In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls
Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated th...
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MDPI AG
2022-03-01
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| author | Elizaveta S. Ershova Galina V. Shmarina Lev N. Porokhovnik Natalia V. Zakharova George P. Kostyuk Pavel E. Umriukhin Sergey I. Kutsev Vasilina A. Sergeeva Natalia N. Veiko Svetlana V. Kostyuk |
| author_facet | Elizaveta S. Ershova Galina V. Shmarina Lev N. Porokhovnik Natalia V. Zakharova George P. Kostyuk Pavel E. Umriukhin Sergey I. Kutsev Vasilina A. Sergeeva Natalia N. Veiko Svetlana V. Kostyuk |
| author_sort | Elizaveta S. Ershova |
| collection | DOAJ |
| description | Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated <i>TLR9</i> and <i>STING</i> gene expression while blocking <i>RIG-I</i> and <i>AIM2</i> expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients’ blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia. |
| first_indexed | 2024-03-09T19:46:57Z |
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| language | English |
| last_indexed | 2024-03-09T19:46:57Z |
| publishDate | 2022-03-01 |
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| spelling | doaj.art-0daf106f099e41a0b7d174d71fa691482023-11-24T01:19:59ZengMDPI AGGenes2073-44252022-03-0113355110.3390/genes13030551In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy ControlsElizaveta S. Ershova0Galina V. Shmarina1Lev N. Porokhovnik2Natalia V. Zakharova3George P. Kostyuk4Pavel E. Umriukhin5Sergey I. Kutsev6Vasilina A. Sergeeva7Natalia N. Veiko8Svetlana V. Kostyuk9Molecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaN.A. Alekseev Clinical Psychiatric Hospital No. 1, 117152 Moscow, RussiaN.A. Alekseev Clinical Psychiatric Hospital No. 1, 117152 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaMolecular Biology Laboratory, Research Centre for Medical Genetics, 115522 Moscow, RussiaSchizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated <i>TLR9</i> and <i>STING</i> gene expression while blocking <i>RIG-I</i> and <i>AIM2</i> expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients’ blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia.https://www.mdpi.com/2073-4425/13/3/551schizophreniacell-free DNATLR9AIM2STINGRIG-I |
| spellingShingle | Elizaveta S. Ershova Galina V. Shmarina Lev N. Porokhovnik Natalia V. Zakharova George P. Kostyuk Pavel E. Umriukhin Sergey I. Kutsev Vasilina A. Sergeeva Natalia N. Veiko Svetlana V. Kostyuk In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls Genes schizophrenia cell-free DNA TLR9 AIM2 STING RIG-I |
| title | In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls |
| title_full | In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls |
| title_fullStr | In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls |
| title_full_unstemmed | In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls |
| title_short | In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls |
| title_sort | in vitro analysis of biological activity of circulating cell free dna isolated from blood plasma of schizophrenic patients and healthy controls |
| topic | schizophrenia cell-free DNA TLR9 AIM2 STING RIG-I |
| url | https://www.mdpi.com/2073-4425/13/3/551 |
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