Biology subjects

Dekaban, G. A.

Publications and source records attributed to Dekaban, G. A..

8 recordsLinked to original sources

bioRxiv2024

Syngeneic adipose-derived stromal cells modulate the immune response but have limited persistence within decellularized adipose tissue implants in C57BL/6 mice

The delivery of adipose-derived stromal cells (ASCs) on cell-instructive decellularized adipose tissue (DAT) scaffolds is a promising strategy for stimulating host-derived soft tissue regeneration. However, a better understanding of the mechanisms through which ASCs modulate regeneration in vivo is needed to harness these cells more effectively. In this study, DAT scaffolds, both with and without seeded syngeneic DsRED+ mouse ASCs, were implanted into immunocompetent C57BL/6 mice. Downstream analyses focused on assessing donor ASC persistence and phenotype, as well as the effects of ASC seeding on host macrophage polarization and the perfused host vascular network. Notably, most donor ASCs were cleared from the scaffolds by 2 weeks. Mass spectrometry-based proteomics indicated that the transplanted ASCs maintained their pre-implantation phenotype up to 1 week in vivo, suggesting that the cells were not undergoing programmed cell death. A higher fraction of the infiltrating host macrophages expressed CD68 and Arginase-1 in the ASC-seeded implants up to 1-week post-implantation. Interestingly, a small population of phagocytic macrophages, identified by uptake of DsRED protein, was present in the DAT implants in the first 2 weeks and showed enhanced expression of CD68, Arginase-1, and CD163, along with reduced expression of iNOS. MicroCT angiography revealed a similar perfused vessel network in the seeded and unseeded groups at 4- and 8-weeks post-implantation. Overall, seeding with syngeneic ASCs modulated the host macrophage response to the DAT bioscaffolds at early timepoints, but did not impact long-term regenerative outcomes, potentially due to the rapid clearance of the donor cell population in this model.

cell biology↗

bioRxiv2023

MERTK Coordinates Efferocytosis by Regulating Integrin Localization and Activation

Efferocytosis - the phagocytic removal of apoptotic cells - is a central component of tissue homeostasis, and in many tissues is mediated by the efferocytic receptor MERTK expressed by macrophages. Although MERTK is critical for efferocytosis, the mechanism by which it directs the engulfment of apoptotic cells is largely unknown. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of [~]180 nm clusters of MERTK, {beta}2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of {beta}2 integrins or their opsonins, while {beta}2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of {beta}2 integrins from the low to high-affinity form via a PI3-kinase-dependent signaling pathway. MERTK and {beta}2 integrins then form a highly structured synapse in which MERTK is retained by ligand-induced clustering in the synapse centre, while {beta}2 integrins and actin form a Src family kinase-, ILK- and FAK-dependent expanding ring which defines the leading edge of the synapse that ultimately engulfs the apoptotic cell. The identification of the MERTK membrane-proximal signaling pathway and the role of {beta}2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.

immunology↗

bioRxiv2023

In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging

BackgroundDespite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo post-injection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Preliminary studies have identified magnetic particle imaging (MPI) as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide-(SPIO)-labeled DC. Herein, we describe a lymph node- (LN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. MethodsBoth adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype using flow cytometry. Ad SPIO+ and SPIO+ DC were fluorescently-labeled and injected into C57BL/6 mouse hind footpads (n=6). Two days later, in vivo DC migration was quantified using whole animal, popliteal LN- (pLN)-focused, and ex vivo pLN MPI scans. ResultsNo significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI successfully quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed MPI signal was pLN-localized and due to originally-injected Ad SPIO+ and SPIO+ DC. ConclusionsWe overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases. With this improved method, we detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable pre-clinical imaging modality to assess DC-based cancer immunotherapeutic efficacy.

immunology↗

bioRxiv2023

SARS-CoV-2 NSP5 Antagonizes the MHC II Antigen Presentation Pathway by Hijacking Histone Deacetylase 2

SARS-CoV-2 interferes with antigen presentation by downregulating MHC II on antigen presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. This downregulation of CIITA is independent of NSP5s proteolytic activity, but rather, NSP5 delivers HDAC2 to IRF3 at an IRF binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response. ImportanceSARS-CoV-2 alters the expression of many immunoregulatory proteins to limit and delay the host antiviral response, thereby producing a more severe and longer-lasting infection. Preventing and limiting the activation of helper T cells by reducing MHC II expression on antigen presenting cells is one of these strategies, but while this mechanism was identified early in the pandemic, the mechanism allowing SARS-CoV-2 to limit MHC II expression has remained unclear. Herein, we demonstrate that this occurs via a tripartite interaction between viral NSP5 and host HDAC2 and IRF3, where a complex of NSP5 and HDAC2 is recruited to IRF3 bound to the promoter of CIITA--the master regulator of MHC II expression--with the delivery of HDAC2 then mediating the deacetylation of the CIITA promoter and the suppression of MHC II expression.

immunology↗

bioRxiv2022

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

microbiology↗

bioRxiv2021

Magnetic Particle Imaging is a sensitive in vivo imaging modality for the quantification of dendritic cell migration

Immunotherapies, such as dendritic cell- (DC-)based therapies, are useful for treating cancer as an alternative to or in combination with traditional therapies. Cells must migrate to lymphoid organs to be effective and the magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With less than 10% of cells expected to reach their destination, there is a need for an imaging modality capable of sensitively and quantitatively detecting cells. MRI has been used to track DC using iron and 19F methods, with limitations. Quantification of iron-induced signal loss is indirect and challenging; 19F signal is directly quantifiable but lacks sensitivity. Magnetic Particle Imaging (MPI) directly detects superparamagnetic iron oxide nanoparticles (SPIO) and enables quantitation of low numbers of SPIO-labeled cells. Here we describe the first study using MPI to track and quantify the migration of DC, injected into the footpads of C57BL/6 mice, to the popliteal lymph nodes (pLNs). As DC migrate from the site of injection to the lymph nodes, we measured a decrease in signal in the footpads and an increase in signal at the pLNs. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI and histology. By measuring the iron mass per cell in samples of labeled cells, we were able to provide an estimate of cell number for each source of signal and we report a sensitivity of approximately 4000 cells in vivo and 2000 cells ex vivo. For some mice, MPI was compared to cellular MRI. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for many pre-clinical studies using a highly concentrated tracer bolus that over shadows nearby lower signals. This study demonstrates the clear advantage of MPI to detect and quantify cells in vivo, bridging the gap left by cellular MRI, and all other in vivo imaging modalities, and opening the door for quantitative imaging of cellular immunotherapies.

biophysics↗

bioRxiv2021

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

immunology↗

bioRxiv2020

Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2

Characterization of the humoral response to SARS-CoV-2, the etiological agent of Covid-19, is essential to help control the infection. In this regard, we and others recently reported that the neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we selected plasma from a cohort of Covid-19 convalescent patients and selectively depleted immunoglobulin A, M or G before testing the remaining neutralizing capacity of the depleted plasma. We found that depletion of immunoglobulin M was associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of IgM.

microbiology↗