Variegate porphyria, caused by monoallelic mutations in protoporphyrinogen oxidase (PPOX), causes acute visceral attacks and skin photosensitivity, presenting in blistering and skin fragility, a more severe phenotype is seen with rare biallelic mutations. This fragility is due to accumulation of intermediate porphyrins from the liver, such as 5’-aminolevulenic acid (ALA), close to the skin surface that are oxidised by sunlight. Oxidised porphyrins cause oxidative stress and degranulation of mast cells resulting in the release of proteases that cause blistering. We hypothesise that PPOX deficiency causes intrinsic pathological changes to keratinocytes that contribute to the skin phenotype.

To test this, we created two PPOX shRNA knockdown cell lines, KD1 and KD2 (50 & 25% residual expression respectively). Both KD lines displayed significantly reduced proliferation with KD2 having reduced migratory capacity. KD clones expressed reduced levels of involucrin in monolayer, proportional to the amount of knockdown, suggesting decreased differentiation. To mimic porphyrin accumulation keratinocytes were treated with ALA and deferoxamine (an iron chelator), resulting in an increase in fluorescent porphyrins in KD2. Treatment of keratinocytes to induce porphyrin accumulation caused intracellular oxidative stress, evidenced by reduced expression of 4 of the 5 electron transport chain complexes, in shC and both KDs. The differentiation phenotype was corroborated by 3D organotypic cultures where KD models expressed less keratin 10 and involucrin, proportional to knockdown, while KD2 models also displayed reduced stratification. Treatment of 3D organoids with ALA and DFO further reduced differentiation and stratification in KD models compared to shC.

In conclusion, we show that keratinocytes have an active haem synthetic pathway and contribute to porphyria pathology with >50% PPOX KD causing significant changes in keratinocyte proliferation, differentiation, and epidermal stratification, with >75% KD also affecting migration and reducing epidermal thickness, which could facilitate the oxidation of porphyrins under the skin. The more severe effects seen with >75% KD reflects the phenotype seen in individuals with biallelic mutations in PPOX. These 3D organotypics could therefore serve as models to trial therapeutic interventions to improve patient skin care.