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. 2003 Mar;10(2):259-66.
doi: 10.1128/cdli.10.2.259-266.2003.

Lipoteichoic Acids From Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages Through Toll-like Receptor 2

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Lipoteichoic Acids From Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages Through Toll-like Receptor 2

Tetsuya Matsuguchi et al. Clin Diagn Lab Immunol. .
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Abstract

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-alpha) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-kappaB activation and TNF-alpha production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall >> polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-alpha secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2(-/-) mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-kappaB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.

Figures

FIG. 1.
FIG. 1.
TNF-α secretion from mouse mononuclear cells induced by various Lactobacillus strains. Mononuclear cells were isolated from spleens of 6-week-old female BALB/c mice. The mononuclear cells (2 × 106/ml) were incubated in RPMI 1640 plus 10% FCS containing 1 μg of heat-killed Lactobacillus bacteria/ml of six different strains. The TNF-α concentration in the culture supernatants was measured by ELISA after 12 h of incubation. The assays were done in triplicate, and the averages and the the standard deviation (SD) values are shown. The same experiment was repeated three times, yielding similar results, and a typical result is shown.
FIG. 2.
FIG. 2.
Proinflammatory activities of Lactobacillus subcellular fractions. Various subcellular fractions of L. casei were prepared as described in Materials and Methods. (A) RAW264.7 cells (5 × 105/ml) were stimulated with subcellular fractions isolated from L. casei. Each bacterial fraction was added to the culture medium at 1 μg/ml, and the TNF-α concentration in the culture supernatants was measured by ELISA after 12 h of incubation. (B) RAW264.7 cells were plated at 7.5 × 105 cells/plate and on the next day were transiently transfected with 1 μg each of a reporter plasmid containing tandem NF-κB consensus binding sites (pGL3-NF-kB-luc) and pRL-SV40 (as an internal control). At 48 h after the transfection, Lactobacillus subcellular fractions were added at 1 μg/ml, and the cell lysates were collected after 8 h of incubation to measure the relative luciferase activities. The experiment was repeated three times with similar results. A typical result is shown.
FIG. 3.
FIG. 3.
TLR2 is essential for TNF-α secretion induced by Lactobacillus LTA. (A) RAW264.7 cells (5 × 105 cells/ml) were stimulated with increasing amounts of LTA purified from L. fermentum (LTA-F) or L. casei (LTA-C). The culture supernatants were collected after 12 h, and the TNF-α concentration was measured by ELISA. The same experiment was repeated three times producing similar results, and a typical result is shown. (B) Splenic mononuclear cells were isolated from TLR2 gene-disrupted mice along with the control (TLR2+/−). The cells (2 × 106 cells/ml) were stimulated with the indicated amounts of purified LTAs from L. fermentum and L. casei, synthetic lipoprotein, or synthetic lipid A. After 12 h of incubation, the TNF-α concentration in the supernatants was measured by ELISA. The experiment was repeated three times, yielding similar results, and a typical result is shown. (C) Splenic mononuclear cells from C3H/HeN and its variant, C3H/HeJ. The cells were treated as in panel B. The TNF-α concentration in the supernatants was measured in triplicate, and the averages and the SD values are shown. The experiment was repeated three times, yielding similar results, and a typical result is shown.
FIG. 4.
FIG. 4.
Signal transduction induced by Lactobacillus spp. (A) HEK293T cells were transiently transfected with 0.5 μg of each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40 (as an internal control), plus 0.5 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2), or p3XFlag-mTLR4 (TLR4). At 48 h after the transfection, the cells were untreated, treated with LTA (1 μg/ml) of L. fermentum (F) or L. casei (C) or with synthetic lipid A (1 μg/ml) (A) for 8 h, and then lysed to measure the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (B) HEK293T cells were transiently transfected with 0.5 μg each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40, plus 1 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2) plus pcDNA3/1(+) (0.5 μg each), or the combination of pEFBOAS-mTLR2/Flag and pEFBOS-mTLR6/Flag (0.5 μg/each) (TLR2 + TLR6). At 48 h after the transfection, the cells were either not treated or treated with heat-killed L. fermentum (1 μg/ml) for 8 h and then lysed for the measurement of the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (C) Lactobacillus LTA induces JNK activation in RAW264.7 cells. RAW264.7 cells were either not treated or treated with 1 μg of either LPS or purified LTA from L. fermentum/ml for 30 min. Cells were lysed, and the JNK1 kinase activity was measured by using GST-c-Jun as the substrate.
FIG. 5.
FIG. 5.
TLR2 is essential for TNF-α secretion induced by Lactobacillus spp. (A) Splenic mononuclear cells were isolated from TLR2 gene-disrupted mice, along with the control (TLR2+/−). The cells (2 × 106 cells/ml) were stimulated with the indicated amounts of heat-killed L. fermentum and L. casei, synthetic lipoprotein, or synthetic lipid A. After 12 h of incubation, the TNF-α concentration in the supernatants was measured by ELISA. Measurements were done in triplicate, and the averages and the SD values are shown. The experiment was repeated three times, yielding similar results, and a typical result is shown. (B) Splenic mononuclear cells from C3H/HeN and its variant, C3H/HeJ. The cells were treated as in panel A. The TNF-α concentration in the supernatants was measured in triplicate, and the averages and the SD values are shown. The experiment was repeated three times producing similar results, and a typical result is shown.

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