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.

Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance

“…Recently, Zhang and coworkers (2018) have shown that ketotic cows with low glucose and high NEFA concentrations have higher blood concentrations of inflammatory cytokines. Those authors related the phenomenon to an overactivation of toll-like receptor 2/4-mediated NF-κB pathway signaling in polymorphonuclear neutrophils as a cause for increasing inflammation in cows suffering from ketosis (Zhang et al, 2018). In the present study, ketosis was treated as soon as ketone bodies were detected in the urine, and mean BHB levels were under the cutoff value for ketosis (p.p.…”

Flunixin Meglumine Reduces Milk Isoprostane Concentrations in Holstein Dairy Cattle Suffering from Acute Coliform Mastitis

“…Elevated NEFA concentrations have been linked to severity of inflammation during coliform mastitis [ 25 , 47 ]. Elevated pre-FM treatment circulating NEFAs among cows that died in this study, when compared to those that survived, may be indicative of an exacerbated inflammatory response since circulating NEFAs and COX-2 enzyme can be increased via upregulation of Toll-like receptor (TLR) signaling [ 21 , 28 , 30 ]. Increased production of pro-inflammatory eicosanoids through the COX pathway can then lead to further adipose mobilization and NEFA release, creating a positive feedback cycle [ 23 , 29 ].…”

“…During inflammation, mediators not only act on localized mammary tissues, but also can have systemic effects, leading to increased mobilization of adipose tissue and increased circulating non-esterified fatty acids (NEFAs) [ 6 , 13 , 23 , 24 , 25 , 26 , 27 ]. Prolonged elevated NEFAs results in systemically increased production of cytokines, adhesion molecules, eicosanoids through increased expression of COX-2 and free radicals, such as reactive oxygen species (ROS) [ 28 , 29 , 30 ].…”

Protease‐dependent excitation of nodose ganglion neurons by commensal gut bacteria

“…Moreover, the toll‐like receptor‐4 (TLR4) inhibitor C‐34 (10 μ m ; Zhang et al . 2018) blocked the effect of LPS on NG neurons (Fig. 6, F (2,65) = 5.31, P < 0.01; one‐way ANOVA followed by Tukey's multiple comparison test).…”