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Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein ( dltD ) knockout CRL1506 strain ( L. plantarum Δ dltD ) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 ( L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarum Δ dltD or L. plantarum WT showed higher levels of IFN-β while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-β and IL-10 and reduced intraepithelial lymphocytes (CD3 + NK1.1 + CD8αα + ) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarum Δ dltD -treated mice showed enhanced levels of IFN-β after poly(I:C) challenge. However, treatment of mice with L. plantarum Δ dltD was not able to increase IL-10 or reduce CD3 + NK1.1 + CD8αα + cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation., (Copyright © 2020 Mizuno, Arce, Tomotsune, Albarracin, Funabashi, Vera, Islam, Vizoso-Pinto, Takahashi, Sasaki, Kitazawa and Villena.)

Complement component 3 (C3) is one of the proteins associated with complement cascades. C3 plays an essential role in three different pathways-the alternative, classical and lectin pathways. It is well known that cytokines activate complement system and increase complement component C3 production. In the current study, we found that lipoteichoic acid isolated from Lactobacillus plantarum K8 (pLTA) inhibited tumor necrosis factor-alpha (TNF-α) or interferon-gamma (IFN-γ)-mediated C3 mRNA and protein expression in HaCaT cells. pLTA inhibited C3 expression through the inhibition of the phosphorylation of p65 and p38 in the TNF-α-treated cells, while the inhibition of STAT1/2 and JAK2 phosphorylation by pLTA contributed to the reduction of C3 in IFN-γ-treated cells. When mice were pre-injected with pLTA followed by re-injection of TNF-α, serum C3 level was decreased as compared to TNF-α-injected only. Further studies revealed that membrane attack complex (MAC) increased by TNF-α injection was lessened in pLTA-pre-injected mice. A bactericidal assay using mouse sera showed that MAC activity in pLTA-pre-injected mice was lower than in TNF-α only-injected mice. These results suggest that pLTA can suppress inflammatory cytokine-mediated complement activation through the inhibition of C3 synthesis. pLTA application has the potential to alleviate complement-mediated diseases caused by excessive inflammation., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Platelet-activating factor receptor (PAFR) plays an important role in bacterial infection and inflammation. We examined the effect of the bacterial cell wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) on PAFR expression in THP-1, a monocyte-like cell line. LPS and aLTA, but not pLTA, significantly increased PAFR expression, whereas priming with pLTA inhibited LPSmediated or aLTA-mediated PAFR expression. Expression of Toll-like receptor (TLR) 2 and 4, and CD14 increased with LPS and aLTA treatments, but was inhibited by pLTA pretreatment. Neutralizing antibodies against TLR2, TLR4, and CD14 showed that these receptors were important in LPS-mediated or aLTA-mediated PAFR expression. PAFR expression is mainly regulated by the nuclear factor kappa B signaling pathway. Blocking PAF binding to PAFR using a PAFR inhibitor indicated that LPS-mediated or aLTA-mediated PAF expression affected TNF-α production. In the mouse small intestine, pLTA inhibited PAFR, TLR2, and TLR4 expression that was induced by heat-labile toxin. Our data suggested that pLTA has an anti-inflammatory effect by inhibiting the expression of PAFR that was induced by pathogenic ligands.

