Biomerker onderzoek voor isocyanaatgeïnduceerd

Transcription

1 CGC en NVAB meeting, 6 Juni 2013, s-hertogenbosch Biomerker onderzoek voor isocyanaatgeïnduceerd astma Jeroen Vanoirbeek

2 Asthma 1. Chronic airway disease: prevalence: 5-10%, people affected world-wide 2. Reversible airflow limitation, airway hyperreactivity & inflammation 3. Wheezing, shortness of breath, coughing, chest tightness

3 Asthma Rapid increase of allergy and asthma in past 40 years Changes in gene pool unlikely Changes in environment? Hygiene, lifestyle, diet,... Air pollution (indoor, outdoor) Specific chemicals? Mechanisms?

4 Occupational asthma Definition: occupational asthma is a disease characterized by variable airflow limitation and/or airway hyperresponsiveness due to causes and conditions attributable to a particular occupational environment and not to stimuli encountered outside the workplace 9-15% of all adult asthma is due to exposures on the work floor The most common cause of work-related lung diseases Bernstein et al., 1999; Mapp et al., 2005

5 Occupational agents High molecular weight (HMW) compounds ( 5 kda) Animal proteins Plant proteins Enzymes Low molecular weight (LMW) compounds (< 5 kda) Chemicals Metals Wood dust Pharmaca IgE mediated HMW allergens Flour, Lab animals, Immune sensitization IgE mediated or non-ige mediated LMW sensitizers Isocyanates, persulphates, No immune sensitization Non-IgE mediated LMW irritants Chlorine, Ammonia,

6 Chemicals Chemicals Agriculture Painting/dye industry Plastic/coating industry Construction

7 Occupational agents Diisocyanates Highly reactive, low molecular weight compounds Most common cause of chemical-induced occupational asthma Used for the production of polyurethanes, foams, paints, etc

8 Redlich C and Herrick C, Curr Opin Allergy Clin Immunol 2008 Skin - lung Development of chemical-induced asthma Primary route of exposure and initiation of immune response = respiratory tract Regulation and prevention of OA almost exclusively focusses on airborne exposures

9 Skin - lung

10 Redlich C and Herrick C, Curr Opin Allergy Clin Immunol 2008 Skin - lung Development of chemical-induced asthma Primary route of exposure and initiation of immune response = respiratory tract Regulation and prevention of OA almost exclusively focusses on airborne exposures Despite reduction in workplace respiratory exposure, diisocyanate asthma continues to occur Focus on skin exposure

11 Skin - lung

12 Skin - lung 1. Workers in car body repair shops Substantial skin exposure to diisocyanates While airborne exposure was minimal Despite personal protective equipment (gloves and masks) 2. Case reports and (limited) cross-sectional studies in humans 3. Prove this in experimental (animal) setting

13 Occupational asthma Mechanisms? Role of chemical properties? chemical reactivity irritant properties Pathways of sensitization? via dermal route? Immunological mechanisms? usually no specific IgE antibodies cellular mechanisms Implications Hazard identification Prediction of asthmogens Prevention Avoid skin contact Surveillance & diagnosis Identification of sensitized subjects Identification of sensitizing agent in affected subjects

14 Aim Provide valid procedures for testing potential asthmogens Predictive toxicology: identification of «asthmogens» high molecular weight agents low molecular weight agents Primary sensitization potential (potent weak) Ability to cause relevant respiratory responses in sensitized subjects Mechanistic toxicology: understanding mechanisms of sensitization (dermal / mucosal; antigen presentation; dose - response relationships; threshold - tolerance; role of irritants; genetics;...) respiratory responses to sensitization (bronchoconstriction, lung inflammation, airway remodelling; resolution / persistence; role of irritants; genetics,...)

15 Mouse model Day Dermal sensitization on dorsum of both ears (20 µl) Intranasal instillation in each nostril (10 µl) 3%TDI 40 min measurement of Penh in whole body plethysmograph

16 Mouse model Day Methacholine provocation (AHR) - whole body plethysmograph Dermal sensitization on dorsum of both ears (20 µl) 3%TDI Intranasal instillation in each nostril (10 µl) 40 min measurement of Penh in whole body plethysmograph Autopsy - BAL - Serum

17 Mouse model Day Methacholine provocation (AHR) - whole body plethysmograph Dermal sensitization on dorsum of both ears (20 µl) 3%TDI Intranasal instillation in each nostril (10 µl) 40 min measurement of Penh in whole body plethysmograph Autopsy - BAL - Serum

18 Mouse model Day Methacholine provocation (AHR) - whole body plethysmograph Dermal sensitization on dorsum of both ears (20 µl) 0.3%TDI Intranasal instillation in each nostril (10 µl) 40 min measurement of Penh in whole body plethysmograph Autopsy - BAL - Serum Vanoirbeek J. et al., Tox Sci 2003 and 2004

