Regulation of gene expression and chemotactic and phagocytic function of bovine neutrophils incubated with citrus oil and lipopolysaccharides
Antibiotic use is coming under increasing public scrutiny due to the possible development of resistant pathogens and risk of residues appearing in the milk. Therefore, new strategies to control or treat mastitis are warranted. Recent studies have shown that citrus derived oil (CO) inhibited growth of major mastitis-causing bacteria. However, the effect of CO on the function of bovine blood neutrophils (BBN) is currently unknown. The objective of this study was to identify the effect of CO and lipopolysaccharide (LPS) endotoxin on BBN by evaluating function and relative expression of genes in vitro. Jugular blood (~150 mL) was collected from 11 healthy Holstein cows in mid-lactation (> 100 DIM). BBN were isolated and incubated with or without 0.01% CO and 50 µg LPS/mL for 2 h at 37◦C, 95% humidity, and 5 % CO2. After incubation, BBN chemotaxis and phagocytosis capabilities were determined in vitro and the cell pellet was recovered and relative gene expression was analyzed via qPCR using the 2^-ΔΔCt method. Three pre-planned non-orthogonal contrasts were evaluated for gene expression. Non-LPS challenged BBN incubated with CO had a 47% increase (P = 0.03) in migration in response to IL-8 and a moderate increase in phagocytic capacity (15.9 vs. 14.2%, P = 0.02). This effect indicates that the CO was not impairing the function of BBN. However, the pattern of gene expression did not reflect the functional response, where BBN incubated with CO, regardless of LPS challenge, reduced expression (P < 0.05, fold-change (FC) ≤ -1.57) of several pro-inflammatory genes (IL1B, NFKB, SOD2, TNFA, and TLR2) with the exception of IL8 which tended to be up-regulated (P < 0.06, FC =1.92) when compared to controls. For controls, expression of TLR4, critical for LPS recognition, was down-regulated (P = 0.03, FC = -1.66) due to LPS although expression of TNFA was up-regulated (P = 0.01, FC = 2.62). In addition, the anti-inflammatory mechanism of CO at the gene-level does not appear to be mediated by IL10, where IL10 was down-regulated (P < 0.01, FC = -3.78) in BBN incubated with CO when compared to controls. In conclusion, CO down-regulated the expression of pro-inflammatory genes in BBN. However, CO does not appear to be inhibitory for overall BBN function in vitro. Future studies examining the effect of CO on BBN during mastitis in dairy cattle are warranted.
Keywords: neutrophil, citrus oil, genes