Streptococcus mutans is a pathogenic Gram-positive bacterium that is closely associated with dental caries and subsequent pulpal inflammation. Although lipoteichoic acid (LTA) is considered a major virulence factor of Gram-positive bacteria, little is known about the innate immunity to S. mutans LTA. In this study, we purified LTA from S. mutans (Sm.LTA) through n-butanol extraction, hydrophobic interaction column chromatography, and ion-exchange column chromatography to investigate its immunological properties using murine macrophages. The Sm.LTA preparation had no detectable contamination with endotoxins, proteins, or nucleic acids. Upon exposure to Sm.LTA, the murine macrophage cell-line RAW 264.7 cells produced TNF-α and nitric oxide (NO) in a dose-dependent manner. Sm.LTA preferentially bound to and activated CHO/CD14/TLR2 cells rather than CHO/CD14/TLR4 cells, which are stable transfectants expressing CD14 and TLR2 or CD14 and TLR4, respectively. Sm.LTA could not induce TNF-α or NO production in macrophages derived from TLR2-deficient mice whereas it dose-dependently induced those inflammatory mediators in wild-type macrophages. TLR2-dependent induction of NO by Sm.LTA was also confirmed in RAW 264.7 cells using specific antibodies blocking TLR2. Furthermore, Sm.LTA deacylated by alkaline hydrolysis neither stimulated TLR2 nor induced TNF-α or NO production, suggesting that Sm.LTA lipid moieties are crucial for the immuno-stimulatory activity of Sm.LTA. Unlike Staphylococcus aureus LTA, which has potent immuno-stimulating activity, Sm.LTA showed a modest induction of NO production comparable to LTAs of other oral bacteria Enterococcus faecalis and Lactobacillus plantarum. In conclusion, our results suggest that the Sm.LTA interacts with TLR2 through the lipid moiety for the induction of inflammatory mediators in macrophages., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lipoteichoic acid (LTA) is a major immuno-stimulating component of Gram-positive bacteria. LTA from the beneficial bacterium Lactobacillus plantarum induces weak nitric oxide (NO) production in murine macrophages. Currently, it is not clear if LTA from L. plantarum is able to stimulate the innate immune response, even in the presence of inflammation. In the present study, we prepared highly pure and structurally intact LTA from L. plantarum and investigated its ability to induce NO in the presence of interferon (IFN)-γ in the RAW 264.7 murine macrophage cell line and bone marrow-derived macrophages (BMMs) from mice. L. plantarum LTA alone was unable to induce NO production, even at 30μg/ml. However, LTA in the presence of IFN-γ significantly induced NO production in RAW 264.7 cells. The observed NO production was inhibited by a NO synthase (NOS) inhibitor l-NAME and an inducible NOS (iNOS) inhibitor l-NIL, suggesting that iNOS is specifically required for this action. Western blot analysis and reverse transcription and polymerase chain reaction further confirmed that L. plantarum LTA increased protein and mRNA levels of iNOS, respectively. However, such induction was substantially inhibited in BMMs from Toll-like receptor 2 (TLR2)-deficient mice and the macrophages treated with an inhibitor blocking platelet-activating factor receptor. In addition, L. plantarum LTA plus IFN-γ induced IFN-β expression and STAT1 phosphorylation, which are key pathways for inducing iNOS expression. Electrophoretic mobility shift assay demonstrated that L. plantarum LTA in the presence of IFN-γ remarkably increased the DNA-binding activity of NF-κB transcription factor, which is known to be involved in the iNOS gene expression. Collectively, these results suggest that LTA from L. plantarum alone has no inflammatory potential but does induce NO production under conditions of inflammation, such as the presence of IFN-γ., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Lipoteichoic acid (LTA) from Staphylococcus aureus (aLTA) and from Lactobacillus plantarum LTA (pLTA) are both recognized by Toll-like receptor 2 (TLR2), but cause different stimulatory effects on the innate immune and inflammatory responses, and their underlying cellular mechanisms are unknown. In this study, comparative proteome analysis was performed using two-dimensional gel electrophoresis and mass spectrometry on protein extracts from human monocyte THP-1 cells stimulated with either aLTA or pLTA. Differentially expressed proteins might be involved in innate immunity and inflammation. Cells treated with aLTA and with pLTA showed different protein expression profiles. Of 60 identified proteins, 10 were present only in treated cells (8 in aLTA-treated only, and 2 in pLTA-treated only), 1 protein (IMPDH2) was suppressed by pLTA, and 49 were up- or down-regulated more than three-fold by aLTA- or pLTA- stimulation. Several proteins involved in immunity or inflammation, antioxidation, or RNA processing were significantly changed in expression by aLTA- or pLTA-stimulation, including cyclophilin A, HLA-B27, D-dopachrome tautomerase, Mn- SOD, hnRNP-C, PSF and KSRP. These data demonstrated that aLTA and pLTA had different effects on the protein profile of THP-1 cells. Comparison of the proteome alterations will provide candidate biomarkers for further investigation of the immunomodulatory effects of aLTA and pLTA, and the involvement of aLTA in the pathogenesis of Staphylococcus aureus sepsis.

Heat-killed cells of Lactobacillus plantarum L-137 are potent inducers of IL-12 in vitro as well as in vivo and have been shown to have antiallergic, antitumor, and antiviral effects through this induction, which leads to a Th1 type immune response. To determine why L-137 cells induce much greater IL-12 production than the type strain Lactobacillus plantarum JCM1149, we examined the differences in their CW components. The L-137 CW was found to have a higher alanine content and IL-12p40 induction was significantly greater in comparison with JCM1149 CW, whereas peptidoglycans isolated from both strains did not cause IL-12p40 induction. Because in purified CW preparations from gram-positive bacteria, the presence of LTA, the major proinflammatory structure on these bacteria, has been known to have high alanine content, we investigated the responsiveness of both strains to anti-LTA antibody by flow cytometry. L-137 cells reacted more with anti-LTA antibody than did JCM1149 cells. Furthermore, derivative strains of L-137, cured of a specific plasmid pLTK11 of the 15 endogenous plasmids in wild-type L-137, had poor responsiveness to anti-LTA antibody and showed lower IL-12p40 inducing activity than the wild-type L-137 with pLTK11. Our results suggest that LTA expression on the cell surface causes IL-12p40 induction, and that the above internal plasmid of L-137 influences LTA synthesis and expression on the cell surface.

