The mutational landscape of the adult healthy parous and nulliparous human breast

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.</p

The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection

<div><p>Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied <i>in vitro</i>, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the <i>Salmonella enterica</i> genotoxin, known as typhoid toxin, in <i>in vivo</i> models of murine infection. Immunocompetent mice were infected with isogenic <i>S</i>. <i>enterica</i>, serovar Typhimurium (<i>S</i>. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.</p></div

Phylogenetic analysis of the intestinal microbiota.

<p>Relative abundance of the significantly altered bacterial families in the intestinal microbiota of mice infected with the MC1-TT (TT) or MC1-Δ<i>cdtB</i> (Δ<i>cdtB</i>) strain compared to uninfected mice. *p value ≤ 0.05.</p

qPCR-Array analysis of colon and liver 180 days p.i.

<p><b>A.</b> Violin plot representing the distribution of the log₂ fold changes of transcripts in the indicated tissues of mice infected with the MC1-TT strain compared to those detected in mice infected with the MC1-∆<i>cdtB</i> strain at 180 days p.i. Statistical analysis was performed with the Wilcoxon rank-sum test. Colon: p-value = 4.989e-13 (distribution of log2 fold changes is significantly shifted below 0). Liver: p-value = 0.03676 (distribution of log2 fold changes is moderately shifted over 0). ****p value ≤ 0.0001, *p ≤ 0.05. <b>B.</b> Heat map and functional annotation analysis for genes down- and up-regulated identified in A.</p

Infection with the toxigenic MC1 strain reduces the inflammatory response in the intestine.

<p><b>A.</b> Haematoxylin and eosin staining of the intestine of uninfected mice or mice infected for 10 days with the control or toxigenic MC1 strains. Black arrows indicate accumulation of neutrophils within the lamina propria, along with invasion of the crypts. The dashed squares indicate the area that was enlarged in the middle panel. <b>B.</b> Histological scores of intestine, liver and spleen of uninfected mice, and mice infected with the control or toxigenic MC1 strains 10 days p.i. The following score was used to grade the lesions: 0: no lesions observed; 1: mild; 2: moderate; 3: severe; 4: very severe. Statistical analysis was performed using the Student t-test (n mice = 5). <b>C.</b> Immunohistochemical analysis performed with a rabbit anti-CD45-specific antibody to detect leukocyte recruitment in the intestinal tissue of uninfected mice and mice infected for 10 days with the control or toxigenic MC1 strains. <b>D.</b> Quantification of the CD45-specific staining, expressed as % of pixel per area. Statistical analysis was performed using the Student t-test (n mice = 5). <b>E.</b> Immunohistochemical analysis performed with rabbit anti-CD14- or mouse anti-CD3-specific antibodies to detect T lymphocyte and macrophage recruitment, respectively, in the intestinal tissue of mice infected for 10 days with the control or toxigenic MC1 strains. <b>F.</b> Quantification of the CD14- and CD3-positive cells (HPF: high power field). Statistical analysis was performed using the One-Way ANOVA test (n mice = 5).</p

The presence of the genotoxin gene promotes the establishment of a persistent infection.

<p><b>A.</b> Dissemination of <i>S</i>. Typhimurium in cecum, mesenteric lymph nodes (MLN), liver, and spleen in mice infected for 30, 60 and 180 with the MC1-∆<i>cdtB</i> or MC1-TT strains. Data are presented as colony forming unit (CFU) per organ. Statistical analysis was performed using the Student t-test. *p value ≤ 0.05 (n mice = 6). <b>B.</b> Immunohistochemical analysis performed with rabbit anti-CD3- or mouse CD14-specific antibodies in the liver of mice infected for 180 days with the control or toxigenic MC1 strains. Arrows indicate the CD3+ and CD14+ immune cells respectively <b>C.</b> Quantification of the CD3- and CD14-positive cells (HPF: high power field). Statistical analysis was performed using the One-Way ANOVA test, **p≤ 0.01, and *p ≤ 0.05 (n mice = 4).</p

The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection - Table 1

<p>The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection</p> - Table

Infection with genotoxic <i>Salmonella</i> strains promotes survival of the host.

<p><b>A.</b> Percentage of survival during the infection with the virulent MC1 (left panel) or the attenuated MC71 (right panel) strain. The Kaplan-Meier method was used to evaluate survival (95% confidence interval). *p value ≤ 0.05. <b>B.</b> Dissemination of <i>S</i>. Typhimurium in cecum, mesenteric lymph nodes (MLNs), liver, and spleen in mice infected for 10 days with the control or toxigenic MC1 or MC71 strains. Data are presented as colony forming unit (CFU) per organ. Statistical analysis was performed using the Student t-test (n mice = 6).</p

The active CdtB subunit is expressed in vivo.

<p>A. Infection model. Female 129S6/SvEvTac mice were randomized into three groups. Mice were infected orally with <i>S</i>. Typhimurium carrying the inactive (∆<i>cdtB</i>) or functional toxin operon (TT) for 10, 30, 60 and 180 days, at an infection dose of 10⁸ bacteria per mouse. Uninfected mice were gavaged with PBS. Groups of 5 to 6 mice were used, unless specified otherwise. <b>B.</b> Immunohistochemical analysis performed with an anti-FLAG-specific antibody to detect the active CdtB subunit of the typhoid toxin in liver tissue of mice infected for 10 days with the control or toxigenic MC1 strains. The black arrows indicate the nuclear localization of the toxin subunit. <b>C.</b> Immunohistochemical analysis performed with a rabbit anti-γH2AX-specific antibody to detect induction of DNA damage in liver tissue of uninfected mice or mice infected for 10 days with the control or toxigenic MC1 strains. <b>D.</b> Quantification of the γH2AX-specific staining, expressed as % of pixel per area. Statistical analysis was performed using the Student t-test, **** p value ≤ 0.0001, ***p value ≤ 0.001 (n mice = 5).</p

Infection with the toxigenic MC1 strain enhances the inflammatory response in the liver.

<p><b>A.</b> Immunohistochemical analysis performed with an anti-LyG6-specific antibody to detect granulocytes and neutrophil recruitment in the liver tissue of uninfected mice and mice infected for 10 days with the control or toxigenic MC1 strains. <b>B.</b> Quantification of the number of inflammatory foci per field (left panel) and the quantification of the LyG6-specific staining (right panel), expressed as % of pixel per area. Statistical analysis was performed using the Student t-test. (n mice = 5). ***p value ≤ 0.001, **p≤ 0.01, and *p ≤ 0.05 (n mice = 5).</p