Lipoteichoic Acid Treatment Research Articles

Transcriptomic analysis of lipoteichoic acid‑treated undifferentiated and neutrophil‑like differentiated HL‑60 cells.

Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

Nrf2/PHB2 alleviates mitochondrial damage and protects against Staphylococcus aureus-induced acute lung injury.

Staphylococcus aureus (SA) is a major cause of sepsis, leading to acute lung injury (ALI) characterized by inflammation and oxidative stress. However, the role of the Nrf2/PHB2 pathway in SA-induced ALI (SA-ALI) remains unclear. In this study, serum samples were collected from SA-sepsis patients, and a SA-ALI mouse model was established by grouping WT and Nrf2-/- mice after 6h of intraperitoneal injection. A cell model simulating SA-ALI was developed using lipoteichoic acid (LTA) treatment. The results showed reduced serum Nrf2 levels in SA-sepsis patients, negatively correlated with the severity of ALI. In SA-ALI mice, downregulation of Nrf2 impaired mitochondrial function and exacerbated inflammation-induced ALI. Moreover, PHB2 translocation from mitochondria to the cytoplasm was observed in SA-ALI. The p-Nrf2/total-Nrf2 ratio increased in A549 cells with LTA concentration and treatment duration. Nrf2 overexpression in LTA-treated A549 cells elevated PHB2 content on the inner mitochondrial membrane, preserving genomic integrity, reducing oxidative stress, and inhibiting excessive mitochondrial division. Bioinformatic analysis and dual-luciferase reporter assay confirmed direct binding of Nrf2 to the PHB2 promoter, resulting in increased PHB2 expression. In conclusion, Nrf2 plays a role in alleviating SA-ALI by directly regulating PHB2 transcription and maintaining mitochondrial function in lung cells.

Probiotic components of Bacillus siamensis LF4 mitigated β-conglycinin caused cell injury via modulating TLR2/MAPKs/NF-κB signaling in Lateolabrax maculatus

β-conglycinin is a recognized factor in leading to intestinal inflammation and limiting application of soybean meal in aquaculture. Our previous study reported that heat-killed B. siamensis LF4 could effectively mitigate inflammatory response and apoptosis caused by β-conglycinin in spotted seabass (Lateolabrax maculatus) enterocytes, but the mechanisms involved are not fully understood. In the present study, therefore, whole cell wall (CW), peptidoglycan (PG) and lipoteichoic acid (LTA) and cell-free supernatant (CFS) have been collected from B. siamensis LF4 and their mitigative function on β-conglycinin-induced adverse impacts and mechanisms underlying were evaluated. The results showed that β-conglycinin-induced cell injury, characterized with significantly decreased cell viability and increased activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, glutamic propylic transaminase (P < 0.05), were reversed by subsequent heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS treatment. Enterocytes co-cultured with heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS (especially PG) significantly increased expressions of anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-β1), tight junction proteins (ZO-1, occludin and claudin-b) and antimicrobial peptides (β-defensin, hepcidin-1, NK-lysin and piscidin-5), and decreased expressions of pro-inflammatory genes (IL-1β, IL-8 and TNF-α) and apoptosis-related genes (caspase 3, caspase 8 and caspase 9) (P < 0.05), indicating their excellent mitigation effects on β-conglycinin-induced cell damages. In addition, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS significantly increased TLR2 mRNA level (especially in PG treatment), and decreased MAPKs (JNK, ERK, p38 and AP-1) and NF-κB related genes expressions. In conclusion, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS could modulating TLR2/MAPKs/NF-κB signaling and alleviating β-conglycinin-induced enterocytes injury in spotted seabass (L. maculatus), and PG presented the best potential.

Determination of the optimal concentration and duration of C5aR antagonist application in an inflammatory model of human dental pulp cells.

