generation-bovine-primary-enteroid-derived-two-dimensional-monolayer

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

Midwestern University

The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric analysis of surface markers (MHC class-II, CD86 and CD14). Data were analyzed using paired t-tests and repeated measures ANOVA. Two MoDC populations that differed in size and phenotype were identified: larger MoDC (LgMoDC) and smaller MoDC (SmMoDC). Medium type, plate chemistry, or length of monocyte adhesion time did not impact MoDC viability or yield. LgMoDC generated in serum-free medium expressed more MHC class-II and CD86 (P ≤ 0.03). A prolonged duration in culture reduced MoDC yield (P ≤ 0.04). MoDC can be consistently and reliably generated using AIM-V serum-free medium in standard tissue culture plates with a recommended culture duration of 3-4 days.

Phenotypic characterization of equine monocyte-derived dendritic cells generated ex vivo utilizing commercially available serum-free medium.

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.

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

Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immune-modulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1α, IL-1β, IL-8, IL-10, IL-17A, IFN-γ, MIP-1α, TNF-α, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II, CD86, CD205, and CD14. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1α, IL-10, MIP-1α, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of bovine MoDC utilizing our well-defined culture protocol offers a serum-free approach to mechanistically investigate mechanisms of diseases and the safety and efficacy of novel therapeutics for both humans and cattle alike.

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

Antimicrobial use in animal agriculture may be contributing to the emerging public health crisis of antimicrobial resistance. The sustained prevalence of infectious diseases driving antimicrobial use industry-wide suggests that traditional methods of bolstering disease resistance are, for some diseases, ineffective. A paradigm shift in our approach to infectious disease control is needed to reduce antimicrobial use and sustain animal and human health and the global economy. Targeting the defensive mechanisms that promote the health of an infected host without impacting pathogen fitness, termed "disease tolerance," is a novel disease control approach ripe for discovery. This article presents examples of disease tolerance dictating clinical outcomes for several infectious diseases in humans, reveals evidence suggesting a similarly critical role of disease tolerance in the progression of infectious diseases plaguing animal agriculture, and thus substantiates the assertion that exploiting disease tolerance mechanisms can positively impact animal and human health.

Can Infectious Disease Control Be Achieved without Antibiotics by Exploiting Mechanisms of Disease Tolerance?

Influenza viruses lead to substantial morbidity and mortality including ~3-5 million cases of severe illness and ~290,000-650,000 deaths annually. One of the major hurdles regarding influenza vaccine efficacy is generating a durable, robust cellular immune response. Appropriate stimulation of the innate immune system is key to generating cellular immunity. Cross-talk between innate dendritic cells (DC) and natural killer (NK) cells plays a key role in activating virus-specific T cells, yet the mechanisms used by influenza A viruses (IAV) to govern this process remain incompletely understood. Here, we used an  autologous human primary immune cell culture system to evaluate the impact of DC-NK cell cross-talk and subsequent naïve T cell activation at steady-state and after exposure to genetically distinct IAV strains-A/California/07/2009 (H1N1) and A/Victoria/361/2011 (H3N2). Using flow cytometry, we found that exposure of DCs to IAV in co-culture with NK cells led to a decreased frequency of CD83 and CD86 cells on DCs and an increased frequency of HLA-DR on both DCs and NK cells. We then assessed the outcome of DC-NK cell cross-talk on T cell activation. At steady-state, DC-NK cell cross-talk increased pan T cell CD69 and CD25 expression while exposure to either IAV strain reduced pan T cell CD25 expression and suppressed CD4 and CD8 T cell IFN-γ and TNF production, following chemical stimulation with PMA/Ionomycin. Moreover, exposure to A/Victoria/361/2011 elicited lower IFN-γ production by CD4 and CD8 T cells compared with A/California/07/2009. Overall, our results indicate a role for DC-NK cell cross-talk in T cell priming in the context of influenza infection, informing the immunological mechanisms that could be manipulated for the next generation of influenza vaccines or immunotherapeutics.

Dendritic cell-natural killer cell cross-talk modulates T cell activation in response to influenza A viral infection.

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations ( = 0.02) and serum CRP concentrations compared to controls ( < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination ( = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α ( = 0.0003), interleukin (IL)-6 ( = 0.04), interferon (IFN)-γ ( = 0.03), monocyte chemoattractant protein (MCP)-1 ( = 0.02), IL-10 ( = 0.02), and lower IL-8 ( = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.

Brina Lopez

Department of Farm Animal Medicine,
College of Veterinary Medicine,
Department of Farm Animal Medicine, College of Veterinary Medicine

Midwestern University

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Brina Lopez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Farm Animal Medicine,College of Veterinary Medicine,Department of Farm Animal Medicine, College of Veterinary Medicine

Midwestern University

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Department of Farm Animal Medicine,
College of Veterinary Medicine,
Department of Farm Animal Medicine, College of Veterinary Medicine