Regulation of IL-8 and IL-1  expression in Crohn's disease associated NOD2/CARD15 mutations

Abstract

Crohn's disease (CD) is a chronic inflammation affecting the gastrointestinal tract. Three mutations (Arg702Trp, Gly908Arg and Leu1007fsinsC) within the NOD2/CARD15 gene increase CD susceptibility. Here, we define cytokine regulation in primary human mononuclear cells, with muramyl dipeptide (MDP), the minimal NOD2/CARD15 activating component of peptidoglycan. By microarray, MDP induces a broad array of transcripts, including interleukin 1beta (IL-1beta) and interleukin 8 (IL-8). Leu1007fsinsC homozygotes demonstrated decreased transcriptional response to MDP. Electromobility shift assay demonstrated that MDP-induced NF-kappaB activation is mediated via p50 and p65 subunits, but not RelB or c-Rel. In wild-type individuals, MDP-induced IL-8 protein expression with a greater response to high dose (1 micro g/ml) compared with low-dose (10 ng/ml) MDP. At low MDP doses, in all homozygotes, we observed no induction of IL-8 protein. With high doses of MDP, Leu1007fsinsC homozygotes showed no induction. Modest induction of IL-8 protein was observed in Gly908Arg and Arg702Trp homozygotes, indicating varying MDP sensitivity of the CD-associated mutations. In wild-type healthy control, CD and ulcerative colitis individuals, low-dose MDP and TNFalpha alone results in only modest IL-1beta protein induction. With MDP plus TNFalpha, there is a synergistic induction of IL-1beta secretion. In Leu1007fsinsC homozygotes, there is a profound defect in IL-1beta secretion, despite marked induction of IL-1beta mRNA. These findings demonstrate post-transcriptional dependency on the NOD2/CARD15 pathway for IL-1beta secretion with MDP and TNFalpha treatment. Taken together, these studies suggest that a signaling defect of innate immunity to MDP may be an essential underlying defect in the pathogenesis of some CD patients.

Engagement of NOD2 has a dual effect on proIL‐1β mRNA transcription and secretion of bioactive IL‐1β

“…Subsequently, the production of the cytokines is abolished. Our quantitative data by real-time PCR, obtained in primary cells, are supported by the results obtained by other groups [36][37][38].…”

“…Subsequently, the production of the cytokines is abolished. Our quantitative data by real-time PCR, obtained in primary cells, are supported by the results obtained by other groups [36][37][38].When MNC are stimulated with low dose MDP and the TLR4 ligand LPS, a remarkable synergy in cytokine production is apparent in MNC from individuals with a intact NOD2 [9,20]. A potential role for caspase-1 in the synergy between TLR4 and NOD2 could be envisaged, since MDP can activate caspase-1 and induce IL-1b, which is known to induce IL-1b production in an autocrine fashion through stimulation of the IL-1R [21].…”

TIR, CARD and PYRIN: three domains for an antimicrobial triad

“…82 In contrast to LPS activation, MDP-induced NF-kB activation in human primary mononuclear cells involves NF-kB p50 and p65 subunits, not RelB nor c-rel. 83 Lack of c-rel subunit involvement in NF-kB complexes has also been observed in murine splenocytes, stimulated with MDP. 84 This could have an important implication, since in splenocytes, PG induces an increased IL-12 production in Nod2-deficient mice.…”

“…TNF synergizes with MDP for the secretion of IL-1b by human monocytes. 83 IL-32, a recently identified proinflammatory cytokine, has been shown to synergize with Nod1 and Nod2 agonists, but not with TLR agonists, for IL-1b and IL-6 secretion. Interestingly, the synergy with MDP is specifically blocked by caspase-1 inhibitors, suggesting a caspase-1-dependent mechanism.…”

“…78 Further examples of synergy between muropeptides and TLR agonists have been reported for cytokine production in human monocytes. 83,[86][87][88][89] Cytokines can also synergize with Nod agonists, for the secretion of other cytokines. TNF synergizes with MDP for the secretion of IL-1b by human monocytes.…”