Triclosan exposure design.

<p>A schematic diagram of the experimental groups and triclosan exposures. Labeled tick marks represent days.</p

Triclosan Exposure Is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

<div><p>Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus <i>Pseudomonas</i> appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiome’s interaction with environmental chemicals.</p></div

Triclosan exposure is associated with alterations in microbial correlation networks.

<p>Interaction networks for microbial communities in (A) unexposed, (B) four-day exposure and (C) seven-day exposure groups. Network communities (subgraphs) are identified by colored polygons and the vertices (OTUs) that comprise these communities are colored identically to indicate network community membership. Each line represents an abundance correlation between two OTUs. The size of each vertex is proportional to its degree. Edges between different network communities are colored red and those within a community are black. (D) Venn diagram of shared vertices between networks. (E) Degree and (F) betweenness centrality distribution for all vertices in network. *** p < 0.001.</p

Triclosan exposure is associated with altered microbial community structure.

<p>(A) Phyla level taxa plot of the most abundant taxa in zebrafish gut microbiomes across exposure groups. (B) Non-metric multidimensional scaling analysis of unexposed (red dots), four-day (blue dots) and seven-day (green dots) exposure group’s microbial communities. Colored ellipses represent the 99.9% confidence interval for standard error of each group. (C) Comparisons of within group Bray-Curtis dissimilarity between groups. (D) Shannon entropy between exposure groups. Significant p-values (p < 0.05) are denoted with an asterisk.</p

Triclosan exposure is associated with unique indicator OTUs.

<p>Indicator values for significant (p < 0.05, q < 0.20) indicator taxa in (A) unexposed and (B) triclosan exposed fish. The size of each point is proportional to its class-wide relative abundance, and its color is proportional to its class-wide frequency.</p

Triclosan exposure is associated with significant alterations in OTU abundance.

<p>Fold change values for OTUs that were significantly altered in abundance in the (A) four-day, and (B) seven day exposure groups. Genus level taxonomic assignments are provided and corresponding OTU IDs indicated inside parentheses.</p

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal <i>Lactobacillus</i>

<div><p>Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to <i>Lactobacillus plantarum</i> WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.</p></div

Enhanced APC inflammatory response to commensal lactobacilli signals predominantly through p38-MAPK.

<p>(<b>A</b>) Representative flow cytometry plot and frequencies of APCs from HIV-infected patients producing proinflammatory cytokines IL-6, IL-12/IL-23p40, and TNFα in response to (<b>B</b>) <i>L. plantarum</i> WCFS1 (n = 16), (<b>C</b>) <i>L. gasseri</i> 1SL4 (n = 12), and (<b>D</b>) <i>L. casei</i> BL23 (n = 11) with or without BIRB796 pretreatment. (<b>E</b>) Relative expression of MKP-1 in unstimulated and bacterial stimulated APCs as determined by real-time PCR (HIV− n = 3–9, HIV+ n = 5–16). Bar graphs represent mean +/− SEM. P values determined using paired t or Mann Whitney U test (*P<0.05, **P<0.01, ***P<0.001).</p

Increased expression of pattern recognition receptors on APCs from HIV-infected patients.

<p>(<b>A</b>) Heatmap and (<b>B</b>) fold changes of PRR gene expression in isolated CD11c+ APCs from HIV-infected patients were compared to HIV-negative controls (HIV− n = 4, HIV+ n = 4) using DNA microarray analysis. P values determined by unpaired t test (*P<0.05, **P<0.01, ***P<0.001). (<b>C</b>) Representative flow cytometry histograms of TLR2 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (<b>D</b>) Median fluorescence intensity of TLR2 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (<b>E</b>) Representative flow cytometry histograms of CD36 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (<b>F</b>) Median fluorescence intensity of CD36 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (<b>G</b>) Frequencies of APCs from HIV-infected patients (n = 7) producing IL-6, IL-12/IL23p40, and TNFα in response to <i>L. plantarum</i> WCFS1 with or without TLR2 blocking antibody. (<b>H</b>) Concentrations of IL-6, IL-12/IL-23p40, and TNFα following stimulation with <i>L. plantarum</i> WCFS1 with or without TLR2 blocking antibody as determined by ELISA (HIV+ n = 3–6). Each dot represents an individual subject. In the HIV+ group, open circles represent therapy-naïve patients, closed circles represent patients on HAART. Bars indicate median value. Bar graphs represent mean +/− SEM. P values determined using Mann Whitney U test or paired t test.</p

Distribution of antigen presenting cells in peripheral blood during chronic HIV infection.

<p>(<b>A</b>) Multicolor flow cytometric gating strategy for the identification of APC populations in peripheral blood. APCs (CD3-CD19-CD56-HLADR+CD11c+CD123-), monocytes (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+), and mDCs (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+) were detected following the exclusion of dead cells and CD3+, CD19+ and CD56+ cells. The HLA-DR-positive cells were gated for CD11c and CD123. Data were analyzed using FlowJo. (<b>B</b>) Percentages of circulating APCs (HIV− n = 37, HIV+ n = 94). (<b>C</b>) Monocyte percentage of the peripheral blood APC population. (<b>D</b>) Myeloid dendritic cell population of the peripheral blood APC population. (<b>E</b>) Plasma sCD14 concentrations as determined by ELISA (HIV− n = 25; HIV+ n = 66). Each dot represents an individual patient. In the HIV+ group, open circles represent therapy-naïve patients and closed circles represent patients on HAART. Bars indicate median value. P values determined using Mann Whitney U test. P values as indicated.</p