| A huge number and diversity of microbes colonize the human gastrointestinal tract. The intestinal epithelial cells serve as the first line of defense against pathogen invading. The antimicrobial peptides secreted by the epithelial cells paly an important role in keeping the microecosystem balance and defending the microorganism infection in the gut. Human β-defensin-2 (hBD-2), one of important antimicrobial peptides, is widely expressed in the skin and mucusal epithelial cells of respiratory, gastrointestinal, and urogenital tracts. In normal condition, hBD-2 does not express in the epithelial cells, howevere it is dramaticlly up-regulated in response to inflammatory stimuli, which implies an essential role in sites of inflammation.The pervious studies have shown that the pathogens such as the Salmonella.enteritidis and the enteroinvasive E.coli which result in the inflammation can induce the hBD-2 expression in the intestinal mucusal tissues. What is the exactly role of the commensal bacterium in the expression of intestinal antimicrobial peptides, however, has not been fully studied. Bifidobacterium, one major normal microbe member of the human intestine, is involved in the digestion, nutrition, metabolism, absorption, immunity and the host defense against infection. Moreover, the regulation and signal transduction of hBD-2 expression is so complex that until now they have not been fully understood. Through evaluating the relation between the Bifidobacterium and the expression of hBD-2 would not only provide a way for treatment of infectionsthrough enhancing the local concentration of antimicrobial peptides, but also be helpful to elucidate the molecular mechanisms of host defense against pathogen invading.In this work, we investgated the induction of hBD-2 gene expression by Bifidobacterium in the human intestinal epithelial cell lines and idenfied the most effective components of Bifidobacterium cell wall in the induction of hBD-2 gene expression. Furthermore, we also evaluated the signaling pathway of the Bifidobacterium-induced hBD-2 gene expression.Firstly, we examined the role of Bifidobacterium in the induction of hBD-2 gene expression in the intestinal epithelial cell line and isolated the effective components of the bacterial cell wall. Bifidobacterium Longum (5. Longum) was cultured in anaerobic incubator and its cell wall was isolated by sonication and centrifugation. The whole cell wall proteins were extracted with SDS. When incubated with the living B. Longum, the heat killing B. Longum, the cell wall and the whole cell wall proteins, the hBD-2 expression was determined in the intestinal epithelia cell line Caco-2 at the mRNA level by the method of RT-PCR and Northern Hybridization, whereas, there was no hBD-2 mRNA signal detected in the un-stimulated cells. Meanwhile, ELISA detection showed that there was a significant increase in hBD-2 peptide expression in the culture supernatant of Caco-2 cells when incubated with heat killing B. longum cells, the cell wall, or the cell wall proteins. In the immocytochemistry detection system, there was a strong hBD-2 reaction in the cytoplasm of Caco-2 cells exposed to B. longum cell wall or cell wall proteins. Coincidence with above determination, hBD-2 signals were detected by Western blotting in the whole cell lysates and the culture supernatants of Caco-2 cells stimulated with B.longum cell wall or cell wall proteins. In contrast, no hBD-2 signal was detected in the cell lysates and the culture supernatant of the un-stimulated Caco-2 cells. Four fractions of B.longum cell wall proteins were obtained via Sephacryl S-100HR chromatography. The second faction with the molecular weight of 35-65kDa was identified to be the main bioactive component that highly stimulated the induction of hBD-2 gene expression in Caco-2 cells.Next, we examined the transcriptional regulation mechanisms and the intracellular signal transduction pathway of the hBD-2 expression after stimulation with the B.longum cell wall proteins. In order to check the possible role of the NF-kB element in the regulation of hBD-2 gene expression, we constructed the recombinant enhanced green fluorescent protein (EGFP) expression plasmids EGFP-1/-510 hBD-2, which contain the binding site for NF-kB in the upstream of hBD-2 gene and a mutational NF-kB binding element plasmid EGFP-l/-510mhBD-2 according to the hBD-2 upstream sequences and the results of the transcription factor database analysis. Those reporter constructs were transfected into the Caco-2 cells, then stimulated with the B.longum cell wall proteins, and the green fluorescence protein (GFP) expression was detected by Flow Cytometry. Results indicated that the NF-kB element is essential for the hBD-2 induction by the B.longum cell wall proteins. Western Blot analysis showed that shortly after the B.longum cell wall proteins stimulation, the IicB-a in the cytoplasm of the Caco-2 cells began to degradation /phoshporylation and the p65 subunit in the nuclear began to increase. Western blot also demonstrated that B.longum cell wall proteins caused phosphorylation of MAPKs pathway including EKR1/2, p38 and JNK in Caco-2 cells.Finally, we investigated the possible role of Toll-like receptor (TLR) in the B.longum cell wall proteins-mediated induction of hBD-2 gene expression in the Caco-2 cells. In order to enhance the transgene efficiency and the target gene expression, based on the AdEasy system we constructed the adenovirus expression vector of mutant pAd-mTLR2 and mutant Ad-mMyD88, which is the key adaptor in TLRs signaling pathway. The Caco-2 cells were infected with these vectors and then exposured to B.longum cell wall proteins. The results indicated that both mutant pAd-mTLR2 and mutant pAd-mMyD88 significantly inhibited B.longum cell wall protein-induced expression of hBD-2 in Caco-2 cells, suggesting that TLR2 receptor may involved in the B.longum cell wall protein-mediated hBD-2 gene expression in human interstinal epithelial cells, and the signaling pathway would be MyD88-dependent.In order to detect hBD-2 expression at protein level, the recombinantprokaryotic expression vector pGEX-lXT-hBD-2 was constructed and ￡.co//-based product of GST-hBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against hBD-2 was produced. After caprylic acid and ammonium sulfate precipitation, high titter of specific polyclonal antibodies against hBD-2 was obtained. This result suggests that recombinant peptide fusion protein could be used to produce antibody instead of using the conjugate of peptide-albumin or peptide-thyroid globulin. The obtained antibodies were used for detection of hBD-2 gene expression at protein level in this study.
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