Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.publishedVersio

Discriminating External and Internal Causes for Heading Changes in Freely Flying Drosophila

As animals move through the world in search of resources, they change course in reaction to both external sensory cues and internally-generated programs. Elucidating the functional logic of complex search algorithms is challenging because the observable actions of the animal cannot be unambiguously assigned to externally- or internally-triggered events. We present a technique that addresses this challenge by assessing quantitatively the contribution of external stimuli and internal processes. We apply this technique to the analysis of rapid turns (“saccades”) of freely flying Drosophila melanogaster. We show that a single scalar feature computed from the visual stimulus experienced by the animal is sufficient to explain a majority (93%) of the turning decisions. We automatically estimate this scalar value from the observable trajectory, without any assumption regarding the sensory processing. A posteriori, we show that the estimated feature field is consistent with previous results measured in other experimental conditions. The remaining turning decisions, not explained by this feature of the visual input, may be attributed to a combination of deterministic processes based on unobservable internal states and purely stochastic behavior. We cannot distinguish these contributions using external observations alone, but we are able to provide a quantitative bound of their relative importance with respect to stimulus-triggered decisions. Our results suggest that comparatively few saccades in free-flying conditions are a result of an intrinsic spontaneous process, contrary to previous suggestions. We discuss how this technique could be generalized for use in other systems and employed as a tool for classifying effects into sensory, decision, and motor categories when used to analyze data from genetic behavioral screens

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age

Discriminating External and Internal Causes for Heading Changes in Freely Flying <em>Drosophila</em>

<div><p>As animals move through the world in search of resources, they change course in reaction to both external sensory cues and internally-generated programs. Elucidating the functional logic of complex search algorithms is challenging because the observable actions of the animal cannot be unambiguously assigned to externally- or internally-triggered events. We present a technique that addresses this challenge by assessing quantitatively the contribution of external stimuli and internal processes. We apply this technique to the analysis of rapid turns (“saccades”) of freely flying <i>Drosophila melanogaster</i>. We show that a single scalar feature computed from the visual stimulus experienced by the animal is sufficient to explain a majority (93%) of the turning decisions. We automatically estimate this scalar value from the observable trajectory, without any assumption regarding the sensory processing. A posteriori, we show that the estimated feature field is consistent with previous results measured in other experimental conditions. The remaining turning decisions, not explained by this feature of the visual input, may be attributed to a combination of deterministic processes based on unobservable internal states and purely stochastic behavior. We cannot distinguish these contributions using external observations alone, but we are able to provide a quantitative bound of their relative importance with respect to stimulus-triggered decisions. Our results suggest that comparatively few saccades in free-flying conditions are a result of an intrinsic spontaneous process, contrary to previous suggestions. We discuss how this technique could be generalized for use in other systems and employed as a tool for classifying effects into sensory, decision, and motor categories when used to analyze data from genetic behavioral screens.</p> </div

Simple models of decision making processes and relative experimental predictions.

<p>This figure shows, on the left, several simplified saccade generation schemes, and their prediction in terms of the observed statistics. All models assume that the visual stimulus is processed as to extract a one-dimensional feature on which the animal decisions are based. The models presented are meant to represent a sample of qualitatively different functional models of behavior generations, and not necessarily biologically plausible models of neural computation. Panel A-i shows a “hard threshold” model: if the feature is below a threshold, no event is generated, otherwise, the event is generated stochastically with a certain rate. Panel A-ii shows what would be the prediction of the model if we were to plot the saccade generation rate (an observable quantity) as a function of the feature , assuming we knew how to compute . Panel B shows the same model, but with noise affecting the computation of the feature. The effect on the observed rate would be to transform the hard threshold in a soft threshold. Panel C shows a model in which there is a parallel saccade generation mechanism, which generates saccades randomly independently of the stimulus. The effect of this on the measured rate is to raise uniformly the curves. Also the contribution of some internal processing based on internal neural states which were not a function of the instantantaneous stimulus would have the same effect on the rate statistics. Panel D shows the case where the behavior depends also on some other feature of the stimulus in addition to . In this case, if we plotted the rates as a function of , ignoring the dependency on , we would see that it is not possible for to explain the rates by itself. Therefore, once we have identified the curves , , and the feature , we are able to identify the contribution of a random generation process (or based on an internal state) as a uniform baseline saccade rate; and we can infer whether another feature is necessary to explain the behavior by the vertical spread of the rates.</p

Estimated saccade generation rates.

<p>This picture shows basic statistics of the processed data. Panel A shows the estimated saccade <i>rate</i> in polar coordinates (i) and fly-centered spatial coordinates (ii), indicated in the text as . This density is obtained by taking the raw number of saccades in each cell (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002891#pcbi-1002891-g001" target="_blank">Figure 1</a>, Panel E), normalizing by the time spent in each cell (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002891#pcbi-1002891-g001" target="_blank">Figure 1</a>, Panel D), and then compensating for the interacting nature of the Poisson processes. Panels B–C show the rates for left and right saccades ( and , respectively), which we plot in red in Panel B (left saccades) and in blue in panel C (right saccades). Note that the left and right saccade ratios appear roughly symmetric.</p

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells.

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter

The estimated decision feature

<p><b>.</b> Panel A shows the estimated one dimensional feature . This is the best one dimensional spatial feature that explains the left and right saccade rates. It is a dimensionless quantity, which we normalize in the interval . Panel B-i shows, for each cell , the rates as a function of the estimated feature ; Panel B-ii shows the same data, but with error bars corresponding to 95% confidence intervals (the bars are not symmetric because the posterior distribution of the estimated rates is not Gaussian; see Supplemental Materials for details). The single feature is sufficient to predict the rate in of the environment, in the sense that 93% of the rates can be considered (with the error bars) as lying on the same curve; these curves are the functions and discussed previously that allow predicting the rates from the feature. The remaining of data that this model cannot fit correspond to configurations with the fly pointing directly against the wall at a small distance (<0.3 m).</p