Deep tooth decay approaching the pulp may develop into pulpitis; to prevent this, pulp cells need to balance the rapid immune response to avoid rapid swelling of the pulp. Current treatment of deep decay that approaches the pulp involves the application of drugs that induce low-level inflammation in the dental pulp to promote its repair, but this treatment is sometimes insufficient. However, the unsuccessful treatment often resulted in pulpitis. The C5a-C5aR is the initial stage of the immune cascade response. Blocking the binding of C5a-C5aR can slow the immune response in the narrow pulp cavity, so that dental pulp cells have enough time to proliferate, migrate, and differentiate. In this study, we compared lipoteichoic acid (LTA) and lipopolysaccharides (LPS) at different concentrations and time points and used the C5aR antagonist W54011 to block the C5a-C5aR axis. The blocking effect was detected by analyzing the expression of C5a, C5aR, interleukin (IL)-6, and Toll-like receptors 2 and 4 (TLR-2, 4). Next, we determined the optimal concentration and duration of LTA and LPS treatment in combination with W54011. Based on our results, we selected 1.0 μg·mL-1 LPS treatment for 48 h to generate an inflammatory model of human dental pulp cells. We then regrouped the cells and conducted expression analyses to monitor the expression of C5a, C5aR, IL-6, and TLR-4 at the protein and mRNA levels. LPS stimulation for 48 h and treatment with W54011 for 48 h effectively inhibited inflammation and did not affect C5a expression. This study provides a basis for follow-up studies of W54011 in dental pulp cells.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 β-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-β release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1β and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

Early effects of lipoteichoic acid from Staphylococcus aureus on milk production-related signaling pathways in mouse mammary epithelial cells

Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased β-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal‐regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.

Akkaraman ve Romanov Kuzularının Alveolar Makrofajlarında Lipopolisakkarit ve Lipoteikoik Asite Yanıtta İmmün İlişkili Genlerin Ekspresyon Profilinin Araştırılması

The alveolar macrophages (AMs) are frontier of defense against foreign materials that initiate immune response in lungs. Knowledge of the expression dynamics of major immune-related genes in the alveolar macrophages in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge can help to understand disease mechanism involved in several respiratory diseases. The aim of this study was to investigate the mRNA expression of selected immune-related genes in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge in sheep alveolar macrophages in vivo. Results revealed that Romanov lambs exhibited higher mRNA expression of TLR2, TLR4, NF-ĸβ, TNFα, IL-1β, IL-6, IL-8, and IL-10 genes as compared to Akkaraman lambs along with the control of all treatments. Moreover, the expression of TLR2, TLR4, NF-ĸβ, TNFα, IL-1β, IL-6, IL-8, and IL-10 genes was higher in combine treatment of LPS and LTA as compared to separate treatments of LPS and LTA in both breeds. The results showed that the mRNA expression of immune-related genes was significantly increased in the sheep AMs in response to LPS and LTA treatment whereas a synergistic effect was observed in LPS+LTS treatment. Also, breed comparison showed that the native Akkaraman was more resistant to disease compared to exotic Romanov.

Combined targeting of vascular endothelial growth factor C (VEGFC) and P65 using miR-27b-3p agomir and lipoteichoic acid in the treatment of gastric cancer.

Gastric cancer is the second leading cancer-related mortality worldwide and more effective treatment strategies are urgently needed to combat the disease. Using lipoteichoic acid (LTA) and miR-27b-3p agomir, we aimed to assess the efficacy of this combination of therapies in treating gastric cancer. The RNA levels of miR-27b-3p, FOXO3, MET, KRAS, vascular endothelial growth factor C (VEGFC), TSC1, and P65 were analyzed by quantified-PCR (Q-PCR) and the cell viability of AGS cells was analyzed by MTT. Confirm Luciferase reporter assays were used to explore the putative miR-27b-3p binding sites and Western blot analyzed the protein level of GAPDH, VEGFC, P65, AKT, and phosphorylated-AKT (p-AKT). The level of P65 in both the cytoplasm and nucleus of AGS cells was visualized by immunofluorescence assay. Subcutaneous xenograft models of gastric cancer were established, and mice were treated with miR-27b-3p agomir, LTA, or both. Hematoxylin-eosin staining and Ki-67 immunohistochemistry analysis of tumor tissues were then performed. The results showed that the decreased expression of miR-27b-3p in gastric cancer cell lines inhibited the viability of AGS cells, and VEGFC was confirmed as the target of miR-27b-3p. In addition, ectopic expression of miR-27b-3p significantly inhibited the AKT pathway in AGS and N87 cells, and LTA suppressed the proliferation of gastric cancer cells by inhibiting the NF-κB pathway. In an established xenograft model, both miR-27b-3p agomir alone and LTA treatment alone inhibited tumor growth and treatment which combined the two showed an even stronger inhibitory effect. Taken together, the combined use of LTA and miR-27b-3p agomir exhibited a synergistic effect in the treatment of gastric cancer.

