Macrophages derived from LPS-stimulated monocytes from individuals with subclinical atherosclerosis were characterized by increased pro-inflammatory activity.

OBJECTIVE: Atherosclerosis is characterized by chronic inflammation in the vascular wall. Currently the violation of immune tolerance of innate immune cells is considered as a possible mechanism of chronification of inflammation. The aim of this study is to assess the inflammatory activity and tolerance of monocytes and macrophages in subclinical atherosclerosis. METHODS: A total of 55 individuals free from clinical manifestations of atherosclerosis-associated cardiovascular disease with a presence or absence of atherosclerotic plaques in the carotid arteries were included in this study. CD14+ monocytes were isolated from individuals' blood and stimulated with a single dose of lipopolysaccharide (LPS) on day 1 or with double doses of LPS on day 1 and day 6. The secretion of cytokines TNF, IL-1ß, IL-6, IL-8, IL-10 and CCL2 were evaluated using ELISA. RESULTS: Our findings demonstrate that macrophages derived from LPS-stimulated monocytes in individuals with subclinical atherosclerosis exhibited increased secretion of IL-6, IL-10 and CCL2, which was associated with intima-media thickness, body mass index, but not with individuals' age. Moreover, macrophages from individuals with atherosclerotic plaques exhibited impaired tolerance towards the second LPS stimulation manifested by elevated secretion of the chemoattractant CCL2. CONCLUSION: Increased secretion of these cytokines by macrophages may contribute to chronic local inflammation in the vascular wall by recruiting other immune cells.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

AAV infection of bovine embryos: Novel, simple and effective tool for genome editing.

Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94-58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis.

The current coronavirus disease (COVID-19) pandemic remains one of the most serious public health problems. Increasing evidence shows that infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a very complex and multifaceted disease that requires detailed study. Nevertheless, experimental research on COVID-19 remains challenging due to the lack of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry hACE2 and GFP transgenes floxed by the STOP cassette, allowing them to be used as breeders for the creation of animals with tissue-specific coexpression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas coexpression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre recombinase. After tamoxifen administration, the copy number of the STOP cassette was decreased, and the offspring expressed hACE2 and GFP, confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the roles of different tissues in SARS-CoV-2-associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.