome research has been undertaken in humans and rodents, and inflammasomes in veterinary species have not been well-characterized. In this study, we observed the effects of well-known inflammasome activators on equine peripheral blood monocytes (PBMCs). The NLRP3 inflammasome triggers include ATP, nigericin, aluminum crystals, and monosodium urate crystals, and NLRP3 activation induces IL-1β secretion in a dose-dependent manner. Activators of NLRC4 and AIM2 inflammasomes include cytosolic flagellin and dsDNA, and their activation induces IL-1β secretion. The bacterial inflammasome triggers Salmonella Typhimurium and Listeria monocytogenes also induce IL-β releases. To elucidate the role of potassium efflux as an upstream signal of NLRP3 inflammasome activation, equine PBMCs were treated with blockers of potassium efflux in the presence of NLRP3 triggers. As a result, the IL-1β secretion stemming from equine NLRP3 inflammasome activation was not completely attenuated by the inhibition of potassium efflux. Taken together, the results indicate that equine PBMCs normally secrete IL-1β in response to well-known inflammasome activators, although equine NLRP3 inflammasome activation might not be dependent on potassium efflux.

Analysis of caecal mucosal inflammation and immune modulation during Anoplocephala perfoliata infection of horses.

Anoplocephala perfoliata is the commonest equine tapeworm, theadult parasites are attached in groups close to the ileocaecal valve causing marked inflammatory pathology. This work aimed to characterize the nature of the in vivo mucosal immune response to Aperfoliata, and to investigate the role of Aperfoliata excretory-secretory components in modulating in vitro immune responses. Real-time PCR detected elevation of IL13 and TGFβ transcription in early-stage Aperfoliata infection. In late-stage infection, IL-13, IL4 and Ifn transcripts were reduced while the regulatory cytokines, TGFβ, IL10 and the transcription factor FOXP3 were increased in tissue close to the site of Aperfoliata attachment; indicating downregulation of T-cell responses to Aperfoliata. In vitro, Aperfoliata excretory-secretory products induced apoptosis of the Jurkat T-cell line and premature cell death of ConA stimulated equine peripheral blood leucocytes. Analysis of cytokine transcription patterns in the leucocyte cultures showed a marked inhibition of IL-1 and IL-2 suggesting that a lack of T-cell growth factor transcription underlies the mechanism of the induced equine T-cell death. These preliminary findings suggest Aperfoliata may have the ability to down-regulate host T-cell responses.