Abstract
CREB and C/EBP β signaling pathways are modulated during inflammation and also targeted by Bacillus anthracis edema toxin (ET), but how these factors individually and jointly contribute to changes in immune cell function is poorly understood. Using CRISPR/Cas9 gene editing, macrophage cell lines lacking CREB and isoforms of C/EBP β were generated and analyzed for changes in responses to LPS, ET, and IL-4. Macrophages lacking C/EBP β suppressed induction of IL-10 and Arg1, while IL-6 was increased in these cells following exposure to LPS. Examination of C/EBP β isoforms indicated the 38 kDa isoform was necessary for the expression of IL-10 and Arg1. ChIP-Seq analysis of CREB and C/EBP β binding to targets on the chromosome of human PBMC identified several regions where both factors overlapped in their binding, suggesting similar gene targeting or cooperative effects. Based on the ChIP-Seq data, a panel of previously unknown targets of CREB and C/EBP β was identified and includes genes such as VNN2, GINS4, CTNNBL1, and SULF2. Isoforms of a transcriptional corepressor, transducin-like enhancer of Split (TLE), were also found to have CREB and C/EBP β binding their promoter and were up regulated by ET. Finally, we explore a possible layer of C/EBP β regulation by a protein complex consisting of adenomatous polyposis coli (APC) and PKA. Collectively, these data provide new insights into the role of CREB and C/EBP β as immunosignaling regulators and targets of an important bacterial virulence factor.
Highlights
All 3 isoforms of C/EBP β were undetectable in macrophages subjected to CRISPR/Cas9-mediated editing of the C/EBP β-encoding gene (Fig. 1C)
RAWΔCREB cells were examined for changes in levels of C/EBP β, and RAWΔtotal C/EBPβ cells were examined for changes in levels of CREB
While much is known about how B. anthracis as well as other pathogens elevate cAMP within host cells[2], less is known about how cAMP reprograms cell such as macrophages
Summary
Panel (B) Immunoblot analysis of C/EBP β and GAPDH expression in RAWΔCREB cells and control cells after exposure to ET (10 nM of EF and 10 nM of PA) for 6 h. Panel (C) RAWΔtotal C/EBPβ cells lack C/EBP β expression as shown by immunoblot analysis of CREB, C/EBP β, and GAPDH. Whether ET exploits physiologically relevant signaling events to promote immunosuppression or if ET causes aberrant signaling events leading to cellular dysfunction is not known This stems in part from an incomplete understanding of the roles CREB and C/EBP β play in modulating immune responses. To this end, in the current study we took a multi-pronged approach to assess the general characteristics of activation of these pathways in response to both inflammatory stimuli and ET.
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