Fig 1.
Treatment scheme of the pbMEC in vitro.
The green arrow indicates the untreated control samples that were taken at every relevant time-point (0 h, 24 h, 30 h and 54 h). The blue arrow indicates the BHBA treatment time-points. The pbMEC that were only treated with 3 mM BHBA were also sampled at every relevant time-point (24 h, 30 h and 54 h). The red arrows indicate the two treatment intervals for the E. coli treatment (6 h and 30 h). It can be clearly seen that within the co-stimulated pbMEC, a 24 h adaptation phase to 3 mM BHBA preceded the bacterial treatment, so that pbMEC stimulated with E. coli for 6 h, obtained a total BHBA treatment period for 30 h and pbMEC stimulated with E. coli for 30 h obtained a total BHBA treatment period of 54 h.
Table 1.
Primer for RT-qPCR measurements.
Fig 2.
The typical cobblestone-like morphology and the epithelial character were confirmed, by the positive cytokeratin staining, the insert shows the negative control. [Leica light microscope, magnification x200].
Table 2.
Fold changes in gene expression upon immune stimulation.
Fig 3.
Attenuation of the gene expression through the co-stimulation of pbMEC with E. coli and 3 mM BHBA.
Fold change of (A) the chemokine CCL2, (B) the milk protein CSN3, (C) the complement component C3, (D) the antimicrobial peptide LF, (E 1–2) the acute phase protein SAA3, and (F) the inflammatory cytokine IL6. Significant changes in the gene expression between the E. coli treated pbMEC and the pbMEC co-stimulated with E. coli and 3 mM BHBA are indicated by stars: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Fig 4.
Comparison of the RT-qPCR data of LF and CCL2 with the LF and CCL2 content in pbMEC cell culture supernatants.
The fold changes of the LF gene expression (A) indicated a significant down-regulation of LF gene expression when pbMEC where co-stimulated with E. coli and 3 mM BHBA. The same effect could be detected within the competitive LF ELISA (B) of pbMEC cell culture supernatants. The amount of secreted LF decreased significantly in case of the co-stimulation. The distinct and significant down-regulation in CCL2 gene expression (C) could also be confirmed by the results of the bovine CCL2 VetSet™ ELISA Kit (D). The CCL2 gene expression and the amount of secreted protein decreased distinctly as well as significantly within the co-stimulatory group. Significant changes: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, distinct changes: + 0.1 ≤ p ≤ 0.05.