Angiotensin II modulates THP-1-like macrophage phenotype and inflammatory signatures via angiotensin II type 1 receptor.

Angiotensin II (Ang II) is a major component of the renin-angiotensin or renin-angiotensin-aldosterone system, which is the main element found to be involved in cardiopathology. Recently, long-term metabolomics studies have linked high levels of angiotensin plasma to inflammatory conditions such as coronary heart disease, obesity, and type 2 diabetes. Monocyte/macrophage cellular function and phenotype orchestrate the inflammatory response in various pathological conditions, most notably cardiometabolic disease. An activation of the Ang II system is usually associated with inflammation and cardiovascular disease; however, the direct effect on monocyte/macrophages has still not been well elucidated. Herein, we have evaluated the cellular effects of Ang II on THP-1-derived macrophages. Ang II stimulated the expression of markers involved in monocyte/macrophage cell differentiation (e.g., CD116), as well as adhesion, cell-cell interaction, chemotaxis, and phagocytosis (CD15, CD44, CD33, and CD49F). Yet, Ang II increased the expression of proinflammatory markers (HLA-DR, TNF-α, CD64, CD11c, and CD38) and decreased CD206 (mannose receptor), an M2 marker. Moreover, Ang II induced cytosolic calcium overload, increased reactive oxygen species, and arrested cells in the G1 phase. Most of these effects were induced via the angiotensin II type 1 receptor (AT1R). Collectively, our results provide new evidence in support of the effect of Ang II in inflammation associated with cardiometabolic diseases.

Quantitative proteomics analysis of COVID-19 patients: Fetuin-A and tetranectin as potential modulators of innate immune responses.

Treatment of severe cases of coronavirus disease 2019 (COVID-19) is extremely important to minimize death and end-organ damage. Here we performed a proteomic analysis of plasma samples from mild, moderate and severe COVID-19 patients. Analysis revealed differentially expressed proteins and different therapeutic potential targets related to innate immune responses such as fetuin-A, tetranectin (TN) and paraoxonase-1 (PON1). Furthermore, protein changes in plasma showed dysregulation of complement and coagulation cascades in COVID-19 patients compared to healthy controls. In conclusion, our proteomics data suggested fetuin-A and TN as potential targets that might be used for diagnosis as well as signatures for a better understanding of the pathogenesis of COVID-19 disease.

Angiotensin II Exaggerates SARS-CoV-2 Specific T-Cell Response in Convalescent Individuals following COVID-19.

Dysregulation of renin-angiotensin systems during coronavirus disease 2019 (COVID-19) infection worsens the symptoms and contributes to COVID-19 severity and mortality. This study sought to investigate the effect of exogenous angiotensin II (Ang-II) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cells response in recovered COVID-19 patients. Human peripheral blood mononuclear cells (PBMCs) were treated with Ang II and then stimulated with a SARS-CoV-2 peptide pool. T-cell responses were measured using flow cytometry, while enzyme-linked immunosorbent assay (ELISA) and intracellular cytokine staining (ICS) assays determined functional capability and polarization. Additionally, the relative level of protein phosphorylation was measured using a phosphokinase array. Our results showed that Ang II treatment significantly increased the magnitude of SARS-CoV-2-specific T-cell response in stimulated PBMCs with a SARS-CoV-2 peptide pool. Moreover, the phosphorylation levels of numerous proteins implicated in cardiovascular diseases, inflammation, and viral infection showed significant increases in the presence of Ang II. The mitogenic stimulation of PBMCs after Ang II and SARS-CoV-2 peptide pool stimulation showed functional polarization of T-cells toward Th1/Th17 and Th17 phenotypes, respectively. Meanwhile, ELISA showed increased productions of IL-1ß and IL-6 in Ang II-stimulated PBMCs without affecting the IL-10 level. To our knowledge, this study is the first to demonstrate that Ang II exaggerates SARS-CoV-2-specific T-cells response. Therefore, during COVID-19 infection, Ang II may aggravate the inflammatory response and change the immune response toward a more inflammatory profile against SARS-CoV-2 infection.

SARS-CoV-2 Coronavirus Spike Protein-Induced Apoptosis, Inflammatory, and Oxidative Stress Responses in THP-1-Like-Macrophages: Potential Role of Angiotensin-Converting Enzyme Inhibitor (Perindopril).

A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process. Graphical: Suggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).

Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues.

PURPOSE: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. METHODS: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. RESULTS: In FFPE tissues, PD-L1 was positive in 7-10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20-30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13-20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. CONCLUSIONS: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.

PD-L1 is overexpressed on breast cancer stem cells through notch3/mTOR axis.

The T-cell inhibitory molecule PD-L1 is expressed on a fraction of breast cancer cells. The distribution of PD-L1 on the different subpopulations of breast cancer cells is not well-defined. Our aim was to study the expression level of PD-L1 on breast cancer stem-like (CSC-like) cells and their differentiated-like counterparts. We used multi-parametric flow cytometry to measure PD-L1 expression in different subpopulations of breast cancer cells. Pathway inhibitors, quantitative immunofluorescence, cell sorting, and western blot were used to investigate the underlying mechanism of PD-L1 upregulation in CSC-like cells. Specifically, PD-L1 was overexpressed up to three folds on breast CSC-like cells compared with more differentiated-like cancer cells. Functional in vitro and in vivo assays show higher stemness of PD-L1hi as compared with PD-L1lo cells. Among different pathways examined, PD-L1 expression on CSCs was partly dependant on Notch, and/or PI3K/AKT pathway activation. The effect of Notch inhibitors on PD-L1 overexpression in CSCs was completely abrogated upon mTOR knockdown. Specific knockdown of different Notch receptors shows Notch3 as a mediator for PD-L1 overexpression on CSCs and important for maintaining their stemness. Indeed, Notch3 was found to be overexpressed on PD-L1hi cells and specific knockdown of Notch3 abolished the effect of notch inhibitors and ligands on PD-L1 expression as well as mTOR activation. Our data demonstrated that overexpression of PD-L1 on CSCs is partly mediated by the notch pathway through Notch3/mTOR axis. We propose that these findings will help in a better design of anti-PD-L1 combination therapies to treat breast cancer effectively.