In vitro and ex vivo expression of serum amyloid A3 in mouse lung epithelia

Background and Purpose Serum amyloid A (SAA), an acute-phase protein whose level tracks infection and inflammation, is the precursor protein of amyloid A (AA) fibrils that is thought to cause AA amyloidosis in human and animals. SAA protein has several isoforms based on the difference of amino acid sequence, such as SAA1 to SAA4 in mice. AA fibrils are associated with chronic inflammation and are mainly originated from SAA1 produced in the liver. SAA3 reportedly contributes to the innate immune response in epithelia; however, little is known about its role at the lung epithelia. Therefore, we investigated SAA3 expression in the lung epithelium activated by bacterial antigens. Materials and Methods The expressions of SAA3 and SAA1 mRNA were investigated using quantitative real-time PCR, in vitro using mouse Clara (Club) cells and ex vivo using surgically removed mouse lungs, after their stimulation by using either lipopolysaccharide (LPS), the major outer membranous antigen of gram-negative bacteria, or lipoteichoic acid (LTA), the major outer membranous antigen of gram-positive bacteria. In addition, SAA3 and SAA1/2 proteins in treated lung samples were detected by immunohistochemistry (IHC). Results SAA3 mRNA expression increased in cells and lungs treated with either LPS or LTA. SAA3 mRNA was more sensitively expressed in LPS than LTA treatment. In contrast, SAA1 mRNA expression did not increase by either LPS or LTA treatment. Furthermore, SAA3 mRNA expression increased in a dose-dependent manner in cells treated with tumor necrosis factor-alpha. By IHC, SAA3 protein was highly expressed in the luminal side of the bronchial epithelium, while SAA1/2 was not expressed. Conclusion These results obtained from in vitro and ex vivo experiments suggest that SAA3 plays an important role in the innate immune response to bacterial infection in the lung epithelia.

Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

Staphylococcus aureus Lipoteichoic Acid has a Dual Effect on Inflammatory Responses in Airway Epithelial Cells via the Transcription Factor NFκB

Staphylococcus aureus is a major human pathogen that causes a wide array of infections ranging in severity from mild skin infections to life-threatening diseases such as sepsis. S. aureus can cause severe inflammation whilst, at the same time, modulating specific innate and humoral immune defense mechanisms. Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive bacteria that is abundantly released by S. aureus. We investigated the dose-dependent effects of LTA on inflammation in airway epithelial cells. Purified S. aureus LTA was applied to airway epithelial (NuLi-1) cells in a range of concentrations, followed by assessment of expression of NFκB p50 and p65/RelA via qPCR, immunofluorescence and western blot. Multiplex analysis of cell culture supernatants was carried out to evaluate cytokine levels. NFκB p50 mRNA varied according to the LTA dose (p = 0.013) whereas NFκB p65/RelA did not (p = 0.90). A dose-dependent increase in NFκB p50 mRNA expression was seen up to 10 μg/ml LTA treatment, before falling at the 100μg/ml treatment. Western blot analysis showed nuclear and cytoplasmic NFκB p50 and cytoplasmic NFκB p65 protein reduced with increasing LTA concentrations. Multiplex analysis demonstrated that cytokine levels increased with LTA dose, but then decreased at the highest LTA dose (100 μg/ml). This study identifies LTA as having a dual dose-dependent effect on immune activation at low dosages and ablation at high dosages, associated with altered expression levels of NFκB. It adds LTA to the extensive repertoire of possible immune modulators produced by S. aureus.

