Revealing the power of innate responses to discriminate sensitizers from irritants.

The innate immune response is often called for being ‘non-specific'. However, a lot of substance-specific information is lost when the ‘specific' adaptive immune response pulls off. Innate responses provide an amazingly detailed picture of the characteristics of the challenging substance. A better understanding of how the early gene changes in keratinocytes, epithelial cells and dendritic cells contribute to the expression of maturation markers may help to understand and resolve the gaps that are currently existing in our understanding of the early events in sensitization. The presented results may have implications for the design of predictive in vitro tests for assessment of sensitization potency, and suggest that -omic approaches contain valuable mechanistic information associated with potency assessment of chemicals.

Introduction

Our understanding of the molecular mechanisms driving chemical skin and respiratory sensitization has reached a level that many consider sufficient for the development of advanced tools for testing and even risk assessment. However, recent genomics and proteomics-based studies targeting early innate events in epithelial cells (including keratinocytes) and dendritic cells have revealed mechanisms that might help filling out the gaps in our understanding of the relation between reactivity rate, mechanism of haptenation, protein target selection, pathway activation and T-cell skewing (1).

From dendritic cell ‘sensing' to activation and maturation

It is generally accepted that activation of DCs results in prominent phenotypic and functional changes including enhanced levels of MHC class I and co-stimulatory molecules (e.g. cluster of differentiation (CD)54, CD80 and CD86, and receptors that are essential for migration) and antigen-presenting capacity. However, DCs have recently been shown to do a lot of sensing and tasting in order to match the right pathways for triggering these phenotypic and functional changes with the chemical challenge.

Preference of amino acid for haptenation. Extensive genomic analysis of monocyte-derived dendritic cells (Mo-DCs), human monocytic leukaemia cell line (THP-1) and MUTZ-3 cells exposed to skin sensitizers exerting cysteine and cysteine/lysine reactivity has identified genes describing the primary pathways of skin sensitization, i.e. signalling through transcription factors Nrf2 and aryl hydrocarbon receptor (AHR), and protein ubiquitination (2, 3). Lysine-reactive chemicals appeared to be less efficient (3). This difference in reactivity may explain the discrimination between small molecule skin and respiratory sensitizers.

Skin and respiratory sensitizers. Johansson et al. (2) published a list of 200 genes that with >95% accuracy (96% sensitivity; 95% specificity) describe skin, but not chemical respiratory, sensitizers (Fig. 1). The primary pathways involved in skin sensitization involved Nrf2, AHR, TLR and PKA signalling. In contrast, DC-based assays measuring phenotypic changes reveal 71-82% sensitivity, 70-75% specificity, and 71-84% accuracy.

Figure 1: 200 genes accurately (>95%) describe skin sensitizers. A. Principle Component Analysis (PCA) of the gene modifications triggered by 90 chemical substances. B. The stimulation index derived after application of the Genomic Allergen Rapide Detection (GARD) algorithm. 

Recently, Forreryd et al. (4) released a list of 389 differentially regulated genes for respiratory sensitizers. Several of these genes were involved in oxidative phosphorylation, ubiquinone metabolism and regulation of innate immune response signalling pathways leading to cell maturation, enhanced antigen presentation and interaction with other immune cells. Preliminary evidence is suggestion that a third gene signature is required to described allergenic proteins. Never the less, the overall key events of sensitization seems to be the same for the three groups of compounds (1).

The genomic data were backed by proteomic data. More than 200 proprietary skin and lung markers emerged from the EU funded FP6 project Sens-it-iv using the mass spectroscopy-based proteomic biomarker discovery platform of Proteome Sciences (5). Specific assays were developed using its Tandem Mass Tagging technology combined with selected-reaction monitoring mass spec. SensiDerm ^TM applies a biomarker panel comprising ten proteins which were shown to be differentially expressed in MUTZ-3 cells in response to sensitisers compared to non-sensitisers.

Chemical reactivity mechanism and rate. By stratifying the sensitizing chemicals into chemical reactivity groups, a number of canonical pathways known to be involved in the biology of sensitization were confirmed, while novel pathways were identified (6). Sensitizers with different reactivity mechanisms were further shown to engage different pathways, indicating that the biological end-point of T-cell priming is achieved through different chemical-specific upstream mechanisms (Fig. 2A).

Sensitizing potency. Assessing sensitizing potency on the basis of the now generally accepted key events for sensitization, remains a challenge. However, 200 genes published by Johansson et al. (2) were found to correlate well with human potency of the tested substances (Fig. 2B). The general trend is that both metabolic and cell cycle associated pathways are engaged gradually, and in correlation with potency (6). In addition, to the genetic differentiation between substances of different potency, there is evidence suggesting that the observed effects are dose dependent. The dose required to observe cellular effects in the GARD are inversely correlating with the in vivo potency of the compound.

Figure 2: Chemical reactivity mechanisms and potency relate to quantitative and qualitative differences in gene responses. A. Genes affected by the different chemical reactivity mechanisms. B. The stimulation index derived after application of the Genomic Allergen Rapide Detection (GARD) algorithm using chemicals of different potency. 

Dendritic cell migration: Where the substance-specific information seems to disappear.

Fibroblasts play a key role both as advisors helping the KCs and Langerhans cells (LCs) to discriminate irritants from sensitizers, which in many cases are irritants themselves, and as guides helping the LCs out of the epidermis into the dermis and further towards lymphatic vessels. Using a full-thickness tissue-engineered skin model containing fully functional MUTZ-3-derived LCs (MUTZ-LC), the MUTZ-LCs were demonstrated to mature and to acquire the ability to migrate towards C-X-C motif ligand (CXCL)12 and C-C motif ligand (CCL)19/21 in a comparable manner with primary LCs in skin explants. The acquired knowledge has resulted in a DC-migration assay which is based on carboxyfluorescein succinimidyl ester (CFSE)-labelled MUTZ-3 cells. The discriminating feature of the assay is that irritant induced migration is CCL5 dependent, while small compound skin and respiratory sensitizers induced migration is CXCL12 dependent (7).

References

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