Investigation of complement system dependent inflammatory pathways in retinal pigment epithelium (RPE) cells

DSpace Repository

Show simple item record

dc.contributor.advisor Ueffing, Marius (Prof. Dr.)
dc.contributor.author Schmidt, Tiziana Luisa
dc.date.accessioned 2023-02-06T10:11:19Z
dc.date.available 2023-02-06T10:11:19Z
dc.date.issued 2023-02-06
dc.identifier.uri http://hdl.handle.net/10900/136097
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1360974 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-77448
dc.description.abstract PURPOSE: Age-related macular degeneration (AMD) constitutes the leading cause of blindness among the elderly worldwide. To date, due to the complex nature of the disease, the exact pathogenesis is not yet fully understood. Ageing, lifestyle and genetic components are considered important risk factors. In particular, a number of single nucleotide polymorphisms (SNP) have been identified in complement system genes, particularly in the complement factor H (CFH) gene, that are associated with an increased risk for AMD. The purpose of this study was to investigate the function of the CFH protein (FH) in the regulation of inflammatory processes and the complement system in retinal pigment epithelium (RPE) cells. Furthermore, we aimed to identify the cellular signaling pathways influenced by endogenous FH dysregulation in RPE cells. METHODS: The CFH gene was knocked-down in human RPE cells (hTERT-RPE1) in order to mimic an almost complete loss of the FH protein. We investigated the effects of FH dysregulation on the gene expression levels of several cytokines (IL6, CXCL8, CCL2) and complement system factors (C3, C5, CFB, CFI). To examine the effects of exogenous complement sources, FH, C3 or C3b were supplemented to the cell culture media. Phosphorylated and total NF-κB p65 levels were measured via Western Blot (WB). To further study the NF-κB pathway under FH deprived conditions, several NF-κB inhibitors were employed (RAP, MG132, CAPE), as well as knock-down of the p65 NF-κB subunit (RELA gene). Besides, activation levels of the Akt and MAPK/Erk signaling pathways under CFH knock-down were examined via WB. RESULTS: Under CFH knock-down (siCFH) in RPE cells, we detected a significant increase in proinflammatory factors at gene and protein level (IL-6, IL-8, MCP-1, GM-CSF, etc.). In addition, we observed a dysregulation in the transcription pattern of several complement system genes. FH dysregulation in RPE cells led to an increase in levels of C3, CFB, CFI, and to a decrease in C5. All of these changes were independent of the addition of exogenous complement factors (FH, C3 or C3b). Activation levels of NF-κB were significantly increased under CFH knock-down. Silencing of NF-κB p65 reversed the observed changes under CFH knock-down in most cytokines and complement components. In addition, we found the activation levels of Akt decreased by siCFH, whereas we did not observe any effect on Erk activation levels. CONCLUSION: In RPE cells, endogenous FH has the intrinsic ability to modulate the expression of several inflammatory and complement system factors. Dysregulation of FH leads to an increase in proinflammatory cytokines and a misbalance of factors of the complement system in a NF-κB dependent manner. Our findings suggest that FH and the NF-κB pathway are involved in maintaining inflammatory and complement system homeostasis in RPE cells. Dysregulation of either one of them, likely causes a loss of balance in the complement system and leads to a shift in RPE cells towards a pro-inflammatory AMD-like phenotype. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podno de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=de de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=en en
dc.subject.ddc 610 de_DE
dc.subject.other age-related macular degeneration (AMD) en
dc.subject.other complement factor H en
dc.subject.other NF-kB signaling pathway en
dc.title Investigation of complement system dependent inflammatory pathways in retinal pigment epithelium (RPE) cells en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2022-04-26
utue.publikation.fachbereich Medizin de_DE
utue.publikation.fakultaet 4 Medizinische Fakultät de_DE
utue.publikation.noppn yes de_DE

Dateien:

This item appears in the following Collection(s)

Show simple item record