A flow cytometric approach to analyzing mature and progenitor endothelial cells following traumatic brain injury

Traumatic brain injury (TBI) continues to be a major source of death and disability worldwide, and one of the earliest and most profound deficits comes from vascular damage and breakdown of the blood-brain barrier (BBB). Cerebral vascular endothelial cells (cvECs) and endothelial progenitor cells (EPCs) have been shown to play essential roles in vessel repair and BBB stability, although their individual contributions remain poorly defined. We employ TruCount beads with flow cytometry to precisely quantify cvECs, EPCs, and peripheral leukocytes in the murine cortex after controlled cortical impact (CCI) injury. We found a significant reduction in the number of cvECs at 3 days post-injury (dpi), whereas the EPCs and invading peripheral leukocytes were significantly increased compared with sham controls. Proliferation studies demonstrate that both cvECs and EPCs are undergoing cell expansion in the first week post-injury. Furthermore, analysis of protein expression using mean fluorescence intensity found increases in PECAM-1, VEGFR-2, and VE-Cadherin expression per cell at 3 dpi, which is consistent with western blot analysis. Classic methods of cell analysis, such as histological cell counts, in the traumatic injured brain are labor intensive, time-consuming, and potentially biased; whereas flow cytometry provides an efficient, non-biased approach to simultaneously quantify multiple cell types. However, conventional flow cytometry that employs capped events can provide misleading results in CNS injured tissues. We demonstrate that TruCount quantification using flow cytometry is a powerful tool for quantifying mature and progenitor endothelial cell changes after TBI

Macrophage-Derived Inflammation Induces a Transcriptome Makeover in Mesenchymal Stromal Cells Enhancing Their Potential for Tissue Repair

Pre-clinical and clinical studies revealed that mesenchymal stromal cell (MSC) transplants elicit tissue repair. Conditioning MSC prior to transplantation may boost their ability to support repair. We investigated macrophage-derived inflammation as a means to condition MSC by comprehensively analyzing their transcriptome and secretome. Conditioning MSC with macrophage-derived inflammation resulted in 3208 differentially expressed genes, which were annotated with significantly enriched GO terms for 1085 biological processes, 85 cellular components, and 79 molecular functions. Inflammation-mediated conditioning increased the secretion of growth factors that are key for tissue repair, including vascular endothelial growth factor, hepatocyte growth factor, nerve growth factor and glial-derived neurotrophic factor. Furthermore, we found that inflammation-mediated conditioning induces transcriptomic changes that challenge the viability and mobility of MSC. Our data support the notion that macrophage-derived inflammation stimulates MSC to augment their paracrine repair-supporting activity. The results suggest that inflammatory pre-conditioning enhances the therapeutic potential of MSC transplants

Small-molecule costimulatory blockade: organic dye inhibitors of the CD40-CD154 interaction

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40-CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC(50) values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-alpha or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40-CD154 costimulatory interaction but can also provide important structure-activity relationship information and can serve as the starting point of a targeted drug discovery program

Suramin inhibits the CD40–CD154 costimulatory interaction: A possible mechanism for immunosuppressive effects

Suramin is a symmetric polysulfonated naphthylamine–benzamide urea derivative approved for the treatment of trypanosomiasis and onchocerciasis and a known P2 (ATP/UTP purine receptor) antagonist. Here, we report its ability to inhibit the important CD40–CD154 costimulatory interaction required for T cell activation and the development of an effective immune response. In vitro, it inhibited the binding of both human and murine CD154 (CD40L) to their receptor (CD40) even in the presence of protein-containing media and prevented the CD154-induced proliferation of human B cells as well as the corresponding increase in surface expression of CD86, CD80, CD40, and MHC class II in a concentration-dependent manner. Furthermore, in isolated human islets, it also decreased the CD154-induced release of inflammatory cytokines such as IFN-γ, interleukin-6 (IL-6), and IL-8. Suramin was selected for investigation because it has been reported to be an inhibitor of the interaction of TNF-α with its receptor and CD154 is a member of the TNF-family. However, it turned out to be a considerably, about 30-fold, more effective inhibitor of the CD40–CD154 protein–protein interaction than of the corresponding TNF interaction. Its median inhibitory concentration (IC 50 ≈ 50 μM) is somewhat higher than for the P2-receptor, but well within the range of its therapeutic concentration levels. Suramin shows considerable polypharmacology, but its interference with the positive costimulatory interaction might provide a possible, not yet identified mechanism for its ability to suppress T cell activity and induce immunosuppression, which might also have limited its clinical usefulness in the treatment of AIDS and cancer

Human Lung Cell Pyroptosis Following Traumatic Brain Injury

Approximately 30% of traumatic brain injured patients suffer from acute lung injury or acute respiratory distress syndrome. Our previous work revealed that extracellular vesicle (EV)-mediated inflammasome signaling plays a crucial role in the pathophysiology of traumatic brain injury (TBI)-induced lung injury. Here, serum-derived EVs from severe TBI patients were analyzed for particle size, concentration, origin, and levels of the inflammasome component, an apoptosis-associated speck-like protein containing a caspase-recruiting domain (ASC). Serum ASC levels were analyzed from EV obtained from patients that presented lung injury after TBI and compared them to EV obtained from patients that did not show any signs of lung injury. EVs were co-cultured with lung human microvascular endothelial cells (HMVEC-L) to evaluate inflammasome activation and endothelial cell pyroptosis. TBI patients had a significant increase in the number of serum-derived EVs and levels of ASC. Severe TBI patients with lung injury had a significantly higher level of ASC in serum and serum-derived EVs compared to individuals without lung injury. Only EVs isolated from head trauma patients with gunshot wounds were of neural origin. Delivery of serum-derived EVs to HMVEC-L activated the inflammasome and resulted in endothelial cell pyroptosis. Thus, serum-derived EVs and inflammasome proteins play a critical role in the pathogenesis of TBI-induced lung injury, supporting activation of an EV-mediated neural-respiratory inflammasome axis in TBI-induced lung injury

Human Lung Cell Pyroptosis Following Traumatic Brain Injury

Approximately 30% of traumatic brain injured patients suffer from acute lung injury or acute respiratory distress syndrome. Our previous work revealed that extracellular vesicle (EV)-mediated inflammasome signaling plays a crucial role in the pathophysiology of traumatic brain injury (TBI)-induced lung injury. Here, serum-derived EVs from severe TBI patients were analyzed for particle size, concentration, origin, and levels of the inflammasome component, an apoptosis-associated speck-like protein containing a caspase-recruiting domain (ASC). Serum ASC levels were analyzed from EV obtained from patients that presented lung injury after TBI and compared them to EV obtained from patients that did not show any signs of lung injury. EVs were co-cultured with lung human microvascular endothelial cells (HMVEC-L) to evaluate inflammasome activation and endothelial cell pyroptosis. TBI patients had a significant increase in the number of serum-derived EVs and levels of ASC. Severe TBI patients with lung injury had a significantly higher level of ASC in serum and serum-derived EVs compared to individuals without lung injury. Only EVs isolated from head trauma patients with gunshot wounds were of neural origin. Delivery of serum-derived EVs to HMVEC-L activated the inflammasome and resulted in endothelial cell pyroptosis. Thus, serum-derived EVs and inflammasome proteins play a critical role in the pathogenesis of TBI-induced lung injury, supporting activation of an EV-mediated neural-respiratory inflammasome axis in TBI-induced lung injury

Radiation-Induced Enhancement of Antitumor T-cell Immunity by VEGF-Targeted 4-1BB Costimulation

Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents.