1. The inhibitory effect of LTA from Lactobacillus plantarum on LPS-mediated cytokine production
Han Geun Kim1, Joo Yun Kim1, Jung Min Lee1, Seung Hyun Han2 and Dae Kyun Chung1.
1 School of Biotechnology and Institute of Life Science and Resources, Kyung Hee university, Suwon, Korea
2 Humoral Immunology Section, International Vaccine Institute, SNU Research Park, Seoul, Korea
Abstract. Lipoteichoic acids (LTAs) are one of the main immunostimulatory components of gram-positive bacteria. In this study, we prepared highly purified LTA from Lactobacillus plantarum (pLTA) using n-butanol extraction followed by chromatography with Octyl-sepharose and DEAE-sepharose, and examined the effects of purified pLTA on the cytokine production. The secretion of IL-12p70, IL-8, IL-23p19, and TNF-α was induced in the manner which is dependent on pLTA concentration. Interestingly, the TNF-α secretion from lipopolysaccharide (LPS) stimulated-THP-1 cells was diminished by pLTA pre-treating. TLR2 mRNA was induced in cells treated with LPS after pLTA stimulation, but nucleotide-binding oligomerization domain (nod) 1 and nod2 mRNA were reduced. Furthermore, up-regulation of NOD proteins by transient transfection reinstates the production of TNF-α after LPS re-stimulation. Taken together, our results suggest that pLTA might induce Th1 polarization and would alleviate inflammation (or septic shock) caused by pathogens through the regulation of NOD. Therefore, pLTA would maintain homeostasis in immune activities.
Introduction. Many lactobacillus species are normal members of the human gut microflora and most are regarded as safe when administered as probitotics. Several strains of Lactobacilli also have effects upon the production of cytokines such as IL-12, IL-10, TNF-α, TGF-β, IL-8 and RANTES, and cell proliferation in human intestinal epithelial cells (1). Lipoteichoic acid (LTA) is one of the main immunostimulatory components of gram-positive bacteria and considered to be analogous to the LPS of Gram-negative bacteria. However, its immunostimulatory potential had been controversial, because the LTA preparations used in earlier studies were either damaged or contaminated (2). Recent studies using highly purified preparations of Lactobacillus plantarum LTA have clearly shown that lactobacillus LTA can efficiently stimulate mononcytes via TLR2 to produce proinflammatory cytokines (3). Some reports have been introduced that Staphylococcus aureus LTA inhibits LPS-induced TNF-α and IL-8 release (4, 5). Another studies using highly purified preparations of Staphylococcus aureus LTA have clearly shown that stahpylococcal LTA can synergize with peptidoglycan to induce septic shock and multi-organ failure in rats (6, 7). Despite potential importance of cross-tolerance in LPS and LTA signaling via TLRs, however, the molecular mechanism of LTA-induced tolerance is not clear. In this study we elucidate the properties for highly purified Lactobacillus plantarum LTA on cytokine production and the molecular mechanism of tolerance induced by LTA.
Fig. 1. LTA preparation from L. plantarum K518 (pLTA).
Fig. 2. Cytokine response of THP-1 cells to stimulation with pLTA.
Fig. 3. Effect of anti-CD14, anti-TLR2, and dnMyD88 transfectants on cytokine induction by pLTA and aLTA.
Fig. 4. pLTA inhibited LPS-mediated TNF-α induction, whereas S. aureus peptidoglycan (PGN), IFN-r, and E. coli K12 PGN synergically induced TNF-α response to LPS (TNF-α ng/ml).
LAL test (to check contamination with endotoxin)
L. plantarum cultivation
Purified LTA
(1.5 mg/ml)
OD (405)
EU/ml
0.0446
0.0584
Relative mRNA expression
Disrupted with glass beads
BCA test (to check contamination with proteins)
Purified LTA
(1.5 mg/ml)
OD (562)
mg/ml
0.0173
0.003
n-butanol extraction
Silver staining (to check contamination with proteins)
Chromatography with Octyl-sepharose
Relative mRNA expression
Chromatography with DEAE-sepharose
Fig. 5. Detection of related gene expressed during pLTA-LPS cross tolerance : pLTA-LPS co-treatment remarkably induced TLR2 and CD14 mRNA, whereas reduced nod1 and nod2 mRNA expression compared with LPS treatment only.
Fig. 6. The synergistic effect of TNF-α production by LPS and endotoxin protein.
Fig. 7. Specific up-regulation of NOD1 and NOD2 reinstated the production of TNF-α after LPS re-stimulation : TNF-α production was inhibited in control cells which were pretreated with pLTA and re-stimulated with LPS, whereas it was reinstated in cells transfected with nod1 or nod2.
a. Antibody treatment against TLR1, TLR2, TLR4, TLR6, CD14, respectively, reduced the TNF-α production, suggesting that phenol extracted LPS (commercial LPS) was still contaminated with endotoxin protein. However, after re-extraction of commercial LPS with phenol once, the purified LPS (phLPS) reduced the TNF-α production only with anti-TLR4 and anti-CD14 treatment.
b. Also, phLPS did not elicit strong response at various concentration, suggesting that endotoxin protein may induce synergistic production of TNF-α with LPS.
transfectant
vector
Nod1
Nod2
pLTA 10ug/ml
LPS 0.5ug/ml - +
Fig. 8. Expression of dominant-positive nod1 and nod2 in transfectants was determined by reverse transcription polymerase chain reaction.
Conclusion
References
Highly purified pLTA was a potent inducer of IL-12, IL-23p19, TNF-α, and IL-8, but not IL-10. Furthermore, pLTA inhibited LPS-mediated TNF-α induction via regulation of NOD level. These results suggest that pLTA might induce Th1 differentiation and would alleviate sepsis caused by Gram-negative bacteria. Therefore, pLTA could maintain homeostasis in immune activities.
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vector
Nod1
Nod2
Transfectant
Beta-actin