Listeriolysin O-induced membrane permeation mediates persistent interleukin-6 production in Caco-2 cells during Listeria monocytogenes infection in vitro

Abstract

Listeriolysin O (LLO), a major virulence factor of Listeria monocytogenes, is a member of the cholesterol-dependent cytolysin family and plays important roles not only in survival of this bacterium in phagocytes but also in induction of various cellular responses, including cytokine production. In this work, we examined the involvement of LLO in induction of the cytokine response in intestinal epithelial cells, the front line of host defense against food-borne listeriosis. Infection of Caco-2 cells with wild-type L. monocytogenes induced persistent expression of interleukin-6 (IL-6) mRNA. In contrast, IL-6 expression was observed only transiently during infection with non-LLO-producing strains. A sublytic dose of recombinant LLO (rLLO) induced the expression of IL-6 via formation of membrane pores. Under conditions of LLO-induced pore formation without extensive cell lysis, Ca2+ influx was observed, and the IL-6 expression induced by rLLO was inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), an intracellular Ca2+ chelator. LLO secreted by cytoplasmic L. monocytogenes appeared to induce pore formation in the membrane and to enable the trafficking of intracellular and extracellular molecules. Pretreatment with BAPTA-AM inhibited persistent IL-6 expression in Caco-2 cells infected with wild-type L. monocytogenes. These results suggest that LLO is involved in IL-6 production in the late phase of infection through the formation of Ca2+-permeable pores and subsequent Ca2+-dependent modulation of signaling and gene expression.

FIG. 1.

L. monocytogenes induces persistent IL-6 expression in Caco-2 cells in an LLO-dependent manner. (A) Kinetics of IL-6 expression in L monocytogenes-infected Caco-2 cells. Caco-2 cells were infected with L. monocytogenes wt (LMwt), the hly mutant, or ATCC 15313, and the expression of IL-6 was analyzed by RT-PCR. The results are representative of the results of three similar experiments. (B) Intracellular growth of L. monocytogenes. Caco-2 cells were infected with L. monocytogenes wt, the hly mutant, a fivefold-higher dose of the hly mutant (5 × hly mutant), or ATCC 15313, and the number of intracellular bacteria was determined by the CFU assay. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations. (C) Cytoplasmic invasion by L. monocytogenes. Caco-2 cells were infected with L. monocytogenes wt and the hly mutant for 5 h. The cells were then fixed and stained with phalloidin-Alexa 488 to detect actin nucleation (green) and with anti-Listeria goat polyclonal antibody and sequential anti-goat immunoglobulin G-Alexa 546 to detect L. monocytogenes (red). (D) Quantitative analysis of IL-6 expression. Caco-2 cells were infected with L. monocytogenes wt in the absence or presence of cytochalashin D (cyt. D) or with a fivefold-higher dose of the hly mutant. The expression of IL-6 at 5 h after infection was analyzed by real-time RT-PCR. The results were expressed relative to the level of β-actin mRNA. The data are the means ± standard deviations from three independent experiments. Two asterisks indicate that there was a significant difference compared to uninfected cells.

FIG. 2.

LLO-induced IL-6 induction in Caco-2 cells is dependent on the pore-forming activity of recombinant protein. (A) Expression of IL-6 in Caco-2 cells. Caco-2 cells were stimulated with different doses of rLLO, cholesterol-treated rLLO [chol. (+) LLO], or rLLO_W492A for 3 h, and the expression of IL-6 was analyzed by RT-PCR. The results are representative of the results of two similar experiments. (B) NF-κB activation induced by rLLO. HEK293 cells transfected with reporter vectors were stimulated with PBS or different doses of rLLO or rLLO_W492A for 6 h, and NF-κB activation was assessed by the luciferase assay. The results are representative of the results of five similar experiments. The data are the means ± standard deviations for three determinations.

FIG. 3.

Recombinant LLO induces IL-6 production only at a sublytic dose. (A) Cytolytic effect of rLLO on Caco-2 cells. Caco-2 cells were stimulated with different doses of rLLO and rLLO_W492A for 3 h, and the LDH activity in culture supernatants was assessed. The results are representative of the results of three similar experiments. The data are the means ± standard deviations for three determinations. (B) Viability of Caco-2 cells after stimulation with rLLO. Caco-2 cells were treated with PBS (P) or 1% Triton X-100 (T) or were stimulated with different doses of rLLO or rLLO_W492A for 3 h. Then the WST-1 reagent was added to the cultures, the cells were incubated for 30 min, and the absorbance at 450 nm of culture supernatants was measured. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations.

FIG. 4.

Involvement of Ca²⁺ in LLO-induced IL-6 expression. (A) Effect of rLLO on intracellular Ca²⁺. Caco-2 cells were treated with Fura 2-AM. Recombinant LLO or rLLO_W492A was added to cells at the time indicated by the arrow. Intracellular Ca²⁺ was assessed by microfluorometry, and the results are expressed as the ratio of fluorescence intensity (510 nm). The y axis is (emission with 340-nm excitation)/(emission with 380-nm excitation). The results are representative of the results of three similar experiments. (B) Inhibition of rLLO-induced IL-6 expression by chelating intracellular Ca²⁺. Caco-2 cells were treated with 10 μM BAPTA-AM for 1 h and then stimulated with different doses of rLLO for 3 h. The expression of IL-6 was analyzed by RT-PCR. The results are representative of the results of three similar experiments. DMSO, dimethyl sulfoxide. (C) Inhibition of rLLO-induced NF-κB activation by chelating intracellular Ca²⁺. HEK293 cells were transfected with reporter vectors and treated with 10 μM BAPTA-AM, 50 μM NiCl₂, and 10 μM verapamil for 1 h. The cells were then stimulated with 4 nM rLLO for 6 h, and NF-κB activation was determined by the luciferase assay. The results are expressed in units relative to the activity of the internal control pRL-SV40. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations. Two asterisks indicate that there was a significant difference compared to cells stimulated with rLLO without any pretreatment.

FIG. 5.

Involvement of Ca²⁺ in L monocytogenes-induced persistent IL-6 expression. (A) LDH release from L. monocytogenes-infected Caco-2 cells. Caco-2 cells were infected with the hly mutant, a fivefold-higher dose of the hly mutant (5 × hly mutant), ATCC 15313, or L. monocytogenes wt (LMwt) in the presence or absence of cytochalashin D (cyt. D). Culture supernatants were collected at different times, and the LDH activity in culture supernatants was assayed. The results are representative of the results of three similar experiments. The data are the means ± standard deviations for three determinations. (B) Gentamicin (GM) influx induced by L. monocytogenes infection. Caco-2 cells were infected with L. monocytogenes wt or a fivefold-higher dose of the hly mutant and incubated in medium containing 5 μg/ml (control) or 50 μg/ml of gentamicin for 5 and 7 h. The number of intracellular bacteria was determined by the CFU assay, and the results were expressed as the number of CFU relative to the control. The solid line indicates the value for the control. The results are representative of the results of two similar experiments. The data are the means ± standard deviations for three determinations. Two asterisks indicate that there was a significant difference compared to cells treated with 5 μg/ml gentamicin. (C) Inhibition of L. monocytogenes-induced persistent IL-6 expression by chelating intracellular Ca²⁺. Caco-2 cells were pretreated with BAPTA-AM for 1 h and then infected with L. monocytogenes wt for 1 or 5 h. Then the expression of IL-6 was analyzed by RT-PCR. The results are representative of the results of two similar experiments.