A team at the Univ of Cologne, led by Manolis Pasparakis, sheds new light on the complexity of RIPK1 and its downstream regulation of cell death and inflammation. This is shown in the context of dermal inflammation and the dysregulatory effects of mutations at the sites of autophosphorylation and how these sites contribute to the pathogenesis of inflammatory disease.
RIPK1 has been shown to be a key regulator of cell death and inflammation but with little knowledge of downstream effects of mutations on its function. Dysregulation of RIPK1 has been implicated in the pathology of inflammatory disease when the discrete control of its phosphorylation is lost, making it a useful target for therapeutic inhibition. The key activation is autophosphorylation, dependent upon two sites, to drive downstream signalling towards apoptosis and necroptosis and ultimately to inflammation. Different mutations enforced at the phosphorylation sites impose a variety of noticeable changes in the downstream outcomes, for example favouring necroptosis over apoptosis.
The work explored in vivo in mice the impact of mutations on keratinocyte biology and resulting skin lesions, examined by histology and for gene expression patterns. Immunoblotting studies validated the use of cell death to be a direct correlate for the changes imposed by the mutations to the phosphorylation sites. From this, extensive in vitro cell-based assays were performed for cell death under different mutations and drug regimes.
In these latter assays performed on the Incucyte S3 live-cell analysis system (Essen Bioscience), the far-red fluorescing cell-impermeant DNA-binding viability dye DRAQ7 was used to dynamically report cell death. Elegantly, the related cell-permeant DRAQ5 was used in replicate wells to give a robust control baseline for the total number of cells and therefore to allow cell death to be reported as a percentage of the total. This means that the readouts for total cells and cell death is from the same fluorescence channel, simplifying instrument set-up while providing capacity for further chromophores, were these deemed necessary. In this context one might suggest a reporter of caspase activity and/or mitochondrial health to further unpick the cell death mechanism.
Koerner, Lioba, et al. "RIPK1 autophosphorylation at S161 mediates cell death and inflammation." Journal of Experimental Medicine 222.12 (2025): e20250279.
