Pharmacological inhibition of the mTOR pathway alters phenotype and cytokine expression in bovine monocyte-derived dendritic cells

Epidemiological studies have long demonstrated the association of nutrient status and immune dysfunction in dairy cows. Postpartum dairy cows experiencing a nutrient deficit show a propensity for increased inflammatory response, decreased pathogen clearance, and increased incidence of infectious disease. Studies in cows and other species show that the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway could be one potential causal pathway connecting the deficit in nutrient availability and the heightened inflammatory response. Our objective was to investigate the effects of pharmacological mTOR pathway inhibition on phenotype and cytokine expression of bovine monocyte derived dendritic cells (moDC). We differentiated CD14+ monocytes from dairy cows (n = 14) into moDC in the presence or absence of first- or second-generation mTOR inhibitor rapamycin and PP242 (both 100 nM), respectively. On day seven cells were matured with E. coli lipopolysaccharide (LPS, 100 ng/mL) or left unstimulated to represent naïve moDC. Surface expression of CD14, CD40, CD80, and MHCII was measured via flow cytometry. We measured mRNA expression of IL10, IL12A, IL12B, and TNFα by rt-qPCR, and protein concentrations of IL-10 and TFN-α in cell culture supernatants with a bead-based multiplex assay. Cultures from ten cows successfully developed the moDC phenotype in culture without inhibitors, defined as increased surface expression of CD40, CD80, and MHCII compared with naïve moDC. Only data from these cows were considered for the results on effects of mTOR inhibitors. In naïve and mature moDC mTOR inhibition increased MHCII expression compared to controls. In mature moDC, in addition to MHCII, CD80 expression was increased compared with untreated LPS-stimulated controls. Expression of IL12A mRNA was upregulated in mature, mTOR inhibited moDC compared with untreated controls. In cell culture supernatants mTOR inhibition reduced IL-10 and increased TNF-α concentrations in naïve and mature moDCs compared with untreated controls. Overall rapamycin had a more consistent effect on altering phenotype and cytokine expression of moDC than PP242. In summary we observed an increased expression of co-stimulatory molecules and antigen presentation potential in mature moDC differentiated under mTOR inhibition, and a cytokine pattern that would potentially favor a Th1 type response. This study provides novel data indicating a role for mTOR signaling in bovine moDC phenotype and mediator profile. This proof-of-concept study demonstrates the role of the mTOR pathway in shaping the bovine immune response and may help to provide mechanistic insight and opportunities for modulation of the immune response during the nutrient deficit of early lactation.