RESUMO
Lipoteichoic acid (LTA) from Gram-positive bacteria exerts different immune effects depending on the bacterial source from which it is isolated. Lacticaseibacillus rhamnosus GG LTA (LGG-LTA) oral administration reduces UVB-induced immunosuppression and skin tumor development in mice. In the present work, we evaluate the immunomodulatory effect exerted by LGG-LTA in dendritic cells (DC) and T cells, both in vitro and in the gut-associated lymphoid tissue (GALT). During cell culture, LTA-stimulated BMDC increased CD86 and MHC-II expression and secreted low levels of pro and anti-inflammatory cytokines. Moreover, LTA-treated BMDC increased T cell priming capacity, promoting the secretion of IL-17A. On the other hand, in orally LTA-treated mice, a decrease in mature DC (lamina propria and Peyer's patches) was observed. Concomitantly, an increase in IL-12p35 and IFN-γ transcription was presented (lamina propria and Peyer's Patches). Finally, an increase in the number of CD103+ DC was observed in Peyer's patches. Together, our data demonstrate that LGG-LTA activates DC and T cells. Moreover, we show that a Th1-biased immune response is triggered in vivo after oral LTA administration. These effects justify the oral LTA activity previously observed.
Assuntos
Células Dendríticas , Linfócitos T , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Ácidos Teicoicos/metabolismo , Ácidos Teicoicos/farmacologiaRESUMO
Dental caries is a diet-biofilm-dependent disease. Streptococcus mutans contributes to cariogenic biofilms by producing an extracellular matrix rich in exopolysaccharides and acids. The study aimed to determine the effect of topical treatments with compound 1771 (modulates lipoteichoic acid (LTA) metabolism) and myricetin (affects the synthesis of exopolysaccharides) on S. mutans biofilms. In vitro S. mutans UA159 biofilms were grown on saliva-coated hydroxyapatite discs, alternating 0.1% sucrose and 0.5% sucrose plus 1% starch. Twice-daily topical treatments were performed with both agents alone and combined with and without fluoride: compound 1771 (2.6 µg/mL), myricetin (500 µg/mL), 1771 + myricetin, fluoride (250 ppm), 1771 + fluoride, myricetin + fluoride, 1771 + myricetin + fluoride, and vehicle. Biofilms were evaluated via microbiological, biochemical, imaging, and gene expression methods. Compound 1771 alone yielded less viable counts, biomass, exopolysaccharides, and extracellular LTA. Moreover, the combination 1771 + myricetin + fluoride decreased three logs of bacterium counts, 60% biomass, >74% exopolysaccharides, and 20% LTA. The effect of treatments on extracellular DNA was not pronounced. The combination strategy affected the size of microcolonies and exopolysaccharides distribution and inhibited the expression of genes linked to insoluble exopolysaccharides synthesis. Therefore, compound 1771 prevented the accumulation of S. mutans biofilm; however, the effect was more pronounced when it was associated with fluoride and myricetin.
Assuntos
Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fluoretos/farmacologia , Saliva/microbiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Streptococcus mutans/crescimento & desenvolvimento , Administração Tópica , Proteínas de Bactérias/genética , Cárie Dentária/microbiologia , Cárie Dentária/prevenção & controle , Sinergismo Farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/metabolismo , Modelos Biológicos , Polissacarídeos Bacterianos/antagonistas & inibidores , Polissacarídeos Bacterianos/metabolismo , Saliva/química , Saliva/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/genética , Ácidos Teicoicos/antagonistas & inibidores , Ácidos Teicoicos/metabolismoRESUMO
PURPOSE: Staphylococcus epidermidis ATCC12228 lipoteichoic acid (LTA) inhibits TNFα production from keratinocytes that are activated with poly I:C. However, this effect has not been proven in clinical or commensal isolates. METHODOLOGY: The <10 kDa fractions of S. epidermidis isolates from ocular infections (n=56), healthy skin (n=35) and healthy conjunctiva (n=32) were obtained. TNFα production was determined by elisa in HaCaT keratinocytes stimulated with poly I:C and with the <10 kDa fractions. LTA in the cytoplasmic membrane and in the <10 kDa fractions of the isolates was determined during bacterial growth by flow cytometry, Western blot and electrospray ionization mass spectrometry. The expression levels of ugtP, ltaA and ltaS were evaluated. RESULTS: Two populations of isolates were found: a population that inhibited TNFα production (TNFα-inhibitor isolates) and a population that did not inhibit it (TNFα non-inhibitor isolates). The cells from the TNFα-inhibitor isolates had less LTA in the cytoplasmic membrane compared to the cells from the TNFα non-inhibitor isolates (P<0.05). Similarly, LTA was detected in the supernatants of TNFα-inhibitor isolates, and it was absent in TNFα non-inhibitor isolates. High expression levels of the ugtP and ltaA genes in the 1850I (TNFα-inhibitor isolate) and 37HS (TNFα non-inhibitor isolate) isolates were found during bacterial growth. However, the ltaS gene had a low expression level (P<0.05) in the 37HS isolate. CONCLUSION: The TNFα-inhibitor isolates release LTA due to high expression of the LTA synthesis genes. By contrast, TNFα non-inhibitor isolates do not release LTA due to low expression level of the ltaS gene.
