Background/Purpose: Through producing inflammatory cytokines and chemokines, keratinocytes play an important role along with hematopoietic cells in mediating an inflammatory response in psoriasis and other inflammatory skin conditions. Keratinocyte-produced cytokines include IL-1a, IL-6, IL-8, IL-23, TNF, IL- 36α, β, and γ, etc. Recently, autosomal dominant mutations in CARD14  have been shown to cause a disease severity spectrum of plaque psoriasis, pustular psoriasis and pityriasis rubra piliaris. CARD14 is mainly expressed in the keratinocytes in skin. As blockade of IL-12/23 has been very successful in inhibiting skin inflammation in psoriasis and in our pediatric patient with CAMPS, and more recently blockade of IL-17A/F in psoriasis, we hypothesize that keratinocyte activation may be the primary trigger of hematopoietic cell recruitment into the skin and that therapy with IL-12/23 inhibitors and more recently with IL-17 inhibitors may either block keratinocytes directly or through the prevention of IL-17 production, and that inhibition of keratinocyte activation might significantly contribute to the treatment success with these agents in controlling skin inflammation.

Methods: We generated primary and immortalized keratinocyte cell lines from a patient with CAMPS, a patient with CARD14 negative plaque psoriasis and 3 foreskin samples. We examined the immune function of keratinocytes in co-stimulation assays with a panel of TLR agonists and pro inflammatory cytokines, measuring IL-8 in cell culture supernatant as initial readout for keratinocyte activation. We assayed cytokine receptor expression on keratinocytes. In some experiments gene expression of other cytokines (CCL20, IL-6 and S100A7 (psoriasin)) were assayed by qRT-PCR. Statistical analysis was performed using paired/unpaired t-tests.

Results: Keratinocytes can be activated by the agonists through TLR3, Poly IC; TLR5, Flagellin; and TLR9, Type B CpG oligonucleotides to produce IL-8. Keratinocytes have a number of cytokine receptors, such as IL-1R, IL-6R, IL-17R, IL-23R, IL-36R, TNF-R, etc, and get stimulated through IL-17A, IL-1a/b, and less through TNF, IL-36γ. However, they do not get stimulated through IL-12 or IL-23. IL17A synergizes with Poly IC, Flagellin, and also with TNF but not with IL-36γ, IL-12 or IL-23. Based on all TLRs and cytokine stimulation at 24hr, the keratinocytes from a patient with the CARD14 mutation have higher IL-8 production than control cells and keratinocytes from a patient with severe plaque psoriasis. Expression studies show that under co-stimulation of TNF and IL-17A, mRNA expression of CCL20 and IL-6 occurs early at 4hr, but S100A7 is maximally expressed later at 24hr.

Conclusion: TLRs stimulating through TLRs 3, 5 and 9 and some hematopoietic-produced cytokines particularly IL-17 have direct synergic effects on keratinocyte activation. CARD14 mutant keratinocytes are more sensitive to these positive stimulants than keratinocytes from a patient with regular psoriasis and health controls. Our data suggest that CARD14 mutations sensitize keratinocytes to heightened responses to TLR agonists and co stimulation with other inflammatory cytokines and is likely the crucial cytokine in promoting skin inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-activating-effects-of-co-stimulation-of-tlr-agonists-and-cytokines-on-primary-and-immortalized-keratinocytes-from-a-patient-with-a-card14-mediated-pustular-psoriasis-camps-and-healthy-co/