Heat killed Helicobacter pylori augments NK cell cytotoxicity through induction of NKG2DL surface expression on gastric adenocarcinoma cells
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1
Universidad de Chile, Programa Disciplinario de Inmunología. Facultad de Medicina, Chile
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2
Universidad de Chile, Departamento de Tecnología Médica. Facultad de Medicina, Chile
Background: NKG2D ligands (NKG2DLs) are expressed on the cell surface in response to cellular stress. The recognition of NKG2DL on damaged cells is a key mechanism of Immunosurveillance to tumors. NKG2D receptor is present on cytotoxic cells, such as NK cells, T gamma delta cells, NKT cells and CD8+ T cells. Inflammation is one stress inductor related to chronic injury on gastric cells infected with Helicobacter pylori (H. pylori). This bacterium can induce cellular damage through its pathogenic factors and pathogen associated molecular patterns (PAMPs), like HSP60 and lipopolysaccharide, which are recognized by Toll like receptors (TLR). The relation between TLR activation and NKG2DL has been studied on antigen presenting cells, but its effect on epithelial cell is not known. Our aim was to evaluate NKG2DLs expression on gastric adenocarcinoma cell lines in response to PAMPs present on H. pylori through TLRs activation and its effect on NK cells cytotoxicity activation.
Methods: NKG2DL expression was assessed at the protein and mRNA levels in MKN-45 gastric adenocarcinoma cells in response to Heat killed H. pylori (HKHP) by flow cytometry and real time PCR respectively. The role of TLR was evaluated by cell treatment with LPS from Rhodobacter sphaeroides, a TLR4 antagonist, while the effect of MyD88 pathway was analyzed with a peptide that inhibits the dimerization of MyD88. Activation of NK cells degranulation was evaluated by CD107a surface expression by flow cytometry after co-culture with MKN-45 cells previously exposed to HKHP. We also assessed NK cell cytotoxicity on target cells by a Lactate dehydrogenase (LDH) release assay. The association between NKG2D receptor and cytotoxic activity was evaluated through a neutralizing assay with a blocking antibody.
Results: HKHP induces a selective modulatory effect on MICA, ULBP2/5/6 and ULBP4 cell surface expression and mRNA levels, but it does not influence other NKG2DL expression on MKN-45 cells. HKHP effect depends on TLR4 activation and MyD88 pathway, since its effect was reverted by TLR4 and MyD88 inhibition. HKHP treatment also results in an increase in TLR4 expression at the cell surface and mRNA levels on MKN-45 cells. Accordingly increased MICA, ULBP2/5/6 and ULBP4 expression on target cells resulted in increased NK cell cytotoxicity, mainly through NKG2D receptor activation.
Conclusion: In our work, we demonstrated that molecules from H. pylori induce NKG2DL surface expression on MKN-45 cells through TLR4 activation, resulting in NKG2D receptor-dependent NK cell cytotoxicity. Not only could this effect contribute to the maintenance of epithelial cell damage, but also to the inflammatory response and chemoattraction of immune effector cells that are associated with the cell damage that generates ulcer and gastritis and causes the events that precedes cancer transformation.
Acknowledgements
Fondecyt regular 1330330
Keywords:
NKG2D receptor,
NKG2D ligands,
TLR4,
NK,
tumor immunology,
Helicobacter pylori,
Gastric cell line
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Tumor immunology
Citation:
Hernández
CJ,
Garrido
M,
Kramm
K,
Ribeiro
CH and
Molina
MC
(2015). Heat killed Helicobacter pylori augments NK cell cytotoxicity through induction of NKG2DL surface expression on gastric adenocarcinoma cells.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00061
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Received:
01 Jun 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Prof. Carolina J Hernández, Universidad de Chile, Programa Disciplinario de Inmunología. Facultad de Medicina, Santiago, Chile, carojimena@gmail.com