Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses

Abstract

Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes.

Deconvolution of the Response to Bacillus Calmette–Guérin Reveals NF-κB-Induced Cytokines As Autocrine Mediators of Innate Immunity

“…Additionally, our recent work has highlighted that in conditions of limiting dose of microbial stimulus, cytokines participate in the spreading of the inflammatory signal. Moreover, they report that the core cytokine-induced signatures are capable of capturing the variable response to stimulation by complex microorganisms (40). Together, our strategy enabled detailed analysis of the response to BCG with the demonstration that a diverse set of immune cells underlay what is a plastic and flexible inflammatory response.…”

Milieu intérieur: Defining the boundaries of a healthy immune response for improved vaccination strategies

“…An additional advantage of TruCulture is its adaptability to diverse immune stimuli and that the biological readout can be modified to monitor alterations in cellular phenotype, 12 induced protein secretion, 12 RNA expression changes, 13 and metabolic or lipidomic functional changes. Transcriptional analysis of stimulated whole blood can be challenging due to multiple technical reasons, but we have developed a high-throughput single-step RNA extraction method from stimulated TruCulture samples allied to a Nanostring hybridization array readout.…”

“…Transcriptional analysis of stimulated whole blood can be challenging due to multiple technical reasons, but we have developed a high-throughput single-step RNA extraction method from stimulated TruCulture samples allied to a Nanostring hybridization array readout. 13 Utilizing the diverse stimulation conditions applied to the Milieu Interieur cohort, we tested the hypothesis that the transcriptional signature of key effector cytokines can capture the responses to Toll-like Receptor (TLR) ligands or microbes. To do this we analyzed the expression of 572 genes in 25 healthy donors following stimulation with 4 key innate cytokines (IFN-β, IFN-γ, IL-1β, and TNF-α) applying unsupervised principal component analysis (PCA) and a linear support vector machine (SVM) algorithm.…”

“…To do this we analyzed the expression of 572 genes in 25 healthy donors following stimulation with 4 key innate cytokines (IFN-β, IFN-γ, IL-1β, and TNF-α) applying unsupervised principal component analysis (PCA) and a linear support vector machine (SVM) algorithm. 13 This allowed the identification of a 44-gene signature that captured the diversity of complex innate immune responses with improved segregation between distinct TLR and microbial stimuli. Furthermore, we can now use this approach to assess how potential vaccine adjuvants such as TLR ligands induce specific cytokine associated gene expression (Fig.…”

Clinical and multi-omics cross-phenotyping of patients with autoimmune and autoinflammatory diseases: the observational TRANSIMMUNOM protocol

“…Using this approach, the authors identified gene expression signatures that could predict the humoral immune response to the yellow fever vaccine. Similarly, several international projects, such as the Human Immunology Project consortium8 funded by National Institute of Health and the Health Human Project emanating from the Milieu Intérieur Consortium,9 have already revealed the power of multiparametric systems immunology studies 10…”