cytokine secretion when co-stimulated with various PAMPs, but no cytokine production was seen in cells only stimulated with MDP in neither CD, nor control monocytes. It has previously been reported that MDP-dependent TNF-a secretion requires costimulation with LPS or other PGN, whereas TNF-a mRNA is greatly increased by MDP stimulation alone, but not translated into TNF-a protein. This is most likely the explanation for the lack of increase in IL-1b protein expression and release seen in our experiments compared to the above sited studies. Thus, we were able to induce secretion of IL-1b with co-stimulation of AN3199 biological activity monocytes with LPS and MDP. Our results suggest that MDP regulated pathways are inhibited in CARD15 non-mutated CD monocytes. It is possible that this lack of response might be overruled by co-activation of other LRR pathways, as some earlier published reports may indicate, but it defines a unique response similar to CARD15 mutated responses. Subsequent analysis of CARD15 dependent pathways suggested that this difference was due to an impaired activation of the classical NFkB pathway in both non-mutated and mutated CARD15 monocytes, since MDP resulted in an impaired IKKa/ b-phosphorylation, the initial step in the activation of the NFkB pathway. Interestingly, the monocytes from controls, CARD15 non-mutated CD patients, and CARD15 mutated CD patients induced p38 phosphorylation in response to MDP, whereas no detectable activation was seen on the JNK pathway. This pattern of activation of MAPK pathways is similar to what previously have been found in whole intestinal biopsies from patients with active IBD. Although the response seems to be normal, it is interesting that it is seen in CARD15 mutated monocytes here. The finding suggests that MDP dependent activation of p38 occurs through other MDP sensing PRRs in human monocytes. It is, however, also reassuring that p38 is activated by MDP in CD monocytes, as it confirms that control and CD cells have been stimulated. It is unlikely that the stimulation could be due to TLR2 dependent signalling, since MDP alone has not been shown to activate TLR2. The results reveal an SU 6668 impairment of a specific NLR pathway in CD monocytes not carrying the disease associated CARD15 variants, and therefore add evidence to the theory of a dysfunctional innate immune response in CD monocytes. It also emphasises that studies into the role of the NLR pathway in CD monocytes should employ CARD15 nonmutated CD monocytes as well
