COST Action TD1206 "StanDerm" Final Workshop, Berlin 18-19 May 2017

Etiology and Prevention of Occupational Contact

Dermatitis: New Challenges

Biomarkers Stefan F. Martin, PhD

Department of Dermatology

Medical Center - University of Freiburg

stefan.martin@uniklinik-freiburg.de

COST is supported by the EU Framework Programme Horizon 2020

Biomarkers

• WHO definition: any substance, structure or process that can be measured in the body or

its products and influence or predict the incidence of outcome or disease

• Can correlate with the nature of a disease, disease state, metabolic activity

• Can be cells, soluble factors (cytokines, hormones etc.), genes

• Ideally measurable in blood, serum, saliva, urine

• Relatively easy to identify: biomarkers for genetic diseases

• More difficult for complex traits such as contact dermatitis (> 4.000 contact allergens

known)

• Biomarkers can be used for diagnosis, prognosis, hazard identification/risk assessment

• Detection by ELISA, antibody staining (FACS, IHC), gene arrays, PCR etc.

Biomarkers for contact dermatitis

• Problem for contact dermatitis: huge diversity of chemicals (irritants, contact

allergens) with diverse physicochemical properties

• Traditionally, focus on immune mediators (e.g. cytokines, chemokines)

• More recent: biomarkers associated with skin barrier, xenobiotic metabolism,

cellular stress responses

• Challenge: identification of biomarker profiles for discrimination of allergic and

irritant contact dermatitis (disease-specific profiles/signatures)

• Due to great overlaps, sometimes biomarkers may only differ quantitatively rather

than qualitatively (e.g. blood glucose)

Biomarker categories

• Alarmins: released from the intracellular and extracellular space or produced de

novo in response to irritants/contact allergens, infection etc. to activate the innate

immune system (e.g. ROS; HMGB1, hyaluronic acid fragments, DNA, RNA,

antimicrobial peptides)

• Cytokines and chemokines: immune mediators released by many different cell

types (e.g. CXCL9, CXCL10 in ACD, not ICD)

• biomarkers associated with skin barrier (e.g. lipids, NMF, proteases, tight

junction proteins)

• xenobiotic metabolism (e.g. aryl hydrocarbon receptor pathway)

• cellular stress responses (e.g. Keap1/Nrf2 pathway)

• Genetic biomarkers: single nucleotide polymorphisms (SNPs) (often associated

with susceptibility)

Goals

• Customized Biomarker Arrays for the improvement of diagnosis and

development of novel therapies

• Prevention (will it be possible to identify biomarkers of susceptibility to identify

individuals at risk?)

• Insight into pathomechanisms to promote further basic research projects

(including mouse studies, 3D human and porcine skin models)

• Contact allergen identification for hazard identification and allergenic potency

assessment (replacement of animal testing, 3R’s)

Prevention of contact dermatitis by identification of hazardous substances

andindividuals at risk

Improved diagnosis (molecular diagnosis) and causative treatment

strategies

Goebel C. et al., Regul. Toxicol. Pharmacol. 63:40-52 (2012)

Reaction mechanistic applicability domains

epidermis

dermis

allergen (sensitization)

Langerhans

cell

lymph node

TC

lymphatic vessel

keratinocyte

Tc Tc

TC

naïve TC

dDC Treg

regulatory

TC

Steps in the sensitization phase of Allergic Contact Dermatitis

skin penetration

activation of keratinocytes

dendritic cell migration

T cell priming

activation of dendritic cells

tissue stress/damage

innate immune response

protein/cell modification

biotransformation

Treg expansion

dermal fibroblast

Teff/Tmem

blood vessel

Teff/Tmem

TC

Rovida, C. et al., ALTEX 30:231-252 (2013)

danger signals: ROS, DAMPs etc.

P2X7R

TLR2/4

NLRP3

Tc1

MHC/hapten/

peptide DC activation, migration

T cell priming

tissue stress/damage

contact allergen irritant

allergic contact dermatitis irritant contact dermatitis

skin inflammation

Allergic and irritant contact dermatitis

Martin S.F. and T.Jakob in Clin. and Basic Immunodermatol., pp. 411-429 Springer (2017)

ASC

Caspase-1

IL-1b

IL-18

TLR4 TLR2

MyD88 Trif

IFNa/b, TNF-a

IL-6, IL-12, IL-23

pro-IL-1b

pro-IL-18

NF-kB IRF3/7 MAPK

HA

P2X7R

ATP

ATP ROS

NLRP3

Dendritic

cell

hTLR4

Ni2+

Co2+

MyD88 Trif

IFNa/b

IL-6, IL-8, IL-12

TNF-a, CCL2

Ni2+

Co2+

NF-kB IRF3/7 MAPK

ROS

pro-IL-1b

pro-IL-18

Orchestration of skin inflammation

by cross-talk of innate immune signaling pathways

Martin S.F. Contact Dermatitis 72:2-10 (2015)

Schmidt M. et al., Nat. Immunol. 11:814-819 (2010)

Raghavan B. et al., EMBO Rep. 13:1109-1115 (2012) Martin S.F. et al., J. Exp. Med. 205:2151-2162 (2008)

Esser P.R. et al., PLoS One 7:e41340 (2012)

ASC

Caspase-1

IL-1b

IL-18

hTLR?

IFNa/b, TNF-a

IL-6, IL-12, IL-23

pro-IL-1b

pro-IL-18

NF-kB IRF3/7 MAPK

mROS

NLRP3

Dendritic

cell

Cr (VI)

Chromium (VI) activates the NLRP3 inflammasome

Adam C. et al., J. Invest. Dermatol. 137:367-376 (2017) )

Cr (VI)

tissue-derived

priming signal?

Tissue-

sress/damage?

Biomarkers for contact allergen identification

• hCLAT assay: up-regulation of CD54 and CD86 on THP1 cells

• KeratinoSens Assay: activation of the transcription factor Nrf2 in HaCaT

keratinocytes

• VITOSENS Assay: CREM and CCR2 up-regulation in CD34+ progenitor

derived DCs

=> single/few biomarkers

• GARD Assay: regulation of 200 genes in MUTZ-3 cells

• => biomarker profiles

Global technologies for biomarker discovery

Genomics Proteomics

in vivo pathway validation

in mouse model (CHS) knockout, knockdwon,

inhibitors/activators

ex vivo/in vitro pathway validation

in humans blood, skin biopsies, 3D cultures

biomarker and pathway identification

prediction signature

chemical class-specific

biomarker panels contact sensitizer-/

irritant-specific biomarker panels

Schoeters E. et al., Mol. Immunol. 44:3222-3233 (2007)

Regulation of gene expression in DCs by contact allergens

(M): Microarray; (P): PCR

Cord blood, CD34+ progenitor cell-derived DCs

The GARD assay for contact allergen identification

• Gene array analysis of human MUTZ-3 cells

• Prediction signature comprises 200 genes

• Ingenuity pathway analysis reveals mechanistic details of sensitization process

(DC activation)

Biomarkers for contact dermatitis

• Serum samples after Patch testing

• Skin biopsies from patients (lesional vs. Non-lesional skin) and non-

allergic controls after Patch testing

Zinkeviciene, A.S. et al., Int. Arch. Allergy Immunol. 168:161-164 (2015)

Zinkeviciene, A.S. et al., Int. Arch. Allergy Immunol. 168:161-164 (2015)

Serum parameters from patients during acute and remission phases

of ACD

Proteome Profiler Human XL Cytokine Array Kit, R&D Systems: 102 cytokines, chemokines, for 16 patients

Gittler J.K. et al., J. Allergy Clin. Immunol. 131:300-313 (2013)

Different forms of eczema

Quaranta, M. et al., Sci. Transl. Med. 6:244ra90 (2014)

Identifying disease-specific signatures by genomic profiling of psoriasis, atopic and induced allergic

eczema (intra-individual comparison!)

Induced eczema (ACD to nickel)

UP:

SPRR family, LCE3 family

HAS3, EPSTI1, ICAM-1

IL-1b, AIM2

CXCL8, CXCL9, CXCL10, CXCL11

Dhingra, N. et al., J. Allergy Clin. Immunol. 134:362-372 (2014)

Contact allergen-specific genomic signatures

Present and future

• Global technologies (genomics, proteomics, metabolomics)

for the identification of hazardous chemicals (important: mixtures and

formulatioms (augmentation, “ cocktail“ effect)

for the identification of predictive and diagnostic biomarkers

- Identification of disease-specific biomarker profiles

- Identification of biomarker profiles to identify individuals at risk (predictive

signatures)

• Identification of chemcial class-specific profiles/signatures

• Qualitative and quantitative differences in profiles for potency assessment

• Pathway analysis for biomarker identification and elucidation of

pathomechanisms

• Development of novel, mechanism-based causative treatment strategies and

diagnostic methods

Koppes, S.A. et al., Contact Dermatitis, epub ahead May 12 (2017)

Allergy Research Group Department of Dermatology

Lab Stefan Martin

Philipp Esser Fabian Gendrisch

Mareike Wegner

Theo Metzger

Usula Voith

Annabelle Buschky

Josephine Völker

Medical Center - University of Freiburg

stefan.martin@uniklinik-freiburg.de

Thank you!

Medical Center - University of Freiburg