Covalent Binding, Protein, leading to Increase, Allergic Respiratory Hypersensitivity Response

Jessica Ponder, Physicians Committee for Responsible Medicine Kristie Sullivan, Institute for In Vitro Sciences

Stella Cochrane, Unilever

Steven Enoch, Liverpool John Moores University

Janine Ezendam, RIVM

Joanna Matheson & Kent Carlson, US CPSC

Grace Patlewicz, US EPA

Erwin Roggen, 3RsMC ApS

Katherina Sewald, Fraunhofer ITEM

The assessment of xenobiotics for potential to induce an allergenic response in the respiratory tract is of great regulatory and industrial interest. Ongoing work in this area has hypothesized some differences between the dermal and respiratory sensitisation pathways; however in some cases a lack of strong empirical evidence on a variety of chemistries to test these hypothesis. This AOP represents the currently available data with the aim of identifying knowledge gaps which may be filled with directed research. (Sullivan, et al., 2017)

Sensitization of the respiratory tract is an important occupational health challenge. Here we build on a previously published skin sensitization AOP (AOP 40), relying on literature evidence linked to low-molecular-weight organic chemicals and excluding other known respiratory sensitizers acting via different molecular initiating events. The established key events (KEs) are as follows: (1) covalent binding of chemicals to proteins, (2) activation of cellular danger signals (inflammatory cytokines and chemokines and cytoprotective gene pathways), (3) dendritic cell activation and migration, (4) activation, proliferation, and polarization of T cells, and (5) sensitization of the respiratory tract. There is some evidence that respiratory sensitizers bind preferentially to lysine moieties, whereas skin sensitizers bind to both cysteine and lysine, however this observation may be biased by the limited number of chemicals investigated. Furthermore, exposure to respiratory sensitizers seems to result in cell behavior for KEs 2 and 3, as well as the effector T cell response, in general skewing toward cytokine secretions predominantly associated with T helper 2 (Th2) response.

Sensitization of the respiratory tract by chemicals is the first stage in the development of chemical respiratory allergy, an immune-mediated hypersensitivity reaction to an exogenous low-molecular-weight chemical, which can result in asthma and rhinitis on repeated exposure and is an important occupational health problem. (Mapp et al., 2005) Due to the severity and irreversibility of the adverse effect, identifying chemical respiratory allergens is of considerable regulatory, industrial, and socioeconomic importance. (Boverhof et al., 2008) Efforts to outline a framework for assessment of potential respiratory-sensitizing chemicals are underway. (North et al., 2016) Currently, however, there are no standardized, validated, and regulatory-accepted models for detecting these chemicals or discriminating them from skin sensitizers, potentially due to remaining gaps within the literature as to the exact mechanistic steps leading to respiratory allergy. (Kimber et al., 2011)

Another important issue in the development of predictive test methods is the route of exposure in the sensitization phase. Inhalation exposure is perhaps the most common exposure route of concern for many substances, but there is evidence that sensitization of the respiratory tract can be induced via skin exposure as well. (Tarlo and Malo, 2006, Heederik et al., 2012, Redlich and Herrick, 2008, Kimber and Dearman, 2002) This knowledge has implications for both the mechanistic understanding of the pathway and the potential test methods that may be used to detect respiratory sensitizers. Therefore, this AOP will include information from models using skin and lung exposure (in vivo) and with dermal and respiratory cells and tissues (in vitro/ex vivo).

The outlines of this pathway follow the already-published skin sensitization AOP 40. However, the divergent AOs of the two pathways reflect differences in the effector response (T helper 1 [Th1] vs. T helper 2 [Th2]) and other mechanistic details of at least some KEs;(Kimber et al., 2014) these differences are the focus of this effort. Therefore, the primary evidence relied on to build this AOP must relate directly to known low-molecular-weight organic chemicals to the exclusion of chemicals that act via other mechanisms and therefore require a separate AOP, for example, chloroplatinates.

In brief, the AOP can be summarized as beginning with covalent protein binding, potentially preferentially to lysine nucleophiles in the lung or skin after respiratory or dermal exposure to a low-molecular-weight organic chemical. This protein binding causes the activation of stress response pathways and cellular danger signals, including oxidative stress, cytokines, and chemokines released by epithelial and other cells, leading to dendritic cell (DC) maturation and migration to the draining lymph nodes (DLN). Haptens can also contribute to DC activation directly. Th2-skewed DCs in the DLN signal activation and maturation of T cells, which characterize the sensitization phase, resulting in chemical respiratory allergy. Consistent with regulatory practice, sensitization is considered the AO. (Vandebriel et al., 2011)

1. Concordance of dose-response relationships

There is fairly sparse evidence wth more than a few respiratory sensitizers that can offer confidence in dose-response concordance. In BALB/c mice, toluene diisocyanate (TDI) applied to the skin led to TDI-haptenated protein (TDI-hp) (skin keratins and albumin) localization in the stratum corneum, hair follicles, and sebaceous glands within 3 hours, with intensity of staining following a dose–response relationship. (Nayak et al., 2014) provides a detailed dose–response analysis of TDI-induced protein binding, colocalization of immune messenger cells, and migration to Delphian lymph nodes.

2. Temporal concordance among the key events and adverse effect;

(Nayak et al., 2014) also provides a detailed temporal analysis of TDI-induced protein binding, colocalization of immune messenger cells, and migration to Delphian lymph nodes.

