Phosphatidylserine Receptor Research Articles

The Low-Density Lipoprotein Receptor-Related Protein-1 Is Essential for Dengue Virus Infection

Dengue virus (DENV) causes the most prevalent and rapidly spreading arboviral disease of humans. It enters human cells by receptor-mediated endocytosis. Numerous cell-surface proteins were proposed as DENV entry factors. Among these, the phosphatidylserine receptor TIM-1 is the only one known to mediate virus internalization. However, several cellular models lacking TIM-1 are permissive to DENV infection, suggesting that other receptors exist. Here, we show that the low-density lipoprotein receptor-related protein-1 (LRP1) binds DENV virions by interacting with the DIII of the viral envelope glycoprotein. DENV infection is effectively inhibited by the purified receptor at 5 × 10−8 mol/L, and the interaction of the envelope protein with LRP1 is also blocked by a natural ligand of LRP1. The depletion of LRP1 causes 100-fold lower production of infectious virus than controls. Our results indicate that LRP1 is another DENV receptor, thus becoming an attractive target to evaluate for the development of effective antiviral drugs against DENV.

TIM-4 Identifies Effector B Cells Expressing a RORγt-Driven Proinflammatory Cytokine Module That Promotes Immune Responsiveness.

B cells can express pro-inflammatory cytokines that promote a wide variety of immune responses. Here we show that B cells expressing the phosphatidylserine receptor TIM-4, preferentially express IL-17A, as well as IL-22, IL-6, IL-1β, and GM-CSF - a collection of cytokines reminiscent of pathogenic Th17 cells. Expression of this proinflammatory module requires IL-23R signaling and selective expression of RORγt and IL-17A by TIM-4+ B cells. TIM-4+ B cell-derived-IL-17A not only enhances the severity of experimental autoimmune encephalomyelitis (EAE) and promotes allograft rejection, but also acts in an autocrine manner to prevent their conversion into IL-10-expressing B cells with regulatory function. Thus, IL-17A acts as an inflammatory mediator and also enforces the proinflammatory activity of TIM-4+ B cells. Thus, TIM-4 serves as a broad marker for RORγt+ effector B cells (Beff) and allows further study of the signals regulating Beff differentiation and effector molecule expression.

Development of betabodies: The next generation of phosphatidylserine targeting agents

Externalized phosphatidylserine (PS) is a phospholipid and a selective marker of the tumor microenvironment (TME). It is exposed on the outer leaflet of the plasma membrane of tumor-associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions in part to suppress immune responses by binding to PS receptors expressed on tumor-infiltrating myeloid cells. PS has been targeted with antibodies, such as bavituximab, that bind the phospholipid via a cofactor, β2-glycoprotein 1 (β2GP1); these antibodies showed excellent specificity for tumor vasculature and induce an immune stimulatory environment. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) constructed by linking PS-binding domain V of β2GP1 to the Fc of an IgG2a. Betabodies bind to externalized PS with high affinity (∼1nM), without the requirement of a co-factor and localize robustly to the TME. We demonstrate that betabodies are a direct PS-targeting agent that has the potential to be used as anti-tumor therapy, drug delivery vehicles, and tools for imaging the TME.

The Role of TIM-1 and CD300a in Zika Virus Infection Investigated with Cell-Based Electrical Impedance.

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.

Cell Membrane-Derived Nanoparticles as Biomimetic Nanotherapeutics to Alleviate Fatty Liver Disease.