Staphylococcus aureus is a common etiologic agent for Gram-positive sepsis, and its lipoteichoic acid (LTA) may be important in causing Gram-positive bacterial septic shock. Here, we demonstrate that highly purified LTA (pLTA) isolated from Lactobacillus plantarum inhibited aureus LTA (aLTA)-induced TNF-alpha production in THP- cells. Whereas pLTA scarcely induced TNF-alpha production, aLTA induced excessive TNF-alpha production. Interestingly, aLTA-induced TNF-alpha production was inhibited by pLTA pretreatment. Compared with pLTA, aLTA induced strong signal transduction through the MyD88, NF-kappaB, and MAP kinases. This signaling, however, was reduced by a pLTA pretreatment, and resulted in the inhibition of aLTA-induced TNF-alpha production. Whereas dealanylated LTAs, as well as native LTAs, contributed to TNF- induction or TNF-alpha reduction, deacylated LTAs did not, indicating that the acyl chain of LTA played an important role in the LTA-mediated immune regulation. These results suggest that pLTA may act as an antagonist for aLTA, and that an antagonistic pLTA may be a useful agent for suppressing the septic shock caused by Gram-positive bacteria.

In this study, the effect of Lactobacillus plantarum lipoteichoic acid (pLTA) on LPS-induced MAPK activation, NF-kappaB activation, and the expression of TNF-alpha and IL-1R-associated kinase M (IRAK-M) was examined. The expression of the pattern recognition receptor and the survival rate of mice were also examined. pLTA pretreatment inhibited the phosphorylation of ERK, JNK, and p38 kinase. It also inhibited the degradation of IkappaBalpha and IkappaBbeta, as well as the activation of the LPS-induced TNF-alpha factor in response to subsequent LPS stimulation. These changes were accompanied by the suppression of the LPS-induced expression of TLR4, NOD1, and NOD2, and the induction of IRAK-M, with a concurrent reduction of TNF-alpha secretion. Furthermore, the overexpression of pattern recognition receptors such as TLR4, NOD1, and NOD2 and the degradation of IRAK-M by transient transfection were found to reinstate the production of TNF-alpha after LPS restimulation. In addition, the i.p. injection of pLTA suppressed fatality, and decreased the level of TNF-alpha in the blood, in LPS-induced endotoxin shock mice. In conclusion, these data extend our understanding of the pLTA tolerance mechanism, which is related to the inhibition of LPS-induced endotoxin shock, and suggest that pLTA may have promise as a new therapeutic agent for LPS-induced septic shock.

In this study, the stimulatory effects of different lactic acid bacteria strains, and their subcellular fractions, on the THP-1 cell line were evaluated. Lactobacillus plantarum was found in particular to induce high levels of IL-23p19 mRNA, but it moderately induced TNF-alpha production. IL-10 production was not entirely affected by L. plantarum stimulation. When subcellular fractions of L. plantarum were used to treat THP-1 cells, IL-23p19 mRNA expression was enhanced in a dose-responsive manner, specifically by lipoteichoic acid (LTA). The cotreatment of THP-1 cells by both L. plantarum and Staphylococcus aureus LTA resulted in decreased IL-10 production when compared with cells treated by S. aureus LTA alone. Taken together, these data suggest that LTA isolated from L. plantarum elicits stimulatory effects upon the expression of IL-23p19 and inhibitory effects on pathogen-mediated IL-10 production.

Teichoic acids (TAs), and especially lipoteichoic acids (LTAs), are one of the main immunostimulatory components of pathogenic Gram-positive bacteria. Their contribution to the immunomodulatory properties of commensal bacteria and especially of lactic acid bacteria has not yet been investigated in detail. To evaluate the role of TAs in the interaction between lactic acid bacteria and the immune system, we analyzed the antiinflammatory properties of a mutant of Lactobacillus plantarum NCIMB8826 affected in the TA biosynthesis pathway both in vitro (mononuclear cells stimulation) and in vivo (murine model of colitis). This Dlt- mutant was found to incorporate much less D-Ala in its TAs than the WT strain. This defect significantly impacted the immunomodulation reactions induced by the bacterium, as shown by a dramatically reduced secretion of proinflammatory cytokines by peripheral blood mononuclear cells and monocytes stimulated by the Dlt- mutant as compared with the parental strain. Concomitantly, a significant increase in IL-10 production was stimulated by the Dlt- mutant in comparison with the WT strain. Moreover, the proinflammatory capacity of L. plantarum-purified LTA was found to be Toll-like receptor 2-dependent. Consistent with the in vitro results, the Dlt- mutant was significantly more protective in a murine colitis model than its WT counterpart. The results indicated that composition of LTA within the whole-cell context of L. plantarum can modulate proinflammatory or antiinflammatory immune responses.