Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria

Abstract

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.

FIG. 1.

Effects of LTA from L. johnsonii strain La1 and L. acidophilus strain La10 on the release of IL-8 by HT29 cells challenged with LPS purified from E. coli. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM in the presence of E. coli LPS at 10 ng/ml (•) or 100 ng/ml (▪) and various amounts of LTA from L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). Activation of HT29 cells by LPS plus HM alone is depicted (dashed lines). The standard deviations are indicated by the error bars. The results are representative of seven independent experiments for La1 LTA and three independent experiments for La10 LTA.

FIG. 2.

Effect of LTA from L. johnsonii strain La1 on the release of IL-8 by HT29 cells challenged with whole E. coli bacteria. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM and whole E. coli bacteria (2.5 × 10⁵/ml) (▪) or E. coli LPS (100 ng/ml) (•) in the presence of various amounts of LTA from L. johnsonii strain La1. Activation of HT29 cells in the absence of LTA is depicted (dashed lines).The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

FIG. 3.

Effects of LTA from L. johnsonii strain La1 and L. acidophilus strain La10 on LPS-induced ENA-78 mRNA expression and TNF-α cytokine release by HT29 cells. (A) ENA-78 expression was assessed by RT-PCR on total RNA of HT29 cells challenged with 100 ng of E. coli LPS per ml in the absence (lane 1) or presence of 2% HM (lanes 2 to 6) with the addition of MY4 anti-CD14 monoclonal antibody (lane 3), isotype-matched antibody control (lane 4), or LTA from L. johnsonii strain La1 at 1 μg/ml (lane 5) or 50 μg/ml (lane 6). The expected PCR product size for ENA-78 transcripts was 220 bp. Amplified bands for β-actin (460 bp) were used as the housekeeping gene. SM, size marker. (B) TNF-α production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM, 100 ng of E. coli LPS per ml, and various amounts of LTA from L. johnsonii strain La1 (▪) or L. acidophilus strain La10 (•). Production of TNF-α by HT29 cells in the absence of LTA is shown (dashed line). The standard deviations are indicated by the error bars. The results are representative of three independent experiments.

FIG. 4.

Effects of deacylation of the LTAs on their antagonistic activity. HT29 cells were challenged with E. coli LPS (100 ng/ml) in medium supplemented with 2% HM in the absence (dashed lines) or presence (solid lines) of various amounts of native LTA (▪) or deacylated LTA (•) purified from either L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). After 24 h, the release of IL-8 in the culture supernatants was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

FIG. 5.

Effects of different sequential treatments with LTA, LPS, and HM on IL-8 production by HT29 cells. (A) HT29 cells were preincubated for 4 h with LTA from L. johnsonii strain La1 (100 μg/ml) in the presence of 2% HM, washed twice with a serum-free medium, and then challenged for 20 h with E. coli LPS (100 ng/ml) in the presence or absence of HM. (B) HT29 cells were incubated with LTA from L. johnsonii strain La1 (50 μg/ml) in the presence of HM for 4 h before the addition of E. coli LPS (100 ng/ml). (C) HT29 cells were challenged with E. coli LPS (100 ng/ml) in the presence of HM for 4 h before the addition of LTA from L. johnsonii strain La1 (1, 10, and 50 μg/ml). IL-8 release in the supernatants after a total of 24 h of culture was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.