19 Mouse model Day Dermal sensitization on dorsum of both ears (20 µl) Intranasal instillation in each nostril (10 µl) Methacholine provocation (AHR) - whole body plethysmograph - forced oscillation (FlexiVent) 40 min measurement of Penh in whole body plethysmograph Autopsy - BAL - Serum - Auricular and cervical lymph nodes Tarkowski M et al., AJP-LCMP 2007, Vanoirbeek J. et al., JACI 2008, De Vooght et al., Tox 2009

20 Overview sensitizer Skin KCs protein protein sensitizer binding to proteins protein protein protein protein protein protein Sensitizer+protein complex binding and internalisation into LC Epidermis protein KCs production of IL-1, TNF-, GM-CSF LC migration and maturation with processing of protein complex T-cell migration into peripheral tissues and blood Y Y Grabbe and Schwarz, 1998 Novak et al., 2004 Redlich et al., 2002 MHC MHC MHC MHC Protein complex presentation to lymphocytes Naive T cell Memory/activated T cells IFN Y Y Ig s Y Y IL-4, IL-13 Memory/activated B cells

21 Overview sensitizer protein protein protein Airways protein protein MHC protein protein MHC protein T-cells Th1 Th2 CD4 CD8 Activated T and B cells Y protein Y Y Y Y Y Y Ig s Y Mast cell protein Cytokines and chemokines MIP-2, TNF-, IL-4, IL-5, IL-13, IFN-, MCP-1, eotaxin Growth and matrix factors (VEGF, MMP,...) Neutrophil Eosinophil Macrophage Basophil Vanoirbeek J et al, 2008, 2009, 2010 Wisnewski A et al, 2001, 2011 Herrick C et al, 2002, 2003 Mathesson J et al, 2001, 2002, 2005 Tarkowski M et al, 2007 Pauluhn J et al, 2008 Ban M et al, 2006

22 Chemical-induced asthma model Ventilatory responses Early response AHR Inflammation Neutrophils and eosinophils Epithelial shedding Immune parameters T and B cell proliferation Th2/Th1 cytokines Total IgE Tarkowski M et al., AJP-LCMP 2007, Vanoirbeek J. et al., JACI 2008, De Vooght et al., Tox 2009

23 Aim To identify (early) biomarkers of chemical-induced asthma and sensitization to chemicals using proteomics techniques

24 Proteomics Proteomics is a global strategy in which all proteins (the proteome) derived from a cell, tissue, body liquid or whole organism, are simultaneously visualized and identified

25 Two-dimensional difference gel electrophoresis: 2D- DIGE Control sample Cy 5 Test sample Cy 3 Internal standard Cy 2

26 Two-dimensional difference gel electrophoresis: 2D- DIGE ph = 3 ph = 10

27 Two-dimensional difference gel electrophoresis: 2D- DIGE ph = 3 ph = 10

28 Two-dimensional difference gel electrophoresis: 2D- DIGE

29 Two-dimensional difference gel electrophoresis: 2D- DIGE SCANNER Cy 2 Cy 3 Overlay Cy 5

30 Two-dimensional difference gel electrophoresis: 2D- DIGE MALDI-TOF MS Trypsin Digest

31 Proteomics in chemical-induced asthma Classical research: focus on immune related cells and cytokines, resulting in many insights, but exact mechanisms of OA still unclear Development of new & sensitive methods new approaches

32 Proteomics in chemical-induced asthma Day Dermal treatment on both ears (20 µl) with 0.3%TDI or AOO (vehicle) sensitization Airway challenge: 0.01% TDI or AOO (vehicle) challenge AHR (methacholine) Sample collection: - auricular lymph nodes - serum - BAL Vanoirbeek et al., 2003, 2004, 2008; De Vooght et al., 2010

33 Proteomics in chemical-induced asthma

34 Proteomics in chemical-induced asthma Auricular lymph nodes BAL Serum p < 0.01 Diff. Spots 53 Identified 27 Up 14 Down 13 p < 0.01 Diff. Spots 210 Identified 72 Up 55 Down 17 p < 0.01 Diff. Spots 40 Identified 18 Up 9 Down 9

35 Proteomics in chemical-induced asthma AOO TDI Auricular lymph nodes

36 Proteomics in chemical-induced asthma

37 Proteomics in chemical-induced asthma auricular lymph nodes bronchoalveolar lavage serum

38 Vanoirbeek et al., 2008, De Vooght et al., 2009 Proteomics in chemical-induced asthma Lymphocyte specific protein-1 Literature: Expression: - lymphocytes - neutrophils - macrophages Chemotaxis & activation neutro Overexpression dysfunction Regulated by IL-4

