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Effects of Human RelA Transgene on Murine Macrophage Inflammatory Responses
Stamatia Papoutsopoulou1,2, Lorna Morris3, Andrew Bayliff4
1Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK.
Abstract:
The NFκB transcription factors are major regulators of innate immune responses, and NFκB signal pathway dysregulation is linked to inflammatory disease. Here, we utilised bone marrow-derived macrophages from the p65-DsRedxp/IκBα-eGFP transgenic strain to study the functional implication of xenogeneic (human) RelA(p65) protein introduced into the mouse genome. Confocal imaging showed that human RelA is expressed in the cells and can translocate to the nucleus following activation of Toll-like receptor 4. RNA sequencing of lipid A-stimulated macrophages, revealed that human RelA impacts on murine gene transcription, affecting both non-NFκB and NFκB target genes, including immediate-early and late response genes, e.g., Fos and Cxcl10. Validation experiments on NFκB targets revealed markedly reduced mRNA levels, but similar kinetic profiles in transgenic cells compared to wild-type. Enrichment pathway analysis of differentially expressed genes revealed interferon and cytokine signaling were affected. These immune response pathways were also affected in macrophages treated with tumor necrosis factor. Data suggests that the presence of xenogeneic RelA protein likely has inhibitory activity, altering specific transcriptional profiles of key molecules involved in immune responses. It is therefore essential that this information be taken into consideration when designing and interpreting future experiments using this transgenic strain.
Insights
Introducing human RelA(p65) into mouse macrophages alters immune gene expression, suggesting inhibitory effects on innate immune responses. This finding is crucial for interpreting experiments with this transgenic model.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Nuclear factor kappa B (NFκB) transcription factors regulate innate immunity.
- Dysregulation of the NFκB pathway is associated with inflammatory diseases.
- Xenogeneic RelA (p65) protein was introduced into the mouse genome for study.
Purpose of the Study:
- To investigate the functional implications of xenogeneic (human) RelA(p65) in mouse macrophages.
- To analyze the impact of human RelA on murine gene transcription and immune signaling pathways.
Main Methods:
- Utilized bone marrow-derived macrophages from a p65-DsRedxp/IκBα-eGFP transgenic mouse strain.
- Employed confocal imaging to track human RelA translocation.
- Performed RNA sequencing on lipid A-stimulated macrophages.
- Conducted validation experiments and enrichment pathway analysis.
Main Results:
- Human RelA expressed and translocated to the nucleus in response to Toll-like receptor 4 activation.
- Human RelA impacted murine gene transcription, affecting both NFκB and non-NFκB targets.
- Transgenic cells showed reduced mRNA levels for NFκB targets but similar kinetics compared to wild-type.
- Interferon and cytokine signaling pathways were significantly affected.
Conclusions:
- Xenogeneic RelA protein exhibits inhibitory activity, altering transcriptional profiles in immune response genes.
- The presence of human RelA modifies key molecular players in immune responses.
- Experimental designs and interpretations using this transgenic strain must consider these findings.
