A Systems Biology Approach to the Analysis of Subset-Specific Responses to Lipopolysaccharide in Dendritic Cells

<div><p>Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 commitment, generating inducible Tregs, and mediating tolerance. It is believed that distinct DC subsets have evolved to control these different immune outcomes. However, how DC subsets mount different responses to inflammatory and/or tolerogenic signals in order to accomplish their divergent functions remains unclear. Lipopolysaccharide (LPS) provides an excellent model for investigating responses in closely related splenic DC subsets, as all subsets express the LPS receptor TLR4 and respond to LPS in vitro. However, previous studies of the LPS-induced DC transcriptome have been performed only on mixed DC populations. Moreover, comparisons of the in vivo response of two closely related DC subsets to LPS stimulation have not been reported in the literature to date. We compared the transcriptomes of murine splenic CD8 and CD11b DC subsets after in vivo LPS stimulation, using RNA-Seq and systems biology approaches. We identified subset-specific gene signatures, which included multiple functional immune mediators unique to each subset. To explain the observed subset-specific differences, we used a network analysis approach. While both DC subsets used a conserved set of transcription factors and major signalling pathways, the subsets showed differential regulation of sets of genes that ‘fine-tune’ the network Hubs expressed in common. We propose a model in which signalling through common pathway components is ‘fine-tuned’ by transcriptional control of subset-specific modulators, thus allowing for distinct functional outcomes in closely related DC subsets. We extend this analysis to comparable datasets from the literature and confirm that our model can account for cell subset-specific responses to LPS stimulation in multiple subpopulations in mouse and man.</p></div

The pathway modulation model.

<p>Schematic representation of our proposed model. LPS-induced signaling occurs through a common set of signaling molecules. LPS induces the expression of subset-specific TLR4 pathway modulators, which ‘fine tune’ signaling and allow for distinct immune outcomes in closely related cell subsets. These pathway modulators likely integrate both signals derived directly from TLR4 and exogenous signals from the microenviroment that contribute to the subset-specificity of the response.</p

Expression of canonical transcription factor-target TLR4-dependent pathways mediating the LPS response in CD8 and CD11b DCs.

<p>Transcription factor-target over-representation analysis of LPS-induced genes in CD8 and CD11b DCs.</p><p>1. The ratio of odds (Odds-Ratio) that a transcription factor-associated pathway is enriched in the selected DC subset was calculated as the odds of differentially expressed genes being regulated by the transcription factor divided by the odds of non-differentially expressed genes being regulated by the same transcription factor.</p><p>2. P-values are adjusted to control for multiple comparisons.</p><p>*denotes not significant (p>0.05).</p

Comparison of steady-state spleen DC subsets.

<p>(A) Steady-state splenic DCs were magnetic bead-enriched for CD19⁻B220⁻CD3⁻Gr-1⁻Ter119⁻CD11c⁺ cells. MHCII⁺CD11c⁺ cells (circled) were then sorted for CD8 (blue gate) and CD11b (orange gate) subsets and RNA prepared and analysed by RNA-Seq. (B) Heatmap showing the relative expression of the 50 most commonly defined and validated markers for CD8 and CD11b subsets. Data are presented as fold changes (CD11b/CD8), all of which were statistically significant with an associated p-value <0.05. Orange denotes genes that were increased in CD11b DCs while blue denotes genes that were decreased in CD11b DCs (and thus increased in CD8 DCs. (C) Overlap between genes that were significantly differentially expressed between CD8 and CD11b DCs in our dataset and in 9 previously published microarray datasets derived from splenic DC subsets (datasets 1–9 listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613.s006" target="_blank">Table S1</a>). The significance of overlap between the gene list from our dataset and those from each of the published datasets was calculated using a hypergeometric test to assess the consistency/quality of our results. Data are presented as 1/p-value on a log scale with all overlaps reaching a significance cut-off <0.05.</p

GO terms annotating LPS-induced genes in CD8 versus CD11b DCs.

<p>GO term over-representation analysis of LPS-induced genes in CD8 and CD11b DCs.</p><p>1. The ratio of odds (Odds-Ratio) that a GO term is enriched in the selected DC subset was calculated as the odds of a differentially expressed gene divided by the odds of a non-differentially expressed gene occurring in the GO term.</p><p>2. P-values are adjusted to control for multiple comparisons.</p><p>*denotes not significant (p>0.05).</p

Reactome pathways enriched within the CD8 and CD11b DC subnetworks.

<p>Reactome pathway over-representation analysis of nodes within the CD8 or CD11b subnetworks. P-values are adjusted to control for multiple comparisons.</p

Subset-specific expression of pathway modulators.

<p>LPS-induced regulation (+LPS/−LPS) of pathway modulators in mouse (A–B) and human (C–D) cell subsets, visualised in Venn diagrams. (A) CD8 and CD11b DCs from our study. (B) Thioglycolate-elicited peritoneal macrophages and bone-marrow derived macrophages <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613-Lattin1" target="_blank">[26]</a>. (C) Vδ1 and Vδ2 γδ T cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613-Kress1" target="_blank">[40]</a>. (D) Retinal vascular endothelium and choroidal endothelial cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613-Smith1" target="_blank">[41]</a>. (E) Cord blood peripheral blood monocytes and neutrophils <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613-Davidson1" target="_blank">[48]</a>. Datasets in (B–E) are listed as 16–19 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613.s006" target="_blank">Table S1</a>.</p

Subset-specific LPS-induced gene signatures.

<p>(A–B) Dendritic cells were stimulated in vivo with an intraperitoneal injection of 25ug LPS and their activation confirmed by measuring upregulation of CD86 24 hours later. (C–E) LPS stimulated DC subsets isolated from a second cohort of mice (n = 10, spleen cells pooled) were magnetic bead enriched, sorted, analysed by RNA-Seq, and then compared to steady-state controls (n = 5, spleen cells pooled). (C) Differences in LPS-induced gene expression were visualised in a Venn diagram. (D–E) Heatmaps showing the expression of key immune effector genes (D) uniquely regulated in either CD8 (left) or CD11b DCs (right) or (E) similarly regulated in both subsets. Data are presented as fold changes (+LPS/−LPS).</p

Population vs. subset-specific LPS-responses.

<p>(A) Data from published studies comparing in vitro stimulated GM-CSF derived BM-DCs +/− LPS stimulation (datasets 10–14 listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613.s006" target="_blank">Table S1</a>) were reanalysed for differential gene expression. All LPS-induced genes identified in BM-DCs at any of the 10 timepoints (p-value <0.05) were compared to LPS-induced genes in our study and visualised in a Venn diagram. (B) Only genes differentially expressed in the 24 hour timepoint sample from the GSE17721 BM-DC dataset (dataset 13 listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613.s006" target="_blank">Table S1</a>) were compared to LPS-induced genes in our study and visualised in a Venn diagram. (C) Genes identified as significantly differentially expressed (p-value <0.05) in splenic CD11c⁺ DCs stimulated in vivo with LPS and anti-CD40 and isolated after 6 hours were compared to in vivo LPS-induced genes in our study (isolated after 24 hours) and visualised in a Venn diagram (dataset 15 listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100613#pone.0100613.s006" target="_blank">Table S1</a>).</p