Ability of Virulence Attenuated Listeria monocytogenes Strains Secreting Truncated Forms of InlA of Different Lengths to Stimulate Cytokine and Chemokine Production by Intestinal Epithelial Cells

Introduction: L. monocytogenes utilizes Internalin A (InlA), a cell wall anchored protein to cross the intestinal barrier. Mutations leading to premature stop codons (PMSC) in inlA result in a truncated and secreted InlA. These mutations are causally associated with attenuated-virulence and are found in 45% of isolates from ready-to-eat foods. Types 3 and 4 are the most prevalent and represent the largest and smallest truncated forms of InlA, respectively. 

Purpose: Compare the ability of L. monocytogenes strains producing full-length and truncated forms of InlA to induce cytokine/chemokine responses during invasion of human intestinal epithelial cells.  

Methods: Caco-2 cells were used to assay cytokine/chemokine response after invasion with L. monocytogenes strains encoding a full-length, truncated InlA or L. innocua as an avirulent control. Caco-2 monolayers were infected with Listeria for 1h.  Cells were harvested, counted, and lysed while stabilizing nucleic acids.  QRT-PCR targeted interleukin-8 (IL-8), interleukin-15 (IL-15), and monocyte-chemotactic-protein-1 (MCP-1).  The DDCt method was used for relative quantification of gene expression to the calibrator (uninfected Caco-2 cells), with Glyceraldehyde-3-phosphate-dehydrogenase as a normalizer.

Results: L. monocytogenes strains encoding either a full-length or truncated and secreted InlA significantly (P < 0.05) increased IL-8 levels as compared to L. innocua and uninfected Caco-2 cells. No strains elicited production of IL-15. Strains carrying PMSC types 3 and 4 produced similar (P > 0.05) levels of IL-8 and MCP-1, showing that the size of a truncated and secreted InlA does not affect cytokine/chemokine responses. All strains produced higher (P < 0.05) levels of MCP-1 compared to the calibrator. L. monocytogenes and L. innocua produced similar levels of MCP-1, indicating InlA does not stimulate MCP-1 production in Caco-2 cells. 

Significance: InlA effects production of IL-8 in Caco-2 cells. Size of secreted InlA product has no effect on the cytokine/chemokines investigated. These results suggest that exposure to virulence-attenuated L. monocytogenes through contaminated foods may stimulate innate immunity.