Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways

Abstract

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Genomic analysis of highly enriched T cells identifies genes whose expression differs between trauma patients and healthy subjects. (a) T cell, monocyte, and granulocyte distribution in the whole-blood leukocyte fraction from trauma patients and healthy subjects. Cell isolation protocols (see Methods) generated highly enriched cell populations. ∗, P < 0.02. (b) Apparent gene expression of leukocytes, T cells, and monocytes reveals different patterns between trauma patients and healthy subjects. Red indicates increased gene expression, and blue indicates reduced expression. (c) Venn diagram of genes whose expression is significantly different (5% false discovery rate) between trauma patients and healthy subjects.

Fig. 2.

Overall network of perturbed T cell-specific gene interactions. Of 749 genes with specific T cell functions identified in the knowledge base, 338 were significantly perturbed in the trauma patients vs. healthy subjects' T cells. A comprehensive interaction network was constructed among these 338 genes plus 40 additional T cell-specific genes that closely interacted with genes in the pathway.

Fig. 3.

An altered balance of proapoptotic and antiapoptotic molecules in trauma patients' T cells could indicate specifically increased vulnerability to apoptotic depletion. A general apoptosis pathway was overlaid with patient genes altered at the 5% false discovery rate. (a) Expression of monocyte proapoptotic genes were unchanged or reduced (caspase 3, caspase 10, BAD, and Apa11). (b) Specific T cell expression of proapoptotic molecules was increased (caspase 3, caspase 10, Apaf1, Faf1, TWEAKR, and TRAIL2), with decreased decoy receptor signaling (TNFRSF25). (c) Analysis of the total leukocyte population failed to reveal T cell-specific changes or increases in proapoptotic molecules. Selected relevant apoptotic genes not in the standard apoptosis pathway were included for comparison.

Fig. 4.

Interface between altered patient monocyte costimulatory/inhibitory gene expression and T cell activation/inhibition pathways. (a) Increased gene expression (red) of inhibitory costimulation receptor/ligand combinations in monocytes and T cells and concordant decreased expression (blue) of stimulatory receptor/ligand combinations. Concordant increases in expression of inhibitory signal transduction pathways (dotted lines) and decreased or unchanged (gray) gene expression in T cell activation pathways (solid lines) are shown. (b) Examples of cell-specific gene expression alternations involved in T cell inhibition/stimulation. T cell depletion in total leukocytes leads to misdetection of some T cell-specific gene expression as depressed or unchanged.

Fig. 5.

Selective validation of patients' gene expression alterations by functional and protein expression analysis. (a) T cells from a matched cohort of 11 trauma patients with MODS and 15 matched healthy subjects were cultured with immobilized anti-CD3/anti-CD4 or anti-CD3/anti-CD28, and proliferation was assessed as a measure of T cell functional competence (P = 0.0037 and 0.0025, respectively). (b and c) Flow-cytometry-assessed expression of proteins presented as the total percentage of T cells (CD2) expressing marker and mean fluorescent intensity (MFI) involved in T cell migration (CXCR3). ∗, P = 0.003 for percent positive and 0.013 for MFI. T cell activation/inhibition markers were assessed. ∗, intracellular CD152, P = 0.021; CD86, P = 0.04; and LAIR, P = 0.015. (d) Comparison of T cells from trauma patients and healthy subjects for expression of inhibitory receptor PD-1, whose mRNA abundance was increased by microarray. Data are presented as the percentage T cells expressing the receptor and MFI. ∗, P = 0.047 and 0.001, respectively. (e and f) Decreased monocyte cell surface expression of costimulatory receptors, CD86 (∗, P = 0.009 for MFI), and HLA-DR as the percentage of monocytes (P = 0.009 expressing the receptor; P = 0.003 for MFI).