Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-KB (NFKB) signaling. In nonruminants, SIRT3 is under the control of PGC1 alpha, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1 alpha in the attenuation of NFKB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 mu g/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFKB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 mu M). Second, SIRT3 was over expressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 mu g/mL LPS. Third, cells were treated with the PGC1 alpha agonist ZLN005 (10 mu g/ mL) for 24 h and then challenged with 150 mu g/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1 alpha inhibitor SR-18292 (100 mu M) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 mu g/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 mu g/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. e. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFKB P65/NFKB P65 ratio and nuclear abundance of NFKB P65. The PGC1 alpha agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFKB P65/NFKB P65 ratio, and prevented NFKB P65 nuclear translocation. Contrary to the agonist, the PGC1 alpha inhibitor had opposite effects, and elevated the concentrations of IL-10, IL-6, and TNF-alpha in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1 alpha-NFKB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.