The most affected up-regulated genes of the eicosanoid pathway after 6 hr of incubation with n-butyrate alone were found to be ALOX5AP, LTB4R, LTB4R2, PLCD1, PTGS2 and TBXA2R. Following 6 hr co-incubation of cells with LPS alone, the major induced genes were ALOX12, LTB4R2, PLA2G4C, PLA2G7,

PTGER2, PTGER3, PTGIR, PTGS2, TBXA2R (Fig. 2a,b). In comparison, ALOX12, LTB4R2, find more PLA2G4C, PTGER2, TBXA2R and massively PTGS2 were found to be further up-regulated after 6 hr co-incubation with LPS and n-butyrate (Fig. 2a,b). To further substantiate alterations in gene expression we first assessed the influence of n-butyrate on the expression of the key enzyme of eicosanoid metabolism COX-2 (PTGS2) at the protein level. Monocytes were incubated with LPS ± n-butyrate for different time periods and expression of COX-1 and COX-2 was assessed by intracellular staining as specified in the Materials and methods. COX-1 was constitutively expressed and not affected by n-butyrate (data not shown). In contrast,

expression of COX-2 was up-regulated APO866 by LPS. Furthermore, we observed an even more pronounced expression of COX-2 after co-incubation with n-butyrate after between 4 and 8 hr of treatment with the maximum detected after 6 hr (Fig. 3). To find out whether the potent enhancement of COX-2 expression was specific for the TLR4 pathway we investigated the effect of n-butyrate also for TLR2 ligation by S. aureus cells. In this experimental setting we also found a significant up-regulation of COX-2 as verified by Western blot (see Supplementary material, Fig. S2). Based on these findings we next elucidated whether enhanced COX-2 expression is accompanied by alterations in the production of mediators Nintedanib order related to the eicosanoid pathway downstream of COX-2. To answer this, release of PGE2 and 15d-PGJ2, two prostaglandins with well-known immunomodulatory effects, was analysed after n-butyrate co-treatment with LPS or with S. aureus cells to trigger TLR4 or TLR2, respectively. Release of PGE2 and 15d-PGJ2

was induced after LPS as well as S. aureus cell stimulation (Fig. 4a,b) and was substantially up-regulated after co-incubation with n-butyrate in both cases. Akin to monocytes we found an increased release of prostaglandins following TLR2 and TLR4 activation and co-incubation with n-butyrate into the supernatants of monocyte-derived dendritic cells (data not shown). Profound up-regulation of genes encoding the key leukotriene synthesizing enzymes was also recorded (Fig. 2a,b), so we next evaluated the impact of n-butyrate on the release of leukotrienes. Here we found that LTB4 and thromboxane B2, both key members of the lipoxygenase pathway, were significantly up-regulated following n-butyrate treatment and LPS activation when compared with LPS stimulation alone (Fig. 5a,b).