Abstract
Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system—the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1β and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1β, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49–0.61) and between neutrophils and cytokine responses to R848 (0.38–0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.
Highlights
Standardized whole blood stimulation assays (TruCulture) were used to define the variability of immune activity in humans, via measurement of immune responses in 25 healthy individuals to 27 different immune stimuli including cytokines and chemokines[8]
Cattle infected with bovine viral diarrhoea virus (BVDV) showed reduced levels of IL-1β and IL-10, correlating with a more severe secondary infection of bovine herpes virus 1 (BHV1)[16]
Significant differences in methodology between laboratories have the potential to introduce artefactual errors into results, as shown in a study in humans examining cytokine responses after PBMC s timulation[18]. It found that PBMC stimulations had higher technical and biological variation compared to the standardized whole blood stimulation assay TruCulture
Summary
Standardized whole blood stimulation assays (TruCulture) were used to define the variability of immune activity in humans, via measurement of immune responses in 25 healthy individuals to 27 different immune stimuli including cytokines and chemokines[8]. Significant inter-individual variation was apparent in both basal and induced responses which is likely to have important relevance for disease susceptibility. Current approaches to assessing immune responses in livestock require detailed laboratory protocols for cell separation and stimulation experiments, which can hamper comparisons between studies or across time These methods can invariably normalize the induced immune response to a control or null response, precluding the analysis of unstimulated or ‘basal’ expression. Significant differences in methodology between laboratories have the potential to introduce artefactual errors into results, as shown in a study in humans examining cytokine responses after PBMC s timulation[18] It found that PBMC stimulations had higher technical and biological variation compared to the standardized whole blood stimulation assay TruCulture. As experimental infection studies are expensive and have obvious animal welfare drawbacks, high throughput immune profiling that can identify natural immune variation could facilitate targeted management, earlier interventions, reduction in antimicrobial usage contributing to antimicrobial resistance and potentially enhance animal selection practices
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