Assuntos
Exocitose , Expressão Gênica , Genes Bacterianos , Lipopolissacarídeos/metabolismo , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Ácidos Teicoicos/metabolismo , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Espectrometria de Massas , Staphylococcus epidermidis/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION: The infectious content of root canals, including bacteria and lipoteichoic acid (LTA), cause injuries to the periapical tissues. The purpose of this clinical study was to quantify the levels of both LTA and cultivable bacteria at the different phases of endodontic retreatment (ER) of teeth with post-treatment apical periodontitis. It also aimed to investigate the presence of gram-positive microorganisms before and after chemomechanical preparation (CMP) and intracanal medication (ICM). METHODS: Twenty infected root canals of single-rooted teeth were randomly assigned into 2 groups according to the chemical substance used for CMP (n = 10 per group): chlorhexidine (CHX) group, 2% CHX gel, and the sodium hypochlorite (NaOCl) group, 6% NaOCl. Root canal samples were taken using paper points before (S1) and after CMP (S2) and after 30 days of ICM with calcium hydroxide + 2% CHX gel (S3). Microorganisms were identified by the culture technique using biochemical tests. Cultivable bacteria were determined by counting the colony-forming unit. LTA levels were measured using the enzyme-linked immunosorbent assay (pg/mL). RESULTS: A total of 70 gram-positive species, out of 102 species isolated, were found in the root canals (54 in S1, 4 in S2, and 12 in S3). Enterococcus faecalis was the most frequent isolated taxon in all phases of the ER. LTA (574.0 ± 94.7) and cultivable bacteria (101.2 ± 79.2) were present in all S1 samples. CMP decreased the overall levels of cultivable bacteria by 99.4% and LTA by 24.8% (P < .05), whereas the total overall reduction level of ICM on viable bacteria was 99.5% and on LTA it was 38.6% (P < .05). CMP with 2% CHX gel (CHX group, 99.3%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 92.1%) on bacterial reduction. Likewise, ICM showed a 100% reduction in the CHX group and 98.5% in the NaOCl group. Regarding the reduction of LTA, CMP with 2% CHX gel (CHX group, 26.9%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 22.6%). In addition, ICM showed a 43.2% reduction in the CHX group and 36.2% in the NaOCl group (P > .05). CONCLUSIONS: The reduction rates of bacteria were higher than the LTA. Moreover, gram-positive microorganisms were present in all phases of the endodontic retreatment.
Assuntos
Bactérias/crescimento & desenvolvimento , Cavidade Pulpar/metabolismo , Cavidade Pulpar/microbiologia , Lipopolissacarídeos/metabolismo , Periodontite Periapical/metabolismo , Periodontite Periapical/microbiologia , Tratamento do Canal Radicular/métodos , Ácidos Teicoicos/metabolismo , Adulto , Bactérias/efeitos dos fármacos , Clorexidina/uso terapêutico , Cavidade Pulpar/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/isolamento & purificação , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Lipopolissacarídeos/análise , Pessoa de Meia-Idade , Distribuição Aleatória , Retratamento , Irrigantes do Canal Radicular/uso terapêutico , Preparo de Canal Radicular/métodos , Hipoclorito de Sódio/uso terapêutico , Ácidos Teicoicos/análiseRESUMO
Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Defensinas/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Ácidos Teicoicos/antagonistas & inibidores , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Crustáceos/química , Crustáceos/fisiologia , Defensinas/química , Defensinas/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Moluscos/química , Moluscos/fisiologia , Alinhamento de Sequência , Relação Estrutura-Atividade , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/antagonistas & inibidores , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
The gut associated lymphoid tissue (GALT) is anatomical and functionally divided in inductive and effectors sites. In previous works we demonstrated that non-pathogenic bacteria with probiotic characteristics can improve the gut mucosal immune system, with an increase in the number of IgA and cytokines producing cells in the effector site of the intestine. In the present work we studied the effect of non-pathogenic Gram(+), Gram(-) bacteria and a Gram(+) probiotic strain on the inductor site (PP) after the oral administration to BALB/c mice. We also studied some signals induced by the assayed strain in the effectors site, such as the enzyme calcineurin and TLR-9 as a way to understand the mechanisms induced in such bacterial stimulation. The implicance of the lipoteichoic acid (LTA) in the immunostimulation was analyzed. All strains increased the number of IFN-gamma and TNF-alpha(+) cells, but not of IL-10(+) cells in the total population of PP. The release of IFN-gamma and TNF-alpha was only induced by LPS stimulation. All assayed strains increased the number of calcineurin(+) cells, while only Gram(+) strains increased the number of TLR-9(+) cells. The immunostimulatory properties of the purified LTA from Gram(+) strains was evaluated on a monocyte-macrophage U937 cell line. These cells showed capacity to release TNF-alpha and IL-10 in response to all LTA assayed in a dose-dependent way. Gram(+) strains induced signals through the calcineurin enzyme able to activate the transcriptional factor NFAT and through TLR-9. The LTA molecule from Gram(+) strains would not be the only structure involved in the immunostimulatory properties observed, specially for the probiotic strain.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Imunidade nas Mucosas , Transdução de Sinais , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/microbiologia , Células Apresentadoras de Antígenos/patologia , Calcineurina/metabolismo , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/patologia , Bactérias Gram-Positivas/patogenicidade , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/patologia , Humanos , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-10/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Probióticos/administração & dosagem , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Células U937RESUMO
Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1microg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10microM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10microM) and Jun N-terminal kinase (JNK; SP600125, 10microM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.