39 Conclusions 1. First systematic & systemic proteomics approach in a model of occupational asthma 2. Dermal sensitization and a single airway challenge leads to profound proteome changes in multiple compartments 3. Physiological and immunological changes are reflected by changes in the proteome Haenen et al., 2010

40 Occupational asthma Mechanisms? Role of chemical properties? chemical reactivity irritant properties Pathways of sensitization? via dermal route? Immunological mechanisms? usually no specific IgE antibodies cellular mechanisms Implications Hazard identification Prediction of asthmogens Prevention Avoid skin contact Surveillance & diagnosis Identification of sensitized subjects Identification of sensitizing agent in affected subjects

41 Proteome changes in auricular lymph nodes and serum after dermal sensitization to toluene diisocyanate in mice

42 Proteome changes after dermal sensitization 1. So far, no study has focused on early time points 2. Human (early) biomarker research is complicated: only when disease has established

43 Proteome changes after dermal sensitization 1. So far, no study has focused on early time points 2. Human (early) biomarker research is complicated: only when disease has established 3. Rationale: investigate changes in the proteome during sensitization to identify (early) markers of sensitization

44 Proteome changes after dermal sensitization Day Dermal treatment on both ears (20 µl) with 0.3%TDI or AOO (vehicle) Oropharyngeal Sample collection: aspiration: - serum 0.01% TDI or AOO - auricular (vehicle) lymph nodes AHR (methacholine) sensitization challenge sample collection: - auricular lymph nodes - serum

45 weight (g) weight (g) Proteome changes after dermal sensitization Auricular lymph node weight application *** 2 application *** AOO TDI AOO TDI

46 x10 6 /ml x10 6 /ml Proteome changes after dermal sensitization Lymphocyte subpopulations * 1 application * * CD3 CD4 CD4 CD25 * * CD8 CD * 2 applications * * CD3 CD4 CD4 CD25 * * CD8 CD19

47 I.S. test control + 1 labeling 2 separation 3 scanning Cy2 Cy5 Cy3 Cy2 Cy5 Cy3 Decyder analysis identification mass spectrometry

48 Proteome changes after dermal sensitization auricular lymph nodes 1 application 2 applications 1 application serum 2 applications p < 0.01 p < 0.01 p < 0.01 p < 0.01 Diff. spots 38 Identified 26 Up 15 Down 11 Diff. spots 58 Identified 35 Up 19 Down 16 Diff. spots 7 Identified 3 Up 3 Down 0 Diff. spots 16 Identified 10 Up 4 Down 6

49 Proteome changes after dermal sensitization auricular lymph nodes 1 application 2 applications AOO TDI

50 Proteome changes after dermal sensitization serum 1 application 2 applications AOO TDI

51 Proteome changes after dermal sensitization auricular lymph nodes 1 application 2 applications

52 Proteome changes after dermal sensitization serum 1 application 2 applications

53 Proteome changes after dermal sensitization auricular lymph nodes serum

54 Proteome changes after dermal sensitization Lymphocyte specific protein-1 Literature: Expression: - lymphocytes - neutrophils - macrophages Chemotaxis & activation neutro Overexpression dysfunction Regulated by IL-4 Down regulated in complete model (Haenen et al., 2010)

55 ratio LSP1/GAPDH Proteome changes after dermal sensitization LSP-1 1 application 2 applications AOO TDI AOO *** TDI LSP-1 GAPDH

56 Proteome changes after dermal sensitization Coronin 1a Literature: Member of well-conserved family of coronin proteins Mediates Ca 2+ release Involvement T cell activation and proliferation

57 ratio Cor1a/GAPDH Proteome changes after dermal sensitization Cor 1a 1 application 2 applications *** ** 0.0 AOO TDI AOO TDI GAPDH Cor1a

58 Proteome changes after dermal sensitization Hemopexin acute phase protein Literature: Involved in innate body defense Upregulated during inflammation Also in complete model (BAL and serum)

59 mg/ml Proteome changes after dermal sensitization Hemopexin 1 application 2 applications 240 * 220 * AOO TDI AOO TDI

60 Conclusions 1. Sensitization causes profound differences in the proteome of TDI-sensitized mice compared to control mice 2. A subset of proteins were confirmed in an independent set of mice 3. Validation needed in human exposed workers Haenen et al., 2012

61 Acknowledgements Center for Environment and Health Dr. STEVEN HAENEN Dr. Vanessa De Vooght Prof. Peter Hoet Prof. Ben Nemery Lab of Functional Genomics & Proteomics Dr. Elke Clynen Prof. Liliane Schoofs Evelyn Maes Prometa Dr. Geert Baggerman