Functional Transcriptomic Studies of Immune Responses and Endotoxin Tolerance in Early Human Sepsis

Abstract

Background: Limited studies have functionally evaluated the heterogeneity in early ex vivo immune responses during sepsis. Our aim was to characterize early sepsis ex vivo functional immune response heterogeneity by studying whole blood endotoxin responses and derive a transcriptional metric of ex vivo endotoxin response.

Methods: Blood collected within 24 h of hospital presentation from 40 septic patients was divided into two fractions and incubated with media (unstimulated) or endotoxin. Supernatants and cells were isolated, and responses measured using: supernatant cytokines, lung endothelial permeability after supernatant exposure, and RNA expression. A transcriptomic signature was derived in unstimulated cells to predict the ex vivo endotoxin response. The signature was tested in a separate cohort of 191 septic patients to evaluate for association with clinical outcome. Plasma biomarkers were quantified to measure in vivo host inflammation.

Results: Ex vivo response to endotoxin varied and was unrelated to immunosuppression, white blood cell count, or the causative pathogen. Thirty-five percent of patients demonstrated a minimal response to endotoxin, suggesting early immunosuppression. High ex vivo cytokine production by stimulated blood cells correlated with increased in vitro pulmonary endothelial cell permeability and was associated with attenuated in vivo host inflammation. A four-gene signature of endotoxin response detectable without the need for a functional assay was identified. When tested in a separate cohort of septic patients, its expression was inversely associated with hospital mortality.

Conclusions: An attenuated ex vivo endotoxin response in early sepsis is associated with greater host in vivo inflammation and a worse clinical outcome.

Figure 1.

Distribution of the fold change in cell culture supernatant cytokines after ex vivo endotoxin stimulation in (A) patients with early sepsis and in (B) healthy controls.

Figure 2.

Heterogeneity in endotoxin response measured by pulmonary endothelial cell permeability. (A) Electric cell-substrate impedance sensing (ECIS) tracings of in vitro pulmonary endothelial cell permeability after exposure to culture supernatants from ex vivo endotoxin-stimulated whole blood of patients with sepsis. *A decrease in resistance across the Human Pulmonary Microvascular Endothelial Cell (HPMEC) monolayer suggested increased permeability and the absolute area under the curve during the 5-hours of ECIS measurements was subsequently used to represent endothelial permeability, as described previously. Marker color represents the magnitude of in vitro endothelial permeability (green: minimal, blue: intermediate, red: maximal). (B) Relationship between fold change in culture supernatant TNFα and in vitro pulmonary endothelial permeability.

Figure 3.

Heterogeneity in endotoxin response: gene expression. (A) Multidimensional-scaling (MDS) clusters samples by sepsis status (dimension 1) and by endotoxin stimulation (dimension 2). * MDS dimension 2 can be interpreted as a measure of the endotoxin response, with positive values corresponding to minimal endotoxin responses and negative values corresponding to maximal endotoxin responses. LPS stimulated FALSE and TRUE refer to unstimulated and stimulated experimental conditions. Within endotoxin-stimulated cells from patients with sepsis, more negative values along dimension 2 correspond to higher in vitro pulmonary endothelial permeability. (B) Differential gene expression among unstimulated and endotoxin-stimulated cells from patients with sepsis and from healthy controls, *4594 differentially expressed genes (1920 genes upregulated, 2674 genes downregulated) (C) Heatmap of the union of top 30 differentially expressed genes related to endotoxin stimulation, *Each column represents an individual sample. Classification by LPS stimulation, blue: stimulated, purple: unstimulated. Classification by sepsis status green: healthy control, orange: sepsis. (D) Heatmap of most significant genes associated with in vitro pulmonary endothelial permeability in endotoxin-stimulated cells from patients with sepsis. *Each column represents an individual patient. (E) Heatmap of most significant genes associated with in vitro pulmonary endothelial permeability in endotoxin-stimulated cells from healthy controls. *Each column represents an individual sample.

Figure 3.

Heterogeneity in endotoxin response: gene expression. (A) Multidimensional-scaling (MDS) clusters samples by sepsis status (dimension 1) and by endotoxin stimulation (dimension 2). * MDS dimension 2 can be interpreted as a measure of the endotoxin response, with positive values corresponding to minimal endotoxin responses and negative values corresponding to maximal endotoxin responses. LPS stimulated FALSE and TRUE refer to unstimulated and stimulated experimental conditions. Within endotoxin-stimulated cells from patients with sepsis, more negative values along dimension 2 correspond to higher in vitro pulmonary endothelial permeability. (B) Differential gene expression among unstimulated and endotoxin-stimulated cells from patients with sepsis and from healthy controls, *4594 differentially expressed genes (1920 genes upregulated, 2674 genes downregulated) (C) Heatmap of the union of top 30 differentially expressed genes related to endotoxin stimulation, *Each column represents an individual sample. Classification by LPS stimulation, blue: stimulated, purple: unstimulated. Classification by sepsis status green: healthy control, orange: sepsis. (D) Heatmap of most significant genes associated with in vitro pulmonary endothelial permeability in endotoxin-stimulated cells from patients with sepsis. *Each column represents an individual patient. (E) Heatmap of most significant genes associated with in vitro pulmonary endothelial permeability in endotoxin-stimulated cells from healthy controls. *Each column represents an individual sample.

Figure 4.

Association between response to endotoxin and gene expression. (A) Pairwise scatter plots demonstrating a linear correlation between complimentary metrics of ex vivo response to endotoxin. *R² values in the lower left panels and are significant at p <1x10⁻⁹ for every pair of variables, based on linear regression. MDS Dim2 refers to Multidimensional-scaling dimension 2 (Figure 3A). Within endotoxin-stimulated cells from patients with sepsis, more negative values along dimension 2 correspond to higher in vitro pulmonary endothelial permeability. (B) Differential gene expression for response to endotoxin. *Response to endotoxin was measured by pulmonary endothelial cell permeability. 2720 significant genes were differentially expressed in stimulated cells from patients with sepsis (1328 upregulated, 1392 downregulated), 47 significant genes in stimulated cells from healthy controls (0 upregulated, 47 downregulated), 4 significant genes in unstimulated cells from patients with sepsis (4 upregulated, 0 downregulated), and no significant genes in unstimulated cells from healthy controls.

Figure 5.

Relationship between the 4 gene signature associated with the endotoxin response and hospital mortality in an external validation cohort of 191 patients with early sepsis. *The four genes included: HLA-DRA, HLA-DPA1, HLA-DPB1, and FUCA1. Values on Y axis represent combined metric for total expression of the four genes (sum of normalized gene expression for each gene). Host gene counts were normalized using DESeq2. All samples included in the analysis had >1 million host gene counts and expression of >10,000 unique genes.

Figure 6.

Association between host inflammatory state (plasma CXCL9/MIG) and ex vivo response to endotoxin measured by (A) the fold change in TNFα in cell culture supernatant, (B) in vitro pulmonary endothelial permeability, and (C) the summary statistic S for the RNAseq endotoxin response size. *Spearman rank correlation was used to compute p-values and included a Bonferroni correction for multiple testing.