Image_1_Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.pdf

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.</p

Table_2_Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.docx

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.</p

Table_3_Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.docx

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.</p

Image_2_Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.pdf

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.</p

Table_1_Overexpression of proinflammatory cytokines in dental pulp tissue and distinct bacterial microbiota in carious teeth of Mexican Individuals.docx

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans. The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella, and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8.</p

Apoptosis induction in <i>Mycobacterium bovis</i>-infected bovine macrophages.

<p>Uninfected macrophages (A), macrophages treated with camptothecin (10 µg/mL for 48 h) (C) and macrophages infected with <i>Mycobacterium bovis</i> field strain 9926 (B, D and E), were washed at 4 h and cultured again for 24 h. Then cells were processed by TUNEL in presence (E) or absence (D) of TdT enzyme. Chromatin condensation was present in no-infected (arrows) and infected cells (arrowheads). TUNEL-positive cells show a green-yellow mark in panel E (BrdUTP-FITC). Magnification for A to C is 63X and for D and E is 40X with a scale bar of 50 µm each.</p

Frequencies of <i>SLC11A1^s</i> and <i>SLC11A1^r</i> genotypes among cattle characterized as susceptible or resistant by BCG killing assay.

a<p>Fisher’s exact test reveals no association between macrophage phenotype and the 3′UTR GT-polymorphism in <i>SLC11A1</i> gene.</p

<i>Mycobacterium bovis</i> induction of bovine macrophage DNA fragmentation is time dependent.

<p>Adherent-macrophages (MØ) were cultured with media alone or with camptothecin (10 µg/mL for 48 h) as negative and positive controls, respectively (A). Also macrophages were cultured in absence (B) or presence of purified <i>E. coli</i> 026:B6 LPS (100 ng/mL) for 22 h (C), and infected with BCG or <i>M. bovis</i> field strain 9926 (MOI of 10 for 4 h), washed and cultured again for 16 and 24 h and stained with TUNEL (BrdUTP-FITC). Histograms are frequency distributions of 1×10⁵ macrophages along FITC signal (log₁₀ scale). Values are percentages of true BrdU-FITC TUNEL-positive cells (M1 gate) of one experiment, representative of two independent experiments with macrophages of three susceptible and three resistant cows. One-way ANOVA showed significant variation in infected versus uninfected controls (<i>P</i><0.05) but not between phenotype (<i>P</i> = 0.4544) regardless of <i>M. bovis</i> strain.</p

Kinetics of apoptosis induction in resistant and susceptible <i>Mycobacterium bovis</i>–infected bovine macrophages, expressed as percentage (mean ± SD) of cells showing chromatin condensation after infection.

a<p>Macrophages were pre-cultured with or without LPS for 18 h then infected with BCG or <i>M. bovis</i> field strain for 4 h, washed and cultured again for indicated times and then stained with propidium iodide. Two hundred macrophages were analyzed per slide in triplicate cultures and those with chromatin condensation were counted. Results are representative of two independent experiments with macrophages of three susceptible (S) and three resistant (R) cattle.</p>*<p>Significant differences (<i>P</i><0.05) are among phenotypes.</p

Bovine macrophage BCG growth control as an indicatior of a natural disease resistance phenotype.

<p>Adherent-macrophages were infected with BCG Danish strain or <i>M. bovis</i> field strain 9926 (MOI of 10) for 4 h, washed and cultured again for 24 h. Bacterial intracellular growth was calculated by dividing the number of intracellular CFU at 24 h post-infection by the number of intracellular CFU at the start of the assay. The figure shows the percentage of <i>M. bovis</i> growth in macrophages of 24 cows. For BCG values below or equal to the cut-off point of 65% growth (dotted line) are indicative of resistant cattle (white symbols) while values higher than 65% are a sign of susceptibility (black symbols). Plots are mean of triplicates of two independent experiments of each animal.</p