Cytokine and chemokine responses as predictive markers for distinct stages of Alveolar Echinococcosis X. Huang1, C.J. Lechner1, B. Grüner2, W.H. Hoffmann1, P. Kern2, P.T. Soboslay1 1Institute for Tropical Medicine, University Clinics of Tübingen 2Division of Infectious Diseases and Clinical Immunology, University Clinics of Ulm Innovation for the management of Echinococcosis. 27th March,2014 1

Study Objective Evaluation of cytokines and chemokines in Alveolar Echinococcosis (AE) patients for their use as predictive markers of regression or progression of AE. Focus of this study is on mediators of inflammation (IL-27, IL-31 and IL-33), as well as chemokines which activate eosinophil granulocytes (Eotaxin-1, Eotaxin-2 and Eotaxin-3). Chemokine genes expression profiles in AE patients Eotaxins can activate and chemoattract eosinophil granulocytes and thus drive Th2 type immunity, which predominates in AE patients with active disease 2

Sample Collections and Methods Peripheral blood mononuclear cells (PBMC) from AE patients with cured (n=12), stable (n=36) or progressive (n=21) diseases and infection-free controls (n=10) PBMC stimulation with E.m. specific antigens ELISA based quantification of cytokines and chemokines in cell culture supernatants Gene Microarrays to study the inducible expression of chemokine genes in PBMC from progressive and cured AE patients.

Production of IL-31 and IL-33 were depressed in AE patients Results Baseline cellular production of IL-27, IL-31 and IL-33 in AE patients (Cured:n=10; Stable n=10; Progressive: n=6; CTRL: n=6) Production of IL-31 and IL-33 were depressed in AE patients

Results Baseline cellular production of Eotaxin-1, Eotaxin-2 and Eotaxin-3 in AE patients (Cured:n=10; Stable n=10; Progressive: n=6; CTRL: n=6) Production of Eotaxin-1, Eotaxin-2 and Eotaxin-3 enhanced with AE progression in patients

Results E.m. metacestode antigen-induced production of Eotaxin-2 by PBMC in AE patients Eotaxin-2 production became strongly activated by E.m. antigen in cured AE patients

Gene Microarrays were applied to study the inducible expression of chemokine genes Peripheral blood mononuclear cells from patients with progressive and cured AE were stimulated in vitro with E. multilocularis metacestode Antigen or remained without stimulation. Gene expression is indicated as stimulation index [Stimulation Index = ]

Results The strongest chemokine gene expression is Liver and Activation Regulated Chemokine (LARC) in Progressive AE patients 8

Results The highest chemokine gene expression is Monocyte Chemo-attractant Protein 4 (MCP4) in Cured AE patients 9

Summary Cellular production of IL-31 and IL-33 were depressed in progressive AE patients; while Eotaxin-1, Eotaxin-2 and Eotaxin-3 levels were enhanced with active and progressive AE. E.m. antigen-specific production of Eotaxin-2 was highly inducible in AE patients. Liver and activation regulated chemokine (LARC) gene expression was highest in progressive AE; while monocyte chemo-attractant protein 4 (MCP4) was strongly expressed in cured AE. Plasma levels of IL-17B is highest in progressive AE and high in all AE patients. Serum level of IL-12 and IL-13 is low in AE patients. Spontaneous releases of Pro-inflammatory MIP-1 alpha/CCL3, MIP-1 beta/CCL4, RANTES/CCL5 are higher in AE patients, and persist after Em antigen stimulation, may contribute to prevent tissue damage. Spontaneous releases of Monokine induced by gamma-inteferon MIG/CXCL9, Th1 type IL-12 and TNF-alpha are low, and persist after Em antigen stimulation. Spontaneous release of MCP4, tissue and activation associatetd chemokines LARC/CCL20, PARC/CCL18 and TARC/CCL17 are low in AE patients IL-31 and the Eotaxin chemokines as well as MCP4/CCL13 and LARC/CCL20 are suggested as biomarkers for staging of Alveolar Echinococcosis 10

Acknowledgements University Clinics of Ulm Prof. Dr. Peter Kern Dr. Beate Grüner University Clinics of Tübingen Prof. Dr. Peter Soboslay Dr. Christian Lechner 11

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