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_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

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

ART-naive and short-term ART experienced individuals have decreased frequency of CD161-expressing gut homing CD8+ T-cells.

<p>Frequencies (expressed as percentages) of peripheral blood (PB) memory gut homing (CD45RO+ CCR9+ B7+) CD4+ (A) and CD8+ (C) T-cells expressing CD161 were determined by flow cytometry on PBMCs of HIV-infected individuals (HIV+), antiretroviral (ART) naïve (n = 18), ART experienced individuals on short-term (n = 15) and long-term treatment (n = 32), HIV controllers (n = 6) and HIV seronegative individuals (SN, n = 5). Scatter plots were used to represent the data. Horizontal lines indicate median values. Each symbol represents one individual. The red symbols represent the females in each group. Groups were compared using Kruskal-Wallis test correcting for multiple comparisons using the Dunnett´s post-test. Only significant corrected p values are shown ***p<0.0005, **p<0.005, *p<0.05. Spearman's rank correlation between PB gut homing CD4+ CD161+ T-cells and plasma viral load (pVL) in the ART naive group (B). Gender was color-coded as follows: red dots, women and black dots, men.</p

Gating strategy.

<p>The gating strategy used for all the samples was to first define singlet and morphology by using forward versus side scatter, followed by the exclusion of dead cells (aqua dye negative events) and the exclusion of CD14, CD19, CD56, CD11c and CD123 positive cells (dump gate). Live cells were gated on CD3+ and CD45RO+ cells. Next, CCR9+ and/or β7+ cells were gated and defined as gut homing T-cells. Then CD4+ and CD8+ T cells were gated and finally the expression of CD161 on the gut homing CD4+ (top) and CD8+ (bottom) T-cells was gated. T cell activation was determined by the simultaneous expression of CD38 and HLADR on gut homing CD4+ and CD8+ T cells.</p

The frequency of PB gut homing CD4+ and CD8+ T-cells is altered in HIV infection.

<p>Frequencies (expressed as percentages) of peripheral blood (PB) memory gut homing (CD45RO+ CCR9+ β7+) CD4+ (A) and CD8+ (B) T-cells was determined by flow cytometry on PBMCs of HIV-infected individuals (HIV+), antiretroviral (ART) naïve (n = 18), ART experienced individuals on short-term (n = 15) and long-term treatment (n = 32), HIV controllers (n = 6) and HIV seronegative individuals (SN, n = 5). Scatter plots were used to represent the data. Horizontal lines indicate median values. Each symbol represents one individual. The red symbols represent the females in each group. Groups were compared using Kruskal-Wallis test correcting for multiple comparisons using the Dunnett´s post-test. Only significant corrected p values are shown ***p<0.0005, **p<0.005, *p<0.05. Gender was color-coded as follows: red dots, women and black dots, men.</p