MD-2

Seksik P, Sokol H, Grondin V, et al. Sera from patients with Crohn's disease break bacterial lipopolysaccharide tolerance of human intestinal epithelial cells via MD-2 activity. Innate Immun. 2010 [Epub ahead of print]. Inflammatory bowel diseases (IBDs) are characterized by inappropriate responses to gut bacteria. Pathogen-associated molecular patterns (PAMPs) derived from intestinal bacteria stimulate innate immune receptors, including Toll-like and NOD-like receptors (TLRs, NLRs). The TLR4 pathway is required for inflammatory responses to lipopolysaccharide (LPS), a molecule that is present in the outer membrane of Gram-negative bacteria. Although TLR4 is the principal signaling receptor, the presence of myeloid differentiation (MD)-2 is necessary to confer LPS responsiveness to a cell. MD-2 is found both as an intracellular protein and as a secreted soluble protein.1 Levels of MD-2 in the serum increase dramatically in conditions such as septic shock.2 Binding of MD-2 to LPS results in the binding of the LPS-MD-2 complex to TLR4, followed by TLR4 signal transduction. In this study, Seksik et al3 tested the hypothesis that IBD patients have increased levels of MD-2 in their serum, which could then drive an inflammatory response by enhancing TLR4 responses to bacterial LPS. This was tested by obtaining sera from Crohn's disease (CD) and ulcerative colitis (UC) patients both in remission and suffering from active disease, and examining the ability of the sera to enhance LPS-dependent inflammatory responses from an LPS-hyporesponsive human epithelial cultured cell line (HT-29) that was stably transfected with TLR4 or MD-2. In support of their hypothesis, they found that sera obtained from both CD and UC patients with active disease significantly enhanced LPS-dependent IL-8 secretion from HT-29 cells as compared with sera from control patients. This enhancement occurred when the sera was placed on either the apical or the basolateral surface of the HT-29 cells. In addition, IL-8 secretion from HT-29 cells was higher when sera from IBD patients with active disease was present, compared with sera from IBD patients in remission or with control serum. This effect of sera was specific to LPS-dependent pathways, in that epithelial responsiveness to tumor necrosis factor alpha (TNF-α) and interleukin beta (IL-1β) was not altered by the presence of sera from IBD or control patients. To confirm that the stimulatory effect of serum was due to the presence of MD-2, experiments were carried out in the presence of neutralizing antibodies, including anti-MD-2, anti-TLR4, anti-CD14, and anti-LBP. Finally, they demonstrated that sera from IBD patients also enhanced LPS-induced Cox-2 expression and PGE2 secretion, which may have a role in the progression from inflammation to colorectal cancer in UC patients. Overall, these studies support the hypothesis that increased levels of biologically active MD-2 are found in the serum of IBD patients, particularly during active disease. Epithelial TLR signaling in the gut plays a significant role in the maintenance of homeostasis. Intestinal epithelial cells in the gut are exposed on a continual basis to high levels of bacterial antigens, and have developed several mechanisms to maintain hyporesponsiveness unless confronted by danger signals. Intestinal epithelial cells express low functional levels of TLR4, along with very low levels of intracellular MD-2, thus remaining relatively nonresponsive to the large amounts of LPS found in the lumen.4 In healthy individuals, TLR4 and MD-2 are primarily expressed by Paneth cells in the ileum, effectively conferring on these cells the ability to detect and respond to LPS by the release of antimicrobial peptides.5 In that soluble MD-2 acts as an opsonin for Gram-negative bacteria and enhances bacterial internalization and intracellular killing,6 it is possible that Paneth cells also release MD-2 into the surrounding area along with antimicrobial peptides. Upregulation of TLR4 expression has been observed in the terminal ileum and rectum of UC patients and in the terminal ileum of CD patients.7 Patients with IBD also demonstrate increased MD-2-positive inflammatory cells within the lamina propria.8 These findings all suggest an enhanced ability of IBD patients to react to luminal bacterial LPS through increased levels of MD-2 and TLR4. As well as demonstrating a possible role for circulating MD-2 in the perpetuation of inflammation in IBD patients, these findings also suggest a mechanism by which IBD may be triggered in susceptible individuals. Numerous studies have demonstrated an increased risk for the development of IBD following an infectious gastroenteritis exposure.9 With the recent findings that the soluble form of MD-2 is an acute phase protein released from the liver,6 the following scenario is envisioned. The release of MD-2 from the liver into the systemic circulation during an infection may inadvertently activate TLR4 in gut epithelial cells through the basolateral surface. Once activated, the TLR4 pathway signals through the key transcriptional regulator nuclear factor kappaB (NF-κB), resulting in the production of proinflammatory cytokines and an inappropriate inflammatory response to normal gut commensal microbes. This inappropriate and exaggerated innate inflammatory response then progresses to a dysregulated adaptive response towards gut microbes. Loss of tolerance to normal gut bacteria is considered a primary event in the pathogenesis of IBD. The findings in this current study that increased levels of biologically active MD-2 are found in the systemic circulation of IBD patients, and that serum from IBD patients effectively activates TLR4 signaling in epithelial cells, suggests that this molecule is a primary mediator in triggering the loss of tolerance to gut microbes and, as such, a potential target for therapeutics. However, it must be kept in mind that TLR4 signaling is also important for repair of the injured gut, so increased expression and signaling through this pathway may also serve a protective role.10

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