<i>In silico</i> simulation of altered commensal bacteria regrowth during <i>Clostridium difficile</i> infection.

<p>Four cases were tested with variations to the inhibition of the commensal bacteria growth: inhibited by both neutrophils and inflamed epithelial cells (N and E_i), by only neutrophils (N), by only inflamed epithelial cells (E_i), and by neither (none). Resulting changes in species populations for each case are shown: (a) <i>baiCD</i>-containing commensal species, (b) <i>C</i>. <i>difficile</i>, (c) activated neutrophils, and (d) iTreg cells in the lamina propria.</p

Commensal bacteria regrowth inhibited with <i>Clostridium difficile</i> infection.

<p>(a) The <i>baiCD</i> content is decreased by antibiotic treatment in both control and <i>C</i>. <i>difficile</i> challenged mice and further decreased post-infection in the <i>C</i>. <i>difficile</i> challenged mice compared to the controls. (b and c) The expression of anti-microbial peptides DefB1 and S100A8 are upregulated with infection. Data points and error bars represent mean ± standard error of the mean (SEM). Asterisks (*) mark significance (p≤0.05) in comparison between control and <i>C</i>. <i>difficile</i> infected mice (n = 10).</p

Simulated dynamics of mucosal immune response to <i>Clostridium difficile</i>.

<p>Modeling results following calibration and validation of the host response model in populations of (a) <i>C</i>. <i>difficile</i>, (b) protective commensal bacteria, (c) infection-exacerbating commensal bacteria, (d) lamina propria T helper 17 cells, (e) effector dendritic cells, (f) infiltrating neutrophils, (g) regulatory T cells, (h) tolerogenic dendritic cells and (i) activated macrophages. Lines represent simulation results, filled points represent experimental calibration data and unfilled points represent experimental validation data.</p

Relative effects of parameters on <i>Clostridium difficile</i> population and epithelial cell death.

<p>(a and b) Histograms showing the distribution of parameter impact on <i>C</i>. <i>difficile</i> population and epithelial cell death, respectively. Measurements are based on sensitivity analysis of the calibrated model. (c and d) Highest impact parameters for each quantity in which positive amounts indicate an increasing effect on the quantity and negative amounts indicate a decreasing effect. For the <i>C</i>. <i>difficile</i> population results, P1 contributes to effector dendritic cell production, P2 to neutrophil activation and migration, P3 to protective commensal bacteria regrowth, P4 to macrophage activation, P5 to commensal bacteria death, P6 to macrophage death, P7 to Th17 cell death, and P8 to neutrophil death. For epithelial cell death, P1 contributes to tolerogenic dendritic cell production, P2 to Th17 to Treg cell plasticity, P3 to commensal bacteria death, P4 to Treg to Th17 cell plasticity, P5 to macrophage activation and P6 to <i>C</i>. <i>difficile</i> growth.</p

Time course of <i>Clostridium difficile</i> infection in mice.

<p>(a and b) Flow cytometry analysis of colonic lamina propria lymphocytes from days 1 to 10 post-infection showing the differences in CD4+ CD25+ FoxP3+ regulatory T (Treg) and CD4+ IL17+ T helper 17 (Th17) cells, respectively, between control and <i>C</i>. <i>difficile</i> challenged wild type mice. (c) Re-isolation data of <i>C</i>. <i>difficile</i> from colonic contents from day 1 to day 8 post-infection. Data points and error bars represent mean ± standard error of the mean (SEM). Asterisks (*) mark significance (<i>P</i>≤0.05) in comparison between control and <i>C</i>. <i>difficile</i> infected mice (n = 10).</p

Network topology of model illustrating mucosal immune responses to <i>Clostridium difficile</i>.

<p>Systems biology markup language (SBML) compliant network of interactions between <i>C</i>. <i>difficile</i> and cellular immune components created in CellDesigner. Reaction modifiers connect cell nodes to reaction arrows with green as indication of activation and red of inhibition. Species consist of <i>C</i>. <i>difficile</i> (<i>Cdiff</i>), infection-exacerbating commensal bacteria (<i>CommH</i>), protective commensal bacteria (<i>CommB</i>), dead commensal bacteria (<i>CommD</i>), epithelial cells (<i>E</i>), inflamed epithelial cells (<i>E</i>_<i>i</i>), neutrophils (<i>N</i>), macrophages (<i>M</i>), dendritic cells (<i>tDC</i> and <i>eDC</i>), T cells (<i>nT</i>, <i>Treg</i>, <i>Th17</i>, <i>Th1</i>) existing in multiple compartments lumen (<i>Lum</i>), epithelium (<i>EP</i>), lamina propria (<i>LP</i>), and mesenteric lymph node (<i>MLN</i>).</p

image_1_Innate Immunity Induced by the Major Allergen Alt a 1 From the Fungus Alternaria Is Dependent Upon Toll-Like Receptors 2/4 in Human Lung Epithelial Cells.jpeg

<p>Allergens are molecules that elicit a hypersensitive inflammatory response in sensitized individuals and are derived from a variety of sources. Alt a 1 is the most clinically important secreted allergen of the ubiquitous fungus, Alternaria. It has been shown to be a major allergen causing IgE-mediated allergic response in the vast majority of Alternaria-sensitized individuals. However, no studies have been conducted in regards to the innate immune eliciting activities of this clinically relevant protein. In this study, recombinant Alt a 1 was produced, purified, labeled, and incubated with BEAS-2B, NHBE, and DHBE human lung epithelial cells. Alt a 1 elicited strong induction of IL-8, MCP-1, and Gro-a/b/g. Using gene-specific siRNAs, blocking antibodies, and chemical inhibitors such as LPS-RS, it was determined that Alt a 1-induced immune responses were dependent upon toll-like receptors (TLRs) 2 and 4, and the adaptor proteins MYD88 and TIRAP. Studies utilizing human embryonic kidney cells engineered to express single receptors on the cell surface such as TLRs, further confirmed that Alt a 1-induced innate immunity is dependent upon TLR4 and to a lesser extent TLR2.</p

image_2_Innate Immunity Induced by the Major Allergen Alt a 1 From the Fungus Alternaria Is Dependent Upon Toll-Like Receptors 2/4 in Human Lung Epithelial Cells.jpeg

<p>Allergens are molecules that elicit a hypersensitive inflammatory response in sensitized individuals and are derived from a variety of sources. Alt a 1 is the most clinically important secreted allergen of the ubiquitous fungus, Alternaria. It has been shown to be a major allergen causing IgE-mediated allergic response in the vast majority of Alternaria-sensitized individuals. However, no studies have been conducted in regards to the innate immune eliciting activities of this clinically relevant protein. In this study, recombinant Alt a 1 was produced, purified, labeled, and incubated with BEAS-2B, NHBE, and DHBE human lung epithelial cells. Alt a 1 elicited strong induction of IL-8, MCP-1, and Gro-a/b/g. Using gene-specific siRNAs, blocking antibodies, and chemical inhibitors such as LPS-RS, it was determined that Alt a 1-induced immune responses were dependent upon toll-like receptors (TLRs) 2 and 4, and the adaptor proteins MYD88 and TIRAP. Studies utilizing human embryonic kidney cells engineered to express single receptors on the cell surface such as TLRs, further confirmed that Alt a 1-induced innate immunity is dependent upon TLR4 and to a lesser extent TLR2.</p