Lipoteichoic acid of Enterococcus faecalis inhibits osteoclastogenesis via transcription factor RBP-J.

Lipoteichoic acid (LTA) of Enterococcus faecalis is a potent stimulator of inflammatory responses, but the effects of E. faecalis LTA on osteoclastogenesis remains far from well understood. This study showed that E. faecalis LTA significantly inhibited osteoclastogenesis of wild type murine bone marrow-derived macrophages (BMMs) in the presence of a high dose of RANKL, while the inhibition of osteoclastogenesis by E. faecalis LTA was significantly removed in BMMs with deficient expression of the transcription factor RBP-J. In addition, a few small osteoclasts were generated in BMMs with only E. faecalis LTA stimulation, presumably due to the production of TNF-α and IL-6. Furthermore, both p38 and ERK1/2 MAPK signaling pathways were activated after 24 h of E. faecalis LTA treatment, but these signaling pathways were not activated after 6 d of treatment with RANKL in mature osteoclasts. In conclusion, E. faecalis LTA, which induces inflammatory response, could inhibit RANKL-induced osteoclastogenesis via RBP-J in BMMs.

Staphylococcal lipoteichoic acid promotes osteogenic differentiation of mouse mesenchymal stem cells by increasing autophagic activity

This study sought to explore the effect of staphylococcal lipoteichoic acid (LTA) on autophagy in mouse mesenchymal stem cells (MSCs), and then influence osteogenesis through the change of autophagy. C3H10T1/2 cells were induced by osteogenic medium with the treatment of LTA at different concentrations (1, 5, 10 μg/mL); 3-methyladenine (3-MA) were used as the autophagy inhibitor, and rapamycin (rapamycin, Rap) were used to activate autophagy; the effects on osteogenesis were detected by alkaline phosphatase staining, alizarin red staining, real-time quantitative PCR, and western blotting; autophagic activity was investigated by the expression of LC3-Ⅱand p62 proteins. Compared with control group, the expression of osteogenesis markers was significantly up-regulated with the LTA treatment on the mRNA and protein level; the positive rate of alkaline phosphatase was enhanced in the LTA groups; and the formation of calcium nodules was increased simultaneously. The expression of LC3-Ⅱ protein was increased in LTA groups, while the expression of p62 protein was decreased. Inhibition of autophagy significantly reduced the effect of LTA on osteogenesis of MSCs; the promotion of LTA on osteogenic differentiation was further enhanced when adding rapamycin to activate autophagic activity. It provides new insight of prevention and treatment for bone infection.

WNT5A-ROR2 is induced by inflammatory mediators and is involved in the migration of human ovarian cancer cell line SKOV-3.

BackgroundWnt5A, which is a member of the non-transforming Wnt protein family, is implicated in inflammatory processes. It is also highly expressed by ovarian cancer cells. ROR2, which is a member of the Ror-family of receptor tyrosine kinases, acts as a receptor or co-receptor for Wnt5A. The Wnt5A–ROR2 signaling pathway plays essential roles in the migration and invasion of several types of tumor cell and influences their cell polarity. We investigated the modulation of Wnt5A–ROR2 by inflammatory mediators and its involvement in the migration of the human ovarian cancer cell line SKOV-3.MethodsSKOV-3 cells were treated with LPS (lipopolysaccharide), LTA (lipoteichoic acid) and recombinant human IL-6 alone or in combination with STAT3 inhibitor (S1155S31-201) or NF-kB inhibitor (BAY11-7082) for 4, 8, 12, 24 and 48 h. The Wnt5A and ROR2 expression levels were determined at the gene and protein levels. Cells were transfected with specific siRNA against Wnt5A in the absence or presence of human anti-ROR2 antibody and cell migration was assessed using transwells.ResultsThere was a strong downregulation of Wnt5A expression in the presence of STAT3 or NF-kB inhibitors. Cell stimulation with LTA or IL-6 for 8 h led to significantly increased levels of Wnt5A (5- and 3-fold higher, respectively). LPS, LTA or IL-6 treatment significantly increased ROR2 expression (2-fold after 48 h). LPS- or LTA-induced Wnt5A or ROR2 expression was abrogated in the presence of STAT3 inhibitor (p < 0.001). IL-6-induced Wnt5A expression was abrogated by both STAT3 and NF-kB inhibitors (p < 0.001). Although not significant, IL-6-induced ROR2 expression showed a modest decrease when STAT3 inhibitor was used. Moreover, cell migration was decreased by 80 % in siRNA Wnt5A-transfected cells in the presence of anti-human ROR2 antibody (p < 0.001).ConclusionsThis study revealed for the first time that inflammatory mediators modulate Wnt5A and ROR2 through NF-kB and STAT3 transcription factors and this may play a role in ovarian cancer cell migration. The results described here provide new insight into the role of the Wnt5A–ROR2 complex in ovarian cancer progression in relation to inflammation.

Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.

Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages.

To study the immune-inflammatory response and signaling mechanism of macrophages to purified Enterococcus faecalis (E. faecalis) lipoteichoic acid (LTA), intact LTA was obtained from an E. faecalis clinical strain P25RC using the butanol method and hydrophobic interaction chromatography purification. The fractions containing LTA were determined using phosphate detection. Contaminations with lipopolysaccharide and proteins were excluded using the Limulus amoebocyte lysate assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. LTA was analyzed using nuclear magnetic resonance. Prior to LTA stimulation assays, THP-1 monocytes were pretreated with phorbol 12-myristate 13-acetate to differentiate into macrophages. Macrophages were treated with LTA in concentration gradients and cells without LTA treatment as the control. Gene expression of TLR2, CD14 and MyD88 were evaluated by quantitative polymerase chain reaction. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 were quantified using ELISA. The activated and total nuclear factor-κB (NF-κB) p65 and three mitogen-activated protein kinases (p38, ERK1/2 and JNK) were assessed using western blot analysis. E. faecalis LTA induced the gene expression of TLR2 and MyD88 whilst it downregulated CD14, suggesting a TLR2-dependent and CD14-independent immune-inflammatory activity. LTA stimulated the expression of pro-inflammatory cytokine TNF-α (P<0.05), but not the anti-inflammatory cytokine IL-10. In conclusion, E. faecalis LTA stimulated the expression of TNF-α in macrophages possibly through the NF-κB and p38 pathways.

Toll-like receptor 2 ligand, lipoteichoic acid is inhibitory against infectious laryngotracheitis virus infection in vitro and in vivo

Lipoteichoic acid (LTA) is one of the pathogen associated molecular patterns (PAMPs) that activates toll-like receptor (TLR)2-cluster of differentiation (CD)14 signalling pathway. This recognition elicits antiviral responses that have been recorded against viruses of mammals although such responses have not been characterized adequately against avian viruses. In this investigation, we characterized the LTA induced antiviral responses against infectious laryntotracheitis virus (ILTV) infection in vitro and in vivo. We found that LTA is capable of up regulating mRNA expression of innate proteins in macrophages such as MyD88, iNOS and IL-1β and reduces the ILTV plaques in vitro. Similarly, we found that LTA treatment of embryonic day 18 (ED18) eggs can lead to the antiviral response against pre-hatch ILTV infection in vivo and is associated with expansion of macrophage populations and expression of IL-1β and MyD88 in the lung. The data highlight that LTA can be a potential innate immune stimulant that can be used against ILTV infection in chickens.

Hydrocortisone Reduces the Beneficial Effects of Toll-Like Receptor 2 Deficiency on Survival in a Mouse Model of Polymicrobial Sepsis