3. Strength, consistency, and specificity of association of adverse effect and initiating event

There are clear connections from chemicals with certain reactivity and binding profiles to cellular- and individual-level downstream Th2-related effects leading to respiratory sensitization. Though the number of chemicals studied is quite low, consistent patterns are identified. A better understanding of how differences in haptenation by these chemicals contribute to distinct cellular and immune-system-level responses, and how early DC gene changes contribute (or not) to the expression of maturation markers, will help to increase the specificity of the available test methods.

4. Biological plausibility, coherence, and consistency of the experimental evidence

Each of the hypothesized KERs is supported by evidence from studies with at least one, and sometimes a few, known respiratory sensitizers. The events fit with what is known in general for sensitization, and the basic KEs outlined here are consistent with established biological knowledge. However, further research is needed to understand, for a larger number of chemicals, the steps leading to a skewing of the effector response toward Th2 and sensitization of the respiratory tract; therefore, the WoE is considered to be ‘‘moderate.’’

5. Alternative mechanisms

Currently, there are about 80 chemicals identified as respiratory allergens. Exposure occurs primarily in occupational settings. AOs are asthma and rhinitis. The biological mechanisms are often Th2 mediated leading to the production of IgE and eosinophilic inflammation. However, this may not always be the case. For example, human studies reveal PPD to be a respiratory sensitizer, (Helaskoski et al., 2014) but it does not cause a Th2 cytokine response in mice. (Rothe et al., 2011) Specific IgE is induced in some subjects, but not in others, particularly for diisocyanate sensitization. Thus, it is unclear whether IgE is mandatory or not.

Notably, it has to be mentioned that for protein-induced respiratory allergy, the clinical understanding of the disease has been changing dramatically during the last years. For many years, asthma has been considered as a single disease with a defined phenotype. It was assumed that the biology of sensitization is based on Th2-mediated IgE production, migration of mast cells, and subsequent eosinophilic infiltration. Nevertheless, clinical studies of cohort revealed that only about 50% of all patients show a Th2-driven eosinophilic inflammation of the airways. It also covers Th17-driven neutrophilic airway inflammation—an asthmatic phenotype that also can be observed with chemical allergens.

Nowadays, asthma is considered as an umbrella disease with multiple heterogeneous phenotypes, depending on the underlying immunology, pathology, symptoms, and the time of elicitation during lifetime. Furthermore, the concept takes other environmental and genetic influences into consideration. The development of animal models reflecting the heterogeneity of asthma phenotypes is still ongoing and shows in particular the (i) irritant properties of the allergen, (ii) the route of exposure during sensitization and elicitation, and (iii) the dose levels of allergen define whether a Th2 or Th17 phenotype develops.

For chemical allergens, less is known about the influence of atopy, viral infections, and indoor and outdoor environmental pollutants such as cigarette smoke. Of interest is the influence of an additional coexposure to irritant if the chemical allergen is present at low dose. Genetic susceptibility is also a variable of interest. (Yucesoy et al., 2012) and (Wisnewski et al., 2008) among others, have determined factors that may affect the potential for a person’s sensitization potential to diisocyanates, including genetic variants in antioxidant defense genes and PRRs.

A number of studies have looked into the sensitization of transition metal complexes, including one which outlines the evidence for these complexes initiating sensitization not through covalent bond formation, but rather through coordination complexes. (Chipinda et al., 2011) The authors provide evidence that these coordination complexes are not stable enough to survive the antigen processing that a covalent hapten undergoes. Instead an alternative MIE is outlined in which these complexes bind to cell surface proteins like MHC, bypassing the intracellular antigen process. This initiating event fits in with the observed cross-reactivity that appears to transcend the trends one would expect based on the periodic table (for example, complexes of Cr, a group 6 metal, cross sensitizing with complexes of Co, a group 9 metal). (Templeton, 2004) It is thought that the surface protein chelates the metal complex and presents it to T-cells directly, requiring a separate AOP from chemicals acting via covalent binding to proteins.

6. Uncertainties, inconsistencies and data gaps.

A better understanding of how differences in haptenation by these chemicals contribute to distinct cellular responses, and how early DC gene changes contribute (or not) to the expression of maturation markers, will help to increase the specificity of the available test methods. A better understanding of human response and population variability is also needed, along with a better quantitative understanding of the linkages between KEs. Additional studies using human cells and tissues are recommended.

Furthermore, as noted in the evaluation section, efforts to fully understand this pathway and develop toxicological test methods and strategies are hampered by a spare data portfolio, as well as a lack of a robust set of harmonized reference chemicals clearly identified as respiratory sensitizers. Previous authors have gathered preliminary chemical sets with supporting rationale, and collating this information and building a set of harmonized reference chemicals, which can be used to optimize and characterize potential test methods or strategies, are the clear next steps. (Enoch et al., 2010, Cochrane et al., 2015, Enoch et al., 2009)

The construction of an AOP for this endpoint would allow the following: (1) organization of available information to identify remaining uncertainties and prioritize further research, (2) highlighting of differences and similarities between skin and respiratory sensitization pathways, and (3) improvement of existing or identification of novel predictive models that, alone or in an integrated approach, could be used to identify respiratory sensitizers.

Given the available (WoE) outlined above, we propose that the AOP for sensitization of the respiratory tract outlined here allows the identification of gaps in knowledge, research needs, and potential test methods that may be developed further using a larger set of respiratory sensitizers.

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