The prevalence of metabolic dysfunction associated-steatotic liver disease (MASLD) (formerly known as nonalcoholic fatty liver disease; NAFLD) is estimated at around 32% of the world's population, resulting in a major healthcare concern in recent times. Current pharmaceutical methods lack efficacy for the treatment of the disease because of suboptimal pharmacokinetic parameters including poor bioavailability, short half-life, and premature clearance. Designing an efficient drug delivery system that provides a protective environment is critical for addressing these challenges. Such a system should aim to enhance the cellular uptake of drugs, improve their bioavailability, and reduce the chances of rapid clearance. Here, we developed nanoengineered natural cell membrane-derived nanoparticles (CMNs) incorporated with a model drug, rosuvastatin, in the bilayer assembly of CMNs to reduce the accumulation of lipids in hepatocytes, a hallmark of MASLD. We used a cell extrusion technique to develop self-assembled CMNs with precise size control compared to the cell shearing method. Interestingly, the prepared CMNs were found to be nonphagocytic, representing around 1.13% of phosphatidylserine receptors on healthy cells, which allows the possibility of their use as stealth nanoparticles for drug delivery. Furthermore, CMNs exhibit higher drug-loading efficiency, excellent cytocompatibility, and enhanced cellular internalization capabilities. Moreover, we show that the delivery of rosuvastatin-loaded CMNs in the in vitro MASLD model efficiently reduced hepatocyte lipid accumulation, including total cholesterol (26.8 ± 3.1%) and triglycerides (11.8 ± 0.8%), compared to the negative control. Taken together, the nanoengineered biomimetic CMNs enhance the drug's bioactivity in hepatic cells, establishing a foundation for further investigation of this drug delivery system in treating MASLD.

Abstract 2078: Integrin α5β1-mediated Interactions With Fibronectin Enhance Tim3 Expression To Drive Efferocytosis By Macrophages

Despite significant advances in diagnosing and treating cardiovascular disease, atherosclerosis remains the leading cause of death worldwide. While most atherosclerotic plaques remain asymptomatic, a subset can lead to myocardial infarction, stroke, or sudden death. A plethora of evidence has demonstrated that rupture-prone atheromas contains a large necrotic core owed to defective efferocytosis, the process by which apoptotic cells (ACs) are engulfed and cleared by macrophages. The efficient clearance of apoptotic cells blocks post-apoptotic necrosis and prevents the release of tissue-degrading enzymes, immunogenic epitopes, and proinflammatory mediators. As atherosclerosis advances, substantial remodeling of the extracellular matrix (ECM) occurs whereby transitional ECM proteins, notably fibronectin (FN), deposit into the subendothelial matrix normally dominated by collagen IV and laminins. However, whether macrophage interactions with the ECM alters efferocytosis, inflammation resolution, and atherosclerosis remains unknown. Surprisingly, we discovered that macrophage interactions with FN enhance efferocytosis compared to their interactions with basement membrane proteins. Furthermore, we demonstrate that the primary FN receptor, integrin α5β1, is required for FN-mediated efferocytosis and TIM3 expression, (encoded by Havcr2 ), a known phosphatidylserine receptor that binds to ACs. Moreover, treating mice with established atherosclerosis with the integrin α5β1 antagonistic peptide ATN-161 reduced TIM3 expression in macrophages and expanded necrotic core formation. We also found lesional macrophages from human unstable atheromas displayed significantly lower TIM3 expression compared to macrophages within stable plaques. In a mouse model of atherosclerosis regression, where atherogenic stimuli dissipate, efferocytosis is restored, and FN remains, lesional macrophages showed a substantial increase in TIM3 expression. Thus, while FN is traditionally considered atherogenic in endothelial and smooth muscle cells in the initial stages of atherosclerosis, our data uncovers that macrophage adhesion to FN upregulates TIM3 expression, mediating efferocytosis and promoting features of plaque stability.

Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens

Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact.

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P<0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P<0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4291.

Macrophage subpopulation identity in Drosophila is modulated by apoptotic cell clearance and related signalling pathways.

In Drosophila blood, plasmatocytes of the haemocyte lineage represent the functional equivalent of vertebrate macrophages and have become an established in vivo model with which to study macrophage function and behaviour. However, the use of plasmatocytes as a macrophage model has been limited by a historical perspective that plasmatocytes represent a homogenous population of cells, in contrast to the high levels of heterogeneity of vertebrate macrophages. Recently, a number of groups have reported transcriptomic approaches which suggest the existence of plasmatocyte heterogeneity, while we identified enhancer elements that identify subpopulations of plasmatocytes which exhibit potentially pro-inflammatory behaviours, suggesting conservation of plasmatocyte heterogeneity in Drosophila. These plasmatocyte subpopulations exhibit enhanced responses to wounds and decreased rates of efferocytosis when compared to the overall plasmatocyte population. Interestingly, increasing the phagocytic requirement placed upon plasmatocytes is sufficient to decrease thesize of these plasmatocyte subpopulations in the embryo. However, the mechanistic basis for this response was unclear. Here, we examine how plasmatocyte subpopulations are modulated by apoptotic cell clearance (efferocytosis) demands and associated signalling pathways. We show that loss of the phosphatidylserine receptor Simu prevents an increased phagocytic burden from modulating specific subpopulation cells, while blocking other apoptotic cell receptors revealed no such rescue. This suggests that Simu-dependent efferocytosis is specifically involved in determining fate of particular subpopulations. Supportive of our original finding, mutations in amo (the Drosophila homolog of PKD2), a calcium-permeable channel which operates downstream of Simu, phenocopy simu mutants. Furthermore, we show that Amo is involved in the acidification of the apoptotic cell-containing phagosomes, suggesting that this reduction in pH may be associated with macrophage reprogramming. Additionally, our results also identify Ecdysone receptor signalling, a pathway related to control of cell death during developmental transitions, as a controller of plasmatocyte subpopulation identity. Overall, these results identify fundamental pathways involved in the specification of plasmatocyte subpopulations and so further validate Drosophila plasmatocytes as a heterogeneous population of macrophage-like cells within this important developmental and immune model.

Immunomodulation of susceptibility to pneumococcal pneumonia infection in mouse lungs exposed to carbon nanoparticles via dysregulation of innate and adaptive immune responses

Carbon nanotubes (CNTs) are emerging pollutants of occupational and environmental health concern. While toxicological mechanisms of CNTs are emerging, there is paucity of information on their modulatory effects on susceptibility to infections. Here, we investigated cellular and molecular events underlying the effect of multi-walled CNT (MWCNT) exposure on susceptibility to Streptococcus pneumoniae infection in our 28-day sub-chronic exposure mouse model. Data indicated reduced phagocytic function in alveolar macrophages (AMs) from MWCNT-exposed lungs evidenced by lower pathogen uptake in 1-h infection assay. At 24-h post-infection, intracellular pathogen count in exposed AMs showed 2.5 times higher net increase (2-fold in vehicle- versus 5-fold in MWCNT-treated), indicating a greater rate of intracellular multiplication and/or survival due to MWCNT exposure. AMs from MWCNT-exposed lungs exhibited downregulation of pathogen-uptake receptors CD163, Phosphatidyl-serine receptor (Ptdsr), and Macrophage scavenger receptors class A type 1 (Msr1) and type 2 (MSr2). In whole lung, MWCNT exposure shifted the macrophage polarization state towards the immunosuppressive phenotype M2b and increased the CD11c+ dendritic cell population required to activate the adaptive immune response. Notably, the MWCNT pre-exposure dysregulated T-cell immunity, evidenced by diminished CD4 and Th17 response, and exacerbated Th1 and Treg responses (skewed Th17/Treg ratio), thereby favoring the pneumococcal infection. Overall, these findings indicated that MWCNT exposure compromises both innate and adaptive immunity leading to diminished host lung defense against pneumonia infection. To our knowledge, this is the first report on an immunomodulatory role of CNT pre-exposure on pneumococcal infection susceptibility due to dysregulation of both innate and adaptive immunity targets.

Red Blood Cell-Derived Extracellular Vesicles Display Endogenous Antiviral Effects and Enhance the Efficacy of Antiviral Oligonucleotide Therapy.

The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.

The phosphatidylserine receptor TIM1 promotes infection of enveloped hepatitis E virus.

The hepatitis E virus (HEV) is an underestimated RNA virus of which the viral life cycle and pathogenicity remain partially understood and for which specific antivirals are lacking. The virus exists in two forms: nonenveloped HEV that is shed in feces and transmits between hosts; and membrane-associated, quasi-enveloped HEV that circulates in the blood. It is suggested that both forms employ different mechanisms for cellular entry and internalization but little is known about the exact mechanisms. Interestingly, the membrane of enveloped HEV is enriched with phosphatidylserine, a natural ligand for the T-cell immunoglobulin and mucin domain-containing protein 1 (TIM1) during apoptosis and involved in 'apoptotic mimicry', a process by which viruseshijackthe apoptosis pathway to promote infection. We here investigated the role of TIM1 in the entry process of HEV. We determined that HEV infection with particles derived from culture supernatant, which are cloaked by host-derived membranes (eHEV), was significantly impaired after knockout of TIM1, whereas infection with intracellular HEV particles (iHEV) was unaffected. eHEV infection was restored upon TIM1 expression; and enhanced after ectopic TIM1 expression. The significance of TIM1 during entry was further confirmed by viral binding assay, and point mutations of the PS-binding pocket diminished eHEV infection. In addition, Annexin V, a PS-binding molecule also significantly reduced infection. Taken together, our findings support a role for TIM1 in eHEV-mediated cell entry, facilitated by the PS present on the viral membrane, a strategy HEV may use to promote viral spread throughout the infected body.

Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

Novel PEGylated cholephytosomes for targeting fisetin to breast cancer: in vitro appraisal and in vivo antitumoral studies.

Fisetin (FIS) is a multifunctional bioactive flavanol that has been recently exploited as anticancer drug against various cancers including breast cancer. However, its poor aqueous solubility has constrained its clinical application. In the current work, fisetin is complexed for the first time with soy phosphatidylcholine in the presence of cholesterol to form a novel biocompatible phytosomal system entitled "cholephytosomes." To improve fisetin antitumor activity against breast cancer, stearylamine bearing cationic cholephytosomes (mPHY) were prepared and furtherly modified with hyaluronic acid (HPHY) to allow their orientation to cancer cells through their surface exposed phosphatidylserine and CD-44 receptors, respectively. In vitro characterization studies revealed promising physicochemical properties of both modified vesicles (mPHY and HPHY) including excellent FIS complexation efficiency (˷100%), improved octanol/water solubility along with a sustained drug release over 24h. In vitro cell line studies against MDA-MB-231 cell line showed about 10- and 3.5-fold inhibition in IC50 of modified vesicles compared with free drug and conventional drug-phospholipid complex, respectively. Preclinical studies revealed that both modified cholephytosomes (mPHY and HPHY) had comparable cytotoxicity that is significantly surpassing free drug cytotoxicity. TGF-β1and its non-canonical related signaling pathway; ERK1/2, NF-κB, and MMP-9 were involved in halting tumorigenesis. Thus, tailoring novel phytosomal nanosystems for FIS could open opportunity for its clinical utility against cancer.

Dual-potential electrochemiluminescence cytosensor based on a metal-organic framework and ABEI-PEI-Au@AgNPs for the simultaneous determination of phosphatidylserine and epidermal growth factor receptors on an apoptotic cell surface.

A new electrochemiluminescence (ECL) cytosensor is proposed for the simultaneous determination of phosphatidylserine (PS) and epidermal growth factor receptor (EGFR) based on the ECL signals of metal-organic framework-5 (MOF-5) loaded CdS quantum dots and N-(aminobutyl)-N-(ethylisoluminol)-polyethylenimine capped Au and Ag nanoparticles. Apoptosis promotes the exposure of PS and reduces the expression of EGFR in cell membranes. Two spatially resolved areas on dual-disk glassy carbon electrodes were designed to eliminate the interference from different ECL probes. Using HepG2 cells treated with resveratrol to induce apoptosis, thecytosensor exhibited high sensitivity, simplicity, and high reproducibility, demonstrating its potential in drug screening and rapid apoptotic cell detection. The strategy reported provides a promising platform for the highly sensitive cytosensing and convenient screening of clinically relevant anticancer drugs.

<i>In silico</i> Determination of Host-Viral Interaction of Apoptotic Mimicry Pathway Proteins During Hepatitis B Viral Pathogenesis

Viruses are the opportunistic pathogens that have developed several elegant strategies to deploy their host systems for a pathogenic invasion. Viral apoptotic mimicry is characterized by the exposure of host cell phospholipid, the phosphatidylserine which marks the host cell for apoptotic activation. The Hepatitis b virus, an enveloped virus has recently been found to interact with Phosphatidylserine (Ptdser) on the host through its large surface protein experimentally. Nonetheless, the employment of apoptotic mimicry during the pathogenesis of HBV has not been determined. Therefore, in the present study, we attempt the in-silico exploration of the interaction of the apoptosis initiating receptors activated by Phosphatidylserine Receptors such as TIM3, AXL, MERTK and GAS6 by Hepatitis B Virus L protein. