Effects of human RelA transgene on murine macrophage inflammatory responses

The NF?B family of transcription factors is a major regulator of the innate immune responses, and its dysregulation has been linked to several inflammatory diseases. In this study we focused on bone marrow derived macrophages from the recently described p65-DsRed/I?Ba-eGFP transgenic strain, in which a human copy of RelA (p65) was introduced into the mouse genome. Confocal imaging analysis showed that the human RelA is expressed these cells and can translocate to the nucleus upon Toll-like receptor 4 activation (TLR4). RNA sequencing analysis of polysaccharide-stimulated macrophage cultures, revealed that the extra copy of human RelA impacts on gene transcription, affecting both NF?B and non-NF?B target genes, including immediate-early and late response NF?B target genes, such as Fos and Cxcl10, respectively. In validation experiments on NF?B targets we observed reduced mRNA levels, but similar expression kinetic profile of the transgenic cells compared to the wild type. Enrichment pathway analysis based on the differentially regulated genes revealed that interferon and cytokine signaling, are affected downstream TLR4. These immune response pathways were also affected when the macrophages were treated with tumor necrosis factor. The impact of genetic manipulation on cell-specific functions is particularly important and highlights the need of understanding the molecular basis on which complicated in vitro and in vivo experiments will be designed on.