The immunomodulatory capacity of helminths on inflammation: Impact of eosinophils on E. coli-induced sepsis and genome-wide transcriptome profiling of human monocytes stimulated with helminth extract and LPS implicate immune functions and diseases DISSERTATION zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms Universität Bonn vorgelegt von BENEDIKT CHRISTIAN BÜRFENT aus Bonn Bonn 2017 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Achim Hörauf 2. Gutachter: Prof. Dr. Sven Burgdorf Tag der Promotion: 06. Oktober 2017 Erscheinungsjahr: 2017 2 Table of Content Table of Content Table of Content......................................................................................................................................... 3 Erklärung ..................................................................................................................................................... 6 Publications in peer-reviewed journals ................................................................................................... 7 Preamble ..................................................................................................................................................... 8 Summary ..................................................................................................................................................... 9 Introduction ............................................................................................................................................... 11 1.1 Human pathogenic parasites ................................................................................................. 11 1.1.1 Helminth-driven human diseases – lymphatic filariasis and onchocerciasis .......... 13 1.1.2 Immunopathogenesis of human lymphatic filariasis and onchocerciasis ................ 18 1.1.3 Immunomodulation by helminths and helminth-derived molecules ......................... 20 1.1.4 Murine Litomosoides sigmodontis infection as model for human filarial infections 23 1.2 Helminth-mediated protection against LPS-driven immune diseases ............................. 24 1.2.1 Immunopathogenesis of bacterial-induced inflammation .......................................... 25 1.2.2 Helminth-mediated immunomodulation against bacterial-induced sepsis .............. 26 1.3 Objectives of the thesis ........................................................................................................... 29 Material & Methods .................................................................................................................................. 30 2.1 Murine studies ................................................................................................................................ 30 2.1.1 Ethics statement and mice .................................................................................................... 30 2.1.2 Chronic filarial infection model – Litomosoides sigmodontis ........................................... 30 2.1.3 Bacterial sepsis model ........................................................................................................... 30 2.1.4 Eosinophil culture ................................................................................................................... 31 2.1.5 In vitro experiments of bone-marrow-derived eosinophil granulocytes ......................... 31 2.1.6 Flow cytometry and immunoassays of murine experiments ............................................ 32 2.1.7 RNA sequencing and transcriptome profiling of sorted eosinophil granulocytes ......... 33 2.2 Human study .................................................................................................................................. 35 2.2.1 Human study population ....................................................................................................... 35 2.2.2 Monocyte isolation ................................................................................................................. 35 2.2.3 Monocyte stimulation ............................................................................................................. 35 2.2.4 Flow cytometry and immunoassays of human monocyte experiments ......................... 36 2.3 RNA extraction ............................................................................................................................... 36 2.4 Transcriptome analysis ................................................................................................................. 37 2.5 Pathway analysis ........................................................................................................................... 37 2.7 Data management and statistical analysis ................................................................................ 38 3 Table of Content Results ....................................................................................................................................................... 39 3.1 Impact of eosinophil granulocytes on E. coli-induced sepsis ................................................. 39 3.1.1 Absence of eosinophil granulocytes exacerbates E. coli-induced sepsis in ∆dblGATA mice ............................................................................................................................................ 39 3.1.2 Ex vivo generation of bone-marrow-derived eosinophil granulocytes ........................... 41 3.1.3 Impact of chemoattractant eotaxins on TLR4 and TLR1/2-driven responses of bmEos ............................................................................................................................................ 42 3.1.4 Anti-bacterial properties of bone-marrow-derived eosinophil granulocytes .................. 45 3.1.5 Genome-wide transcriptome profiling of filarial-induced and non-induced murine eosinophil granulocytes in the absence or presence of an E. coli challenge ......................... 47 3.2 Genome-wide transcriptional profiling of human monocytes stimulated with Brugia malayi crude extract and LPS reveals link to IL-17 associated pathways and diseases ...................... 52 3.2.1 Experimental design and human CD14+ monocyte purification ..................................... 52 3.2.2 BmA stimulation alone significantly induced transcripts in human monocytes compared to unstimulated controls ............................................................................................... 53 3.2.3 High similarity in the transcriptional profiles of LPS-only and BmA+LPS re-stimulated monocytes compared to unstimulated controls ........................................................................... 55 3.2.4 BmA treatment modulates gene expression in human monocytes after LPS stimulation ......................................................................................................................................... 56 3.2.5 BmA priming reduces CD86 and HLA-DR expression of LPS-stimulated human monocytes and triggers CXCL5 and CXCL6 release ................................................................. 59 3.2.6 Pathway analysis of transcriptome profiles of BmA primed and LPS stimulated human monocytes reveals link to IL-17 associated pathways and diseases ....................................... 61 Discussion ................................................................................................................................................. 70 4.1 Eosinophil granulocytes are involved in the protection against sepsis ................................. 71 4.1.1 Eosinophil-deficiency exacerbates inflammation during E. coli-induced sepsis ........... 71 4.1.2 Eosinophils release pro-inflammatory recruitment factors to TLR2 and TLR4 ligands 72 4.1.3 Eosinophils independently regulate CCR3 receptor and ICAM-1 expression .............. 74 4.1.4 Eosinophil granulocytes execute important roles during sepsis – a hypothesized model ............................................................................................................................................ 75 4.1.5 Outlook: Immunomodulatory impact of helminths on eosinophils .................................. 77 4.2 Genome-wide transcriptional profiling of human monocytes stimulated with filarial extract and LPS reveals link to IL-17 associated pathways and diseases .............................................. 78 4.2.1 BmA induces cellular protective mechanisms in human non-endemic monocytes ..... 79 4.2.2 BmA modulates noncoding RNA expression with diverse cellular functions ................ 81 4.2.3 BmA priming before LPS treatment affects chemotaxis and cell movement of myeloid cells ............................................................................................................................................ 82 4.2.4 BmA priming before LPS re-stimulation reveals link to inflammatory disorders including IL-17 dependent diseases .............................................................................................. 85 4 Table of Content 4.2.5 Expression quantitative trait loci (e²QTLs) analysis of human monocytes to determine DNA variations that contribute to different gene expressions of anti-filarial responses and helminth-induced immunomodulation ..........................................................................................