We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-B in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-B activation, confirming the central role of NF-B in contact allergen-induced keratinocyte activation

Study on the inflammasome NLRP3 and BLIMP-1/nlrp12 after keratinocyte exposure to contact allergens / V. Galbiati, L. Cornaghi, A. Papale, E. Donetti, M. Marinovich, E. Corsini. - In: TOXICOLOGY LETTERS. - ISSN 0378-4274. - 313(2019 Oct 01), pp. 130-136. [10.1016/j.toxlet.2019.07.003]

Study on the inflammasome NLRP3 and BLIMP-1/nlrp12 after keratinocyte exposure to contact allergens

V. Galbiati
Primo
Membro del Collaboration Group
;
L. Cornaghi
Secondo
Membro del Collaboration Group
;
A. Papale
Membro del Collaboration Group
;
E. Donetti
Membro del Collaboration Group
;
M. Marinovich
Penultimo
Membro del Collaboration Group
;
E. Corsini
Ultimo
2019

Abstract

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-B in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-B activation, confirming the central role of NF-B in contact allergen-induced keratinocyte activation
keratinocytes, contact allergen, allergen potency, NLRP3 inflammasome, NF-B, in vitro
Settore BIO/14 - Farmacologia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/654175
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