Toll-like receptors (TLRs) play a crucial role in early host defense against microorganisms. Toll-like receptor 2 (TLR2) polymorphisms have a prevalence of 10%; functional defects of TLR2 are associated with higher susceptibility toward gram-positive bacteria, and TLR2 deficiency has been associated with an impaired adrenal stress response. In the present study, we compared endogenous corticosterone production of wild-type (WT) and TLR2-deficient (TLR2) mice and analyzed survival after hydrocortisone therapy during sepsis induced by cecal ligation and puncture (CLP). Male C57BL/6J (WT); and B6.129-Tlr2tm1Kir/J (TLR2) mice were subjected to CLP or sham operation and randomly assigned to postoperative treatment with either hydrocortisone (5 mg/kg) or vehicle (n = 10 mice/group). Survival was documented for an observation period of 48 h. Endogenous corticosterone production following hydrocortisone treatment and lipoteichoic acid (LTA) exposure, interleukin 6 (IL-6) and IL-1β plasma levels, and blood counts were determined following sham operation or CLP using another n = 5 mice/group. Statistical analysis was performed using analysis of variance/Bonferroni. TLR2 mice exhibited a lack of suppression and an attenuated increase in endogenous corticosterone production following hydrocortisone or LTA treatment, respectively. After CLP, TLR2 mice exhibited an uncompromised adrenal stress response, higher IL-6 levels, and increased survival compared with WT controls (75 vs. 35%; P < 0.05). Hydrocortisone therapy of TLR2 mice completely abolished this advantage (decrease in survival to 45%, P < 0.05 vs. vehicle-treated TLR2 mice) and was associated with decreased IL-1β plasma concentrations. Toll-like receptor 2 deficiency is associated with an uncompromised adrenal stress response and increased survival rates during polymicrobial sepsis. Hydrocortisone treatment increases mortality of septic TLR2 mice, suggesting that hydrocortisone therapy might be harmful for individuals with functional TLR2 polymorphisms.

Efecto del ácido lipoteicoico sobre la expresión de genes en cardiomiocitos de ratón (H9c2)

Most dental pulp diseases and diseases of tissues surrounding the root are somehow related to micro-organisms. Peptidoglycans and lipoteichoic acid are two of the main Gram-positive bacteria components with activities related to sepsis development. When tissues sustain microbial invasion the host responds with both unspecific inflammatory defenses and specific immunological reactions. Surgical and non surgical endodontic treatments are essentially debridement procedures intended to destroy and eliminate the microbial eco-system associated to the pathological process. It is essential for clinicians to understand the intimate relationship existing between micro-organisms and endodontic disease, so as to be able to tailor a rational and effective treatment especially in subjects susceptible to infective endocarditis processes. In the present study research was conducted on TNFα, IL-1 COX-2 expression through the effect of lipoteichoic acid (LTA) of Streptococcus sanguinis by characterizing intra-cellular signals involved in H9c2” cardiomyocytes. The cell line was treated with LTA at different concentrations during 30 minutes. When compared to control group, responses to LTA treatment were dependent on dosage. That expression was assessed by means of a One Step RT-PCR (Invitrogen) analysis. It was noted that the afore mentioned expression resembled the organisms’s physiological response during an infective endocarditis episode and to exacerbation observed during an endodontic procedure.

Lipoteichoic Acid Suppresses Effector T Cells Induced by Staphylococcus aureus-Pulsed Dendritic Cells

Lipoteichoic acid (LTA), uniquely expressed on gram-positive bacteria, is recognized by Tolllike receptor 2 (TLR2) on not only antigen-presenting cells but also activated T cells. Therefore, it is reasonable to assume that LTA is acting on T cells. However, little is known about the effect of LTA on T-cell regulation. In the present study, we investigated the immunomodulatory effects of LTA on CD4⁺ T cells. Effector CD4⁺ T cells, induced after co-culture with S. aureuspulsed dendritic cells, produced high levels of interferon-γ, CD25, CD69, and TLRs 2 and 4. When effector CD4⁺ T cells were treated with LTA, the expressions of the membrane-bound form of transforming growth factor (TGF)-β and forkhead box P3 increased. Coincidently, the proliferation of effector CD4⁺ T cells was declined after LTA treatment. When TGF-β signaling was blocked by the TGF-β receptor 1 kinase inhibitor, LTA failed to suppress the proliferation of effector CD4⁺ T cells. Therefore, the present results suggest that LTA suppresses the activity of effector CD4⁺ T cells by enhancing TGF-β production.