Rapid Detection of Neutrophil Oxidative Burst Capacity is Predictive of Whole Blood Cytokine Responses

Abstract

Background: Maladaptive immune responses, particularly cytokine and chemokine-driven, are a significant contributor to the deleterious inflammation present in many types of injury and infection. Widely available applications to rapidly assess individual inflammatory capacity could permit identification of patients at risk for exacerbated immune responses and guide therapy. Here we evaluate neutrophil oxidative burst (NOX) capacity measured by plate reader to immuno-type Rhesus Macaques as an acute strategy to rapidly detect inflammatory capacity and predict maladaptive immune responses as assayed by cytokine array.

Methods: Whole blood was collected from anesthetized Rhesus Macaques (n = 25) and analyzed for plasma cytokine secretion (23-plex Luminex assay) and NOX capacity. For cytokine secretion, paired samples were either unstimulated or ex-vivo lipopolysaccharide (LPS)-stimulated (100μg/mL/24h). NOX capacity was measured in dihydrorhodamine-123 loaded samples following phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment. Pearson's test was utilized to correlate NOX capacity with cytokine secretion, p<0.05 considered significant.

Results: LPS stimulation induced secretion of the inflammatory molecules G-CSF, IL-1β, IL-1RA, IL-6, IL-10, IL-12/23(p40), IL-18, MIP-1α, MIP-1β, and TNFα. Although values were variable, several cytokines correlated with NOX capacity, p-values≤0.0001. Specifically, IL-1β (r = 0.66), IL-6 (r = 0.74), the Th1-polarizing cytokine IL-12/23(p40) (r = 0.78), and TNFα (r = 0.76) were strongly associated with NOX.

Conclusion: NOX capacity correlated with Th1-polarizing cytokine secretion, indicating its ability to rapidly predict inflammatory responses. These data suggest that NOX capacity may quickly identify patients at risk for maladaptive immune responses and who may benefit from immuno-modulatory therapies. Future studies will assess the in-vivo predictive value of NOX in animal models of immune-mediated pathologies.

Fig 1. Cytokine secretion in response to LPS stimulation in Rhesus Macaque whole blood.

Scatter plots depicting the individual responses (pg/ml) in each of the 23 cytokines measured in Rhesus Macaque whole blood stimulated with 100ng/ml LPS for 24 hours or not (UT). Cytokines were assayed by multiplex analysis of plasma supernatant.

Fig 2. Immuno-typing Non-human primate baseline neutrophil oxidative burst capacity and whole blood cytokine production in response to LPS stimulation.

(A) 5 x 10⁵ Rhesus Macaque neutrophils were stimulated or not with 10ng/ml PMA and 1μg/ml ionomycin for 30 minutes. During the final 5 minutes of incubation, cells were loaded with 1μg/ml DHR-123 and analyzed by microplate reader at 488nm. The fold changes in mean fluorescence intensity (MFI) between stimulated and unstimulated samples from each non-human primate are depicted. (B) The fold changes of cytokines determined by multiplex analysis between LPS-stimulated and unstimulated whole blood samples from each non-human primate assayed in Fig 1 are depicted.

Fig 3. Baseline neutrophil oxidative burst capacity is predictive of ex-vivo whole blood production of IL-1β, IL-6, TNFα, and the Th1-polarizing cytokine, IL-12/23(p40).

(A) Scatter plots of cytokine fold change (y-axis) and neutrophil oxidative burst fold change in MFI (x-axis) that were determined to have statistically significant correlative coefficient (r) values (p≤0.0001) by Pearson’s Test. (B) Scatter plots of cytokine fold change and neutrophil oxidative burst capacity fold change for two cytokines found to be not statistically significant: IL-4 (Th2 cytokine), IL-17 (Th17 cytokine).