TESTICULAR NEOPLASIA: FUNCTIONAL POLARIZATION OF MACROPHAGES AND DENDRITIC CELLS INAUGURAL-DISSERTATION submitted in partial fulfilment of the requirements for the degree of Dr.rer.nat./ Ph.D. to the Faculty of Biology at the Justus-Liebig-University, Giessen as part of the joint award Ph.D. program with Monash University Submitted by Dana Püschl, M.Sc. (Biology) from Zeitz Gießen, 2018 Mit Genehmigung des Fachbereiches Biologie der Justus-Liebig-Universität Gießen Dekan/Dean: Prof. Dr. Jürgen Janek_________________________________ Gutachter/Reviewer: Prof. Dr. Martin Bergmann Gutachter/Reviewer: Prof. Dr. Tina Trenczek Tag der Disputation/date of disputation: 28.01.2019 II TABLE OF CONTENTS CHAPTER 1: Literature Review: The Contribution of Innate Immune Cells to Testis Function in the Healthy Testis and Testicular Neoplasia ................................................. 2 1.1 Introduction to cells of the innate and adaptive immune system ...................................... 4 1.2 The testis under physiological conditions ......................................................................... 5 1.2.1 The immune privilege of the testis ................................................................................ 5 1.2.2 Contribution of testicular macrophages and other immune cells to the immune privilege of the testis ............................................................................................................... 8 1.2.3 The immunosuppressive role of cytokines and chemokines in the testis .................. 10 1.2.4 Complexity of signaling pathways: TGF-β and IL-10 influence pro-inflammatory cytokine expression .............................................................................................................. 11 1.2.5 Functional macrophage polarization by cytokines, chemokines and hormones ........ 14 1.3. Testicular germ cell tumours ..........................................................................................15 1.3.1 Classification and epidemiology .................................................................................. 15 1.3.2 Pathogenesis ............................................................................................................... 16 1.3.3 Immune cells associated with testicular cancer .......................................................... 18 1.3.4 Function of tumour-associated macrophages and dendritic cells .............................. 20 1.3.5 Contribution of tumour cells to macrophage and DC polarization .............................. 21 1.3.6 Immune checkpoints ................................................................................................... 22 CONCLUSION .....................................................................................................................25 CHAPTER 2: Detailed analysis of immune cells in the human testis under physiological and pathological conditions, with a focus on macrophage and dendritic cell subsets .........................................................................................................................27 2. INTRODUCTION ..............................................................................................................27 2.1 MATERIAL AND METHODS .........................................................................................28 2.1.1 Patients ........................................................................................................................ 28 2.1.1.1 Routine Histology .................................................................................................. 28 2.1.2 Immunohistochemistry ................................................................................................ 29 2.1.4 Immunofluorescence ................................................................................................... 32 2.1.5 RNA extraction, cDNA synthesis, RT-PCR and quantitative real-time PCR .............. 32 2.1.6 Protein isolation, SDS-PAGE and western blot .......................................................... 36 2.1.6.1 Protein isolation from cryopreserved testis tissue ............................................... 36 2.1.6.2 Protein isolation from paraffin-embedded testis tissue ........................................ 36 2.1.6.3 Gel electrophoresis ............................................................................................... 36 2.1.6.4 Protein transfer ..................................................................................................... 37 2.1.7 Analysis of immune cells in testes of Gilz KO and WT mice ...................................... 38 2.1.7.1 Histology ............................................................................................................... 38 III 2.1.7.2 Flow cytometry ...................................................................................................... 39 2.2 RESULTS ......................................................................................................................40 2.2.1 Human testis: Assessment of immune cells under physiological and pathologic conditions .............................................................................................................................. 40 2.2.1.1 Anti-inflammatory M2 macrophages detectable in the normal testis whereas pro- inflammatory M1 macrophages were absent ................................................................... 40 2.2.1.2 IHC analysis revealed increased immune cell populations associated with testicular germ cell neoplasia ........................................................................................... 41 2.2.1.3 Identification of DC and macrophage subsets associated with testicular germ cell neoplasia by immunofluorescence (IF) double-staining ................................................... 44 2.2.2 Potential function of immune cells in the human testis ............................................... 46 2.2.2.1 M2 macrophages as contributors of the immune suppressive milieu in the testis ........................................................................................................................................... 46 2.2.2.2 M1 and M2 macrophages balance pro- and anti-inflammatory cytokine milieu in testicular germ cell neoplasia ........................................................................................... 47 2.2.2.3 M1 macrophages: Indication of phagocytosis and antigen presenting capabilities ........................................................................................................................................... 48 2.2.3 Recruitment of immune cells by chemokine signalling in the human testis ............... 50 2.2.3.1 Possible recruitment of CD68+ macrophage precursors by CCL15 ................... 50 2.2.3.2 Chemokine and chemokine receptor expression analysis in testicular germ cell neoplasia ........................................................................................................................... 51 2.2.4 Tumour-/immune cell interaction and functional polarization of macrophages .......... 53 2.2.4.1 Potential macrophage polarization factors ........................................................... 53 2.2.4.2 Identification of potential prognostic testicular cancer marker: Signalling pathways (p65, NF-κB) and immune checkpoints T-cell immunoglobulin mucin-3 (TIM-3) and Galectin-9 associated with testicular cancer ............................................................. 55 2.2.4.3 Impacted spermatogenesis in glucocorticoid-induced leucine zipper protein (GILZ) KO mice influenced testicular immune cell populations ....................................... 56 2.3 DISCUSSION .................................................................................................................59 CONCLUSION .....................................................................................................................64 CHAPTER 3: Assessment of mono-cultured tumour and immune cells of relevance to testicular neoplasia: the TCam-2 seminoma and THP-1 macrophage cell lines ............69 3 INTRODUCTION ...............................................................................................................69 3.1 MATERIAL AND METHODS .........................................................................................71 3.1.1 Cell cultures ................................................................................................................. 71 3.1.1.1 TCam-2 (human seminoma cell line) ................................................................... 71 3.1.1.2 THP-1 (human monocytic leukemia cell line) ....................................................... 71 3.1.2 Cell migration ............................................................................................................... 73 IV 3.1.3 Reverse transcriptase PCR (with RNA isolation) ....................................................... 75 3.1.4 Quantitative RT-PCR ................................................................................................... 75 3.1.5 Western blot ................................................................................................................ 77 3.1.5.1 Protein Isolation .................................................................................................... 77 3.1.5.2 SDS-PAGE ........................................................................................................... 78 3.1.5.3 Protein transfer