Eczema relevant inflammation in primary human keratinocytes
In this open innovation assay primary human keratinocyte are stimulated using an eczema-specific cytokine cocktail which induce an inflammatory response measured as an increase in CCL5 secretion.
Eczema, or Atopic dermatitis, is a common skin disease affecting about of 30% of children and 10% of adults in industrialized countries. Clinical symptoms include itchy plaques often affecting the face, head and limbs with significant negative impact on the quality of life (1). A typical aspect of eczema is the chronic and relapsing form of skin inflammation, a disease mechanism addressed by the described assay. The specific cytokine environment in the skin plays an important role in both induction and maintenance of chronic skin inflammation. In particular e.g. IL-4 and IL-13 are both critical and specific for the induction of an eczema phenotype in human skin and keratinocytes (2).
Figure. IL-4, IL-13, IL-22 and IFN-gamma is used to induce an eczema-like inflammatory response in primary human keratinocytes. The test compound's ability to inhibit the induced CCL5 release is measured as well as the effect on the cell viability.
Assay protocol outline
Primary human keratinocytes are seeded in 384-well plates (5000 cells/well), and stimulated with a cocktail of eczema-relevant cytokines (IL-4, IL-13, IL-22, IFN-gamma) for two days. The inflammatory response is measured by CCL5 release and compounds are tested for ability to inhibit the inflammatory response. Cell viability is used as counter readout to determine if CCL5 inhibition is due to a specific pathway or due to a general cytotoxic effect.
The curve to the left in figure illustrates the potent effect of the steroid Dexamethasone inhibiting the detected levels of CCL5 by approximately 80% efficacy (Emax). Calculated relative EC50 (Effective Concentration at 50% effect) is 6 nM. A toxic compound will reduce cell viability while a specific CCL5 release inhibitor might not. In general it is preferred to have a potent and efficacious compound which reduced the CCL% levels without effect on the cell viability. The EC50 and Emax values are calculated using a 4-parameter sigmoidal curve fitting algorithm. Y-axis shows effect of CCL% release inhibition in percent (%), where 100% represent a full reduction of CCL5 and 0% has no effect on the CCL5 release respectively.
- Bieber et al. Atopic dermatitis. N Engl J Med. 2008 Apr 3;358(14):1483-94. PubMed PMID: 18385500.
- Bernard et al. Keratinocytes under Fire of Proinflammatory Cytokines: Bona Fide Innate Immune Cells Involved in the Physiopathology of Chronic Atopic Dermatitis and Psoriasis. J Allergy (Cairo). 2012;2012:718725. PubMed PMID: 23193414.