biomarkers stefan f. martin, phd · 2017-06-20 · biomarkers • who definition: any substance,...
TRANSCRIPT
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
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)
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
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
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
Thank you!
Medical Center - University of Freiburg