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Maren Depke Results Gene Expression Pattern of Bone-Marrow Derived Macrophages after Interferon-gamma Treatment The peptide transporters Tap1 and Tap2 translocate peptides from cytosolic proteasome into the endoplasmatic reticulum, where they bind to MHC-I molecules, while the peptide-MHCcomplexes are lateron transferred to the cell surface. Tap1 and Tap2 are described to be induced by IFN-γ (Brucet et al. 2004, e. g. human, Ma W et al. 1997, Schiffer et al. 2002). In this study, Tap1 (5.0 / 5.2) and Tap2 (3.3 / 3.0) were higher expressed after IFN-γ treatment in BMM of both strains BALB/c and C57BL/6 (Fig. R.3.4 A, B). Erap 1, an endoplasmic reticulum aminopeptidase that takes part in further processing and N-terminal trimming of the peptides in the ER (Chang et al. 2005, Jung et al. 2009), was additionally induced after IFN-γ treatment in BALB/c BMM (2.1) and C57BL/6 BMM (2.2) [data not shown]. Finally, several genes of MHC class I and class II molecules were induced after IFN-γ treatment in BMM of both strains BALB/c and C57BL/6 [data not shown]. Comparison of differentially expressed gene lists (Fig. R.3.2 A), comparison of log 2 -ratio data (Fig. R.3.2 B), and Ingenuity Pathway Analyis (Fig. R.3.4) revealed highly similar gene expression signatures in BALB/c and C57BL/6 BMM after IFN-γ treatment even for genes which were only differentially expressed in one of the two comparisons. Therefore, further analysis of biological effects resulting from IFN-γ treatment was performed using the union list of IFN-γ effects in BALB/c and C57BL/6 BMM. First, the list of in total 531 IFN-γ dependent genes was subjected to a global functional analysis using Ingenuity Pathway Analysis (IPA, www.ingenuity.com), which compared functional categories within the data set with those of the complete array and assigned a p-value to rate on significant overrepresentation (Table R.3.3). Table R.3.3: Global functional analysis of IFN-γ dependent genes in BMM of at least one strain, BALB/c or C57BL/6, using Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com). Diseases and Disorders category p-value a of number genes Inflammatory Response Immunological Disease Inflammatory Disease Connective Tissue Disorders Skeletal and Muscular Disorders Infection Mechanism Cancer Respiratory Disease Antimicrobial Response Infectious Disease 2.06E-06 - 3.53E-40 5.28E-06 - 1.39E-23 5.61E-06 - 5.75E-23 8.25E-07 - 5.12E-21 8.25E-07 - 5.12E-21 2.83E-06 - 3.51E-19 7.13E-06 - 4.05E-17 2.20E-06 - 2.20E-16 4.46E-08 - 6.66E-15 2.20E-06 - 1.14E-14 146 150 157 Molecular and Cellular Functions category p-value a of number genes Cellular Growth and Proliferation Cellular Development Cell-To-Cell Signaling and Interaction 111 Cellular Movement 150 Cell Death 43 141 Cellular Function and Maintenance Antigen Presentation 56 Cell Cycle 29 Gene Expression 87 Cell Signaling a Ten categories with the smallest p-values are cited. D. a. F. – Development and Function 6.38E-06 - 4.55E-25 6.28E-06 - 8.22E-25 6.78E-06 - 2.73E-23 1.36E-09 - 5.54E-21 5.67E-06 - 1.21E-18 4.90E-06 - 1.76E-18 6.38E-06 - 3.53E-16 1.04E-06 - 4.82E-10 1.98E-07 - 1.00E-09 9.08E-07 - 3.04E-09 154 117 Physiological System Development and Function category p-value a of number genes Hematological System D. a. F. Immune Cell Trafficking 127 Hematopoiesis 101 150 84 56 71 69 62 Tissue Morphology Cell-mediated Immune Response Organismal Survival Tissue Development Humoral Immune Response Cardiovascular System D. a. F. Organismal Development 6.38E-06 - 4.55E-25 4.90E-06 - 6.53E-21 2.06E-06 - 2.65E-20 9.94E-07 - 1.36E-18 4.01E-07 - 2.27E-18 2.50E-07 - 3.64E-15 6.82E-06 - 4.43E-14 9.94E-07 - 2.03E-10 4.05E-06 - 2.39E-09 3.05E-06 - 9.33E-09 138 93 82 70 66 76 71 37 39 41 98
Maren Depke Results Gene Expression Pattern of Bone-Marrow Derived Macrophages after Interferon-gamma Treatment This analysis revealed expected categories within the IFN-γ dependently regulated genes in BMM like “Inflammatory Response”, “Antigen Presentation”, “Immune Cell Trafficking” and other immune response related functions. Furthermore, growth, proliferation, but also cell cyle and cell death associated genes were included in the set of differentially expressed genes. For a more detailed view, IPA’s canonical pathway analysis was applied (Table R.3.4). Here, the pathway “Interferon Signaling” appeared with the highest ratio value, i. e. the fraction of genes from the pathway, which was also included in the list of IFN-γ dependent genes. This observation nicely proved the relevance of the gene expression signature which was recorded after stimulation of BMM with interferon. Further pathways from the group with the highest ratio values were among others “Antigen Presentation Pathway”, “Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses”, and “Activation of IRF by Cytosolic Pattern Recognition Receptors” which fits well into the expected characterization of the experimental system. Again, also cell death associated features appeared with the “Retinoic acid Mediated Apoptosis Signaling” pathway. Table R.3.4: Canonical pathway analysis of IFN-γ dependent genes in BMM of at least one strain, BALB/c or C57BL/6, using Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com). Pathway ratio a p-value Interferon Signaling 11/30 = 0.367 8.34E-12 Antigen Presentation Pathway 11/39 = 0.282 1.61E-09 Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses 20/86 = 0.233 5.03E-15 Complement System 8/36 = 0.222 4.09E-07 Retinoic acid Mediated Apoptosis Signaling 9/44 = 0.205 2.42E-08 Role of PKR in Interferon Induction and Antiviral Response 9/46 = 0.196 2.95E-07 T Helper Cell Differentiation 8/41 = 0.195 7.42E-06 Activation of IRF by Cytosolic Pattern Recognition Receptors 14/74 = 0.189 1.51E-11 Allograft Rejection Signaling 8/45 = 0.178 2.34E-05 TREM1 Signaling 12/69 = 0.174 3.84E-09 a Ten pathways with the highest ratio values are cited. Manual data mining revealed different cytokines and cytokine receptors, which were differentially expressed in BMM upon treatment with IFN-γ. Here, the induction of several chemotactic chemokines (Ccl5, Ccl12, Ccl22, Cxcl9, Cxcl10, Cxcl11, Cxcl16) was noticeable. Among the receptors, Ccr5 induction fitted to the induction of one of its ligands, Ccl5. The induced receptor Ccrl2 is known to be induced after activation and during differentiation from monocytes to macrophages, but its function is not described yet. Contrarily, the receptor Cxcr4 was repressed after treatment of BMM with IFN-γ. Two interleukins were included in the differentially expressed cytokines: IL-15, a regulatory, anti-apoptotic cytokine with structural similarity to IL-2, and the subunit IL27-p28 of the heterodimeric IL-27, a regulatory cytokine produced by antigen presenting cells. Several interleukin receptors or receptor subunits were induced after IFN-γ treatment. The induction of the IL-15 receptor alpha-chain (Il15ra) corresponded to the already mentioned induction of IL-15. Furthermore, the strongly interdependent receptor subunits Il4ra, Il13ra1, Il21r, and Il2rg were induced. The alpha-chain of IL-4 receptor (Il4ra) and the IL-21 receptor (Il21r) employ the IL-2 receptor gamma-chain (Il2rg) as their second subunit. Similarly, the primary IL-13 binding subunit of the IL-13 receptor (Il13ra1) builds a receptor complex together with the alpha-chain of IL-4 receptor (Il4ra). Also Il12rb1, coding for the low-affinity binding subunit of the IL-12 receptor, was induced. The induction of Il10ra, high-affinity binding subunit of the IL-10 receptor, and of Il18bp, cytokine IL-18 binding protein, is an example for inhibitory processes in BMM after IFN-γ treatment. Repression of interleukin receptor subunits was also found after IFN-γ treatment: Il1rl2, receptor for interleukin 1 family member 9, and Il6st alias gp130, signal transducer for several cytokines, e. g. IL-6, were 99
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G E N O M E W I D E C H A R A C T E
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Maren Depke C O N T E N T S GENOMEW
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Maren Depke Z U S A M M E N F A S S
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Maren Depke Zusammenfassung der Dis
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Maren Depke S U M M A R Y O F D I S
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Maren Depke Summary of Dissertation
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Maren Depke I N T R O D U C T I O N
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Maren Depke Introduction Besides op
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Maren Depke Introduction Extracellu
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Maren Depke Introduction 2005). SaP
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Maren Depke Introduction STUDIES OF
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Maren Depke M A T E R I A L A N D M
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Maren Depke Material and Methods Li
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Maren Depke D I S C U S S I O N A N
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Maren Depke Discussion and Conclusi
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Maren Depke References Athanasopoul
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Maren Depke References Byrne GI, Le
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Maren Depke References Demling R. T
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Maren Depke References Jin T, Bokar
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Maren Depke A F F I D A V I T / E R
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Maren Depke Acknowledgments Kidney
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