Differential Gene Expression in Thrombomodulin (TM; CD141)⁺ and TM⁻ Dendritic Cell Subsets

<div><p>Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin⁺ dendritic cells are tolerogenic while thrombomodulin⁻ dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin⁺ and thrombomodulin⁻ dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin⁺ dendritic cells compared to control dendritic cells and thrombomodulin⁻ dendritic cells. Thrombomodulin-treated and thrombomodulin⁺ dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin⁺ dendritic cells produced more lipoxins than thrombomodulin⁻ dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin⁺ dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin⁺ dendritic cells contained more cells actively dividing than those of thrombomodulin⁻ dendritic cells. Production of IL-10 is increased in thrombomodulin⁺ dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin⁺ dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin⁺ dendritic cells, which have significantly altered gene expression compared to thrombomodulin⁻ dendritic cells in key immune function pathways.</p></div

Gene expression changes in DCs following sTM treatment or sorting into TM⁺ or TM⁻ DCs.

<p>(A) The mean (black) and 95th percentile (red) of PWF values for each probe rank from 500 permutations of the sample-labels as described in Materials and Methods show that the ‘sorted’ term PWF values (blue) were extraordinarily large compared to the simulations. For the ‘treatment’ term, the PWF values of fewer than 1000 probes were well above the simulated values, and for the ‘sorted:treatment’ interaction term, few probes showed a significantly higher PWF than by permuted sample-label computations. (B) PWF statistics permitted identification of genes exhibiting the strongest association with sorting into TM⁺ and TM⁻ DC, for example <i>Alox15</i>. Similarly, genes exhibiting strong association with TM treatment included <i>Copa</i>. <i>Serpinb8</i> exhibited a large PWF value for the sorted:treatment interaction, as seen by the contrasting effect of treatment on gene expression in TM⁻ and TM⁺ DCs. <i>Fdft1</i> gave large values of PWF for both treatment and sorted:treatment interaction, and shows both down-regulation with TM treatment and notably higher expression in TM⁺ DCs, specifically in untreated cells. (C) Correlation between gene expression changes determined by microarray to those determined by qPCR. A panel of genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072392#pone-0072392-t001" target="_blank">Table 1</a>) whose expression was changed when analyzed by microarray, their fold change was also determined by qPCR. The qPCR data was normalized to GAPDH resulting in this equation for the best fit line: log y = (0.99 *log x) +1.16 with R² = 0.79.</p

Characterization of TM⁺ DCs.

<p>Mouse bone marrow cells were differentiated in GM-CSF for 6 days in the presence of 200 nM sTM from day 4 to 6. DCs were then analyzed for the expression of cell surface markers by flow cytometry cells gated on CD11c⁺ and TM⁺ for the expression of other DC markers. Isotype control is shown in gray.</p

TM⁺ DCs can activate PC and proCPB2.

<p>DCs were cultured in the presence of rhTM before sorting into TM⁺ and TM⁻ DCs. (A) Thrombin and PC were incubated with the cells and the generation of aPC was measured. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni correction. *p<0.05; ***p<0.001 and ****p<0.0001. The mean of 3 experiments is shown with error bars indicating ± sem. (B) Thrombin and proCPB2 were incubated with the cells and the generation of CPB2 was measured. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni correction. *p<0.05; ***p<0.001 and ****p<0.0001. The mean of 3 experiments is shown with error bars indicating ± sem.</p

Antagonism of IL-10 prevents induction of TM on DCs treated with sTM.

<p>DCs were cultured in the presence of sTM from day 4. Neutralizing anti-mouse IL-10 mAb (clone: 2A5) or irrelevant control IgG were added on day 4 at a final concentration of 10<b> µ</b>g/ml. Cells were collected on day 6 and stained with mAb to CD11c and TM. CD11c⁺ cells were gated and expression of TM was analyzed by flow cytometry. Isotype control is shown in gray. (A) Untreated DCs with added control rat IgG. (B) Untreated DCs with added anti-IL-10 mAb. (C) sTM treated DCs with added control IgG. (D) sTM treated DCs with added anti-IL-10 mAb. (E) The mean percentage of TM⁺ DCs was calculated from three experiments is shown with error bars indicating ± sd. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni correction. *p<0.05 and ****p<0.0001.</p

TM⁺ DCs from control cultures have lower expression of maturation markers than TM⁻ DCs.

<p>Mouse DC cultures were sorted into TM⁺ and TM⁻ DCs before analysis of MHC II, CD80 and CD86 expression by flow cytometry. (A) Representative experiment is shown with isotype control in gray. (B) The mean fluorescent intensity (MFI) from three independent experiments is shown. Error bars indicate s.d.</p

List of miRNAs modulated in TM⁺ DCs compared to TM⁻ DCs.

<p>List of miRs whose expression is altered, identified from microarray analysis by comparison of levels in TM⁺ and TM⁻ DCs which change >2 fold with p<0.0001.</p

Altered expression of hemostasis genes’, EPCR and Sphingosine phosphate receptors’ mRNA.

<p>Change in levels of expression of mRNA encoding some genes involved with hemostasis and down stream effects on inflammation, identified from the pathway analysis as part of the “Hemostasis” set.</p

Gene expression is significantly altered in TM⁺ DCs compared to TM⁻ DCs.

<p>(A) A heat map was constructed of the top 100 genes that were changed between the TM⁺ and TM⁻ DCs identified as described in Materials and Methods. The list of genes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072392#pone.0072392.s002" target="_blank">Table S1a</a>. (B) A heat map of the top 100 genes that were changed between the sTM treated and untreated DCs. Down-regulated genes are in blue; up-regulated genes are in yellow. The depth of the colors is based on rescaled Z-values, with high values being yellow and low values blue. The list of genes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072392#pone.0072392.s002" target="_blank">Table S1b</a>.</p

Alterations in phosphorylation of proteins in TM⁺ compared to TM⁻ DCs.

<p>Cell lysates were analyzed by Western blots in which individual tracks were probed with a panel of specific anti-phosphoprotein antibodies, developed and scanned. The values from TM⁺ DCs were compared to those from TM⁻ DCs as a ratio. The antibodies used are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072392#pone.0072392.s003" target="_blank">Table S2</a>. The lines in lanes 1 and 21 represent the migration of the marker proteins. (A) representative Western blot of phosphoproteins from TM⁺ DCs. (B) representative Western blot of phosphoproteins from TM⁺ DCs. (C) Quantitation of Western blots showing phosphoproteins whose level had changed by >25%.</p