Abstract

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-kappaB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521-30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.

Highlights

  • Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces

  • We examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells

  • Asialo-GM1 on epithelial cells interacted with P. aeruginosa flagellin, leading to activation of phospholipase C, Ca2ϩ mobilization, phosphorylation of ERK1/2, and activation of mucin transcription [32]

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Summary

Introduction

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase. HBD-1 was expressed constitutively, hBD-2, hBD-3, and hBD-4 were induced following bacterial infections (10 –12, 14, 15) In addition to their antimicrobial activity, hBD-2 and murine ␤-defensins can potentially function as chemokines for immature dendritic cells and memory T cells through interaction with chemokine receptor CCR6 [16, 17]. From our previous work to evaluate the role of hBD-2 in Salmonella enteritidis infection, we concluded that S. enteritidis flagellin (FliC) increased hBD-2 promoter activity and mRNA levels in Caco-2 cells via NF-␬B. Other studies demonstrated that the flagellin of Salmonella dublin [22] or Salmonella typhimurium [23] induced nitric-oxide synthase and IL-8 secretion, respectively

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