Iterative Modeling Reveals Evidence of Sequential Transcriptional Control Mechanisms.

Combinatorial control of gene expression is presumed to be mediated by molecular interactions between coincident transcription factors (TFs). While information on the genome-wide locations of TFs is available, the genes they regulate and whether they function combinatorially often remain open questions. Here, we developed a mechanistic, rather than statistical, modeling approach to elucidate TF control logic from gene expression data. Applying this approach to hundreds of genes in 85 datasets measuring the transcriptional responses of murine fibroblasts and macrophages to cytokines and pathogens, we found that stimulus-responsive TFs generally function sequentially in logical OR gates or singly. Logical AND gates were found between NF-κB-responsive mRNA synthesis and MAPKp38-responsive control of mRNA half-life, but not between temporally coincident TFs. Our analyses identified the functional target genes of each of the pathogen-responsive TFs and prompt a revision of the conceptual underpinnings of combinatorial control of gene expression to include sequentially acting molecular mechanisms that govern mRNA synthesis and decay.

Ex vivo innate immune cytokine signature of enhanced risk of relapsing brucellosis.

BACKGROUND: Brucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5-40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis. METHODOLOGY/PRINCIPAL FINDINGS: Blood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM) isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively. Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1ß, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1ß, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM. CONCLUSIONS/SIGNIFICANCE: This work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may allow for better follow-up care of brucellosis patients through improved identification of patients at risk for relapse.

The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50.

The specific binding of transcription factors to cognate sequence elements is thought to be critical for the generation of specific gene expression programs. Members of the nuclear factor κB (NF-κB) and interferon (IFN) regulatory factor (IRF) transcription factor families bind to the κB site and the IFN response element (IRE), respectively, of target genes, and they are activated in macrophages after exposure to pathogens. However, how these factors produce pathogen-specific inflammatory and immune responses remains poorly understood. Combining top-down and bottom-up systems biology approaches, we have identified the NF-κB p50 homodimer as a regulator of IRF responses. Unbiased genome-wide expression and biochemical and structural analyses revealed that the p50 homodimer repressed a subset of IFN-inducible genes through a previously uncharacterized subclass of guanine-rich IRE (G-IRE) sequences. Mathematical modeling predicted that the p50 homodimer might enforce the stimulus specificity of composite promoters. Indeed, the production of the antiviral regulator IFN-ß was rendered stimulus-specific by the binding of the p50 homodimer to the G-IRE-containing IFNß enhancer to suppress cytotoxic IFN signaling. Specifically, a deficiency in p50 resulted in the inappropriate production of IFN-ß in response to bacterial DNA sensed by Toll-like receptor 9. This role for the NF-κB p50 homodimer in enforcing the specificity of the cellular response to pathogens by binding to a subset of IRE sequences alters our understanding of how the NF-κB and IRF signaling systems cooperate to regulate antimicrobial immunity.