Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001). Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity. The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the ‘universe’ of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity. Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equivalent potency assay with the release of IL-18 lead to the development of an in vitro model able to identify contact allergens and rank them according to their potency. One other objective of this thesis was to study the signal transduction pathways involved in PPD, DNCB and citral-induced IL-18. For such purpose several inhibitors were used. To investigate the intracellular source of ROS, specific inhibitors of the three main cellular sources of ROS, namely DPI, a NADPH synthetase inhibitor; rotenone, a mitochondrial electron transport inhibitor; allopurinol, a xanthine oxidase inhibitor, were used. Z-VAD-FMK, a cell-permeant pan caspase inhibitor, that irreversible binds to the catalytic site of caspases, and a neutralizing anti-TLR4 antibody were used to investigate the role of the inflammasome and TLR4. Glycirrizic acid, a direct inhibitor of HMGB1 protein, was used to establish the role of HMGB1 as possible DAMP associated with allergen-induced IL-18. To specifically investigate the signal transduction pathway involved in PPD-induced IL-18 production selective inhibitors were used: GF109203X to inhibit PKC, PDTC and Bay 11-70-85 to inhibit NF-κB, and SB203580, as p38 MAPK inhibitor. The results obtained during this three year of research activity have clearly shown that the in vitro methods based on NCTC2544 and IL-18 production are able to discriminate contact/photocontact allergens from irritants/photoirritants and respiratory allergens. Furthermore, the combined use of the epidermis in vitro model with the IL-18 production, beside the ability to identify sensitising compounds, is able to rank them according to their potency. With respect to the molecular mechanisms behind skin sensitization I could demonstrate that different intracellular sources of ROS are triggered by different contact allergens. Allergens-induced IL-18 production is dependent upon NF-κB and p38 MAPK activation and TLR4 and inflammasome activation. Among the DAMPs, the evolutionarily conserved non-hystone chromatin-binding protein HMGB1 is released into the extracellular space following exposure to contact allergens, resulting in TLR4 activation and IL-18 neosynthesis. Even if more studies are necessary to elucidate the mechanisms that are involved in chemical allergens-induced oxidative stress, the signalling pathways activated and their role in contact allergy, data clearly indicated a pivotal role of ROS in chemical allergy. Consequently, the redox state of the cell becomes imbalanced, with the activation of several pathways, including MAPK, such as SAPK/JNK, ERK1/2 and p38, NF-κB, Akt/ASK1 or Keap1/Nrf2 pathways, resulting in a inflammatory and cytotoxic response with the production of costimulatory molecules, cytokines, chemokines, and phase 1 detoxifying enzymes. On the basis of the results obtained, the following scenario could be imagined: chemical sensitisers can induce oxidative stress owing to their elecrophilicity, which in turn activates the inflammasome and HMGB1 release (and possible other DAMPs), which can activate TLR4. Activation of TLR4 will results in NF-κB and p38 MAPK activation and in the neosynthesis of IL-18.

ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH / V. Galbiati ; tutor: C.L. Galli ; correlatore: A. Panerai. - : . Università degli Studi di Milano, 2014 Dec 15. ((27. ciclo, Anno Accademico 2014. [10.13130/galbiati-valentina_phd2014-12-15].

ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH

V. Galbiati
2014-12-15

Abstract

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001). Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity. The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the ‘universe’ of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity. Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equivalent potency assay with the release of IL-18 lead to the development of an in vitro model able to identify contact allergens and rank them according to their potency. One other objective of this thesis was to study the signal transduction pathways involved in PPD, DNCB and citral-induced IL-18. For such purpose several inhibitors were used. To investigate the intracellular source of ROS, specific inhibitors of the three main cellular sources of ROS, namely DPI, a NADPH synthetase inhibitor; rotenone, a mitochondrial electron transport inhibitor; allopurinol, a xanthine oxidase inhibitor, were used. Z-VAD-FMK, a cell-permeant pan caspase inhibitor, that irreversible binds to the catalytic site of caspases, and a neutralizing anti-TLR4 antibody were used to investigate the role of the inflammasome and TLR4. Glycirrizic acid, a direct inhibitor of HMGB1 protein, was used to establish the role of HMGB1 as possible DAMP associated with allergen-induced IL-18. To specifically investigate the signal transduction pathway involved in PPD-induced IL-18 production selective inhibitors were used: GF109203X to inhibit PKC, PDTC and Bay 11-70-85 to inhibit NF-κB, and SB203580, as p38 MAPK inhibitor. The results obtained during this three year of research activity have clearly shown that the in vitro methods based on NCTC2544 and IL-18 production are able to discriminate contact/photocontact allergens from irritants/photoirritants and respiratory allergens. Furthermore, the combined use of the epidermis in vitro model with the IL-18 production, beside the ability to identify sensitising compounds, is able to rank them according to their potency. With respect to the molecular mechanisms behind skin sensitization I could demonstrate that different intracellular sources of ROS are triggered by different contact allergens. Allergens-induced IL-18 production is dependent upon NF-κB and p38 MAPK activation and TLR4 and inflammasome activation. Among the DAMPs, the evolutionarily conserved non-hystone chromatin-binding protein HMGB1 is released into the extracellular space following exposure to contact allergens, resulting in TLR4 activation and IL-18 neosynthesis. Even if more studies are necessary to elucidate the mechanisms that are involved in chemical allergens-induced oxidative stress, the signalling pathways activated and their role in contact allergy, data clearly indicated a pivotal role of ROS in chemical allergy. Consequently, the redox state of the cell becomes imbalanced, with the activation of several pathways, including MAPK, such as SAPK/JNK, ERK1/2 and p38, NF-κB, Akt/ASK1 or Keap1/Nrf2 pathways, resulting in a inflammatory and cytotoxic response with the production of costimulatory molecules, cytokines, chemokines, and phase 1 detoxifying enzymes. On the basis of the results obtained, the following scenario could be imagined: chemical sensitisers can induce oxidative stress owing to their elecrophilicity, which in turn activates the inflammasome and HMGB1 release (and possible other DAMPs), which can activate TLR4. Activation of TLR4 will results in NF-κB and p38 MAPK activation and in the neosynthesis of IL-18.
CORSINI, EMANUELA
Settore BIO/14 - Farmacologia
ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH / V. Galbiati ; tutor: C.L. Galli ; correlatore: A. Panerai. - : . Università degli Studi di Milano, 2014 Dec 15. ((27. ciclo, Anno Accademico 2014. [10.13130/galbiati-valentina_phd2014-12-15].
Doctoral Thesis
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