Non-Esterified Fatty Acids Over-Activate the TLR2/4-NF-Κb Signaling Pathway to Increase Inflammatory Cytokine Synthesis in Neutrophils from Ketotic Cows

Abstract

Background/Aims: Dairy cows with clinical ketosis display a negative energy balance and high blood concentrations of non-esterified fatty acids (NEFAs), the latter of which is an important pathological factor of ketosis in cows. The aims of this study were to investigate the inflammatory status of ketotic cows and to determine whether and through what underlying mechanism high levels of NEFAs induce an inflammatory response. Methods: Proinflammatory factors and the nuclear factor kappa B (NF-κB) signaling pathway were evaluated in neutrophils from clinical ketotic and control cows, using methods including western blotting, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. In vitro, the effects of NEFAs on the NF-κB signaling pathway in cow neutrophils were also evaluated using the above experimental techniques. Results: Ketotic cows displayed low blood concentrations of glucose and high blood NEFA and β-hydroxybutyrate concentrations. Importantly, Toll-like receptor 2 (TLR2) and TLR4 expression and IκBα and NF-κB p65 phosphorylation levels in neutrophils (PMNs) were significantly higher in ketotic cows than in control cows, indicating over-activation of the TLR2/4-induced NF-κB inflammatory pathway in PMNs in ketotic cows. The blood concentrations of the inflammatory cytokines interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) were also significantly increased in ketotic cows. Interestingly, we found that NEFAs were positively correlated with proinflammatory cytokines. In vitro, after pharmacological inhibition of TLR2 and TLR4 expression in cow neutrophils, TLR2 and TLR4 expression was significantly decreased, and the phosphorylation level of NF-κB p65 was also reduced. Cow neutrophils were treated with different concentrations of NEFAs and pyrrolidine dithiocarbamate (PDTC; an NF-κB inhibitor). High concentrations of NEFAs (0.5 and 1 mM) significantly increased TLR2 and TLR4 expression, IκBα and NF-κB p65 phosphorylation levels, NF-κB p65 transcriptional activity, and IL-6, IL-1β, and TNF-α synthesis in cow neutrophils. The inhibition of NF-κB by PDTC suppressed the NEFA-induced synthesis of proinflammatory cytokines. Conclusions: High concentrations of NEFAs can over-activate the TLR2/4-mediated NF-κB signaling pathway to induce the over-production of proinflammatory cytokines, thereby increasing inflammation in cows with clinical ketosis.