Trypsin-Sensitive Modulation of Intestinal Epithelial MD-2 as Mechanism of Lipopolysaccharide Tolerance

Abstract

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn’s disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.

Loss of TLR2 Worsens Spontaneous Colitis in MDR1A Deficiency through Commensally Induced Pyroptosis

“…Soluble MD-2 may act as an acute-phase reactant in sepsis (61). Expression of MD-2 protein is significantly increased in the inflamed intestinal mucosa of patients with active IBD colitis (62). Here the exaggerated inflammatory response seen in TLR2/MDR1A double deficiency resulted from broken tolerance to commensal LPS via MD-2, as deletion of MD-2 markedly abrogated colitis exacerbation.…”

Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis

“…Additionally, expression of MD-2 is thought to be another important factor that determines the LPS responsiveness because optimal LPS recognition by TLR4 requires MD-2 as a coreceptor. It has been reported that MD-2 expression in healthy, normal intestinal mucosa is minimal, while it is increased in active inflammatory bowel disease colitis (33,34). Moreover, inhibition of CpG methylation and histone deacetylation was shown to result in increased mRNA expression of MD-2 gene in IECs just recently (35).…”

TLR2 Mediates Gap Junctional Intercellular Communication through Connexin-43 in Intestinal Epithelial Barrier Injury

“…For preparation of subcellular fractions, the ProteoExtract subcellular proteome extraction kit from Merck was used according to the manufacturer's instructions. Immunoprecipitation/immunoblotting and mouse 62-cyto/chemokine antibody array III (RayBiotech) were performed as described previously (18,33). All of the experiments were repeated at least twice; representative results are shown for each experiment.…”

“…Confocal Laser Microscopy-After mounting with Vectashield mounting medium with propidium iodide or DAPI (Vector Laboratories), immunofluorescent sections were assessed using a laser-scanning confocal microscope (Plan-Neofluar 40ϫ/1.3 (oil) or Plan-Apochromat 63ϫ/1.4 (oil) differential interference contrast objectives; Zeiss Axiovert 100M-LSM 510), as described previously (18,33). Briefly, the multitrack option of the microscope and sequential scanning for each channel were used to eliminate any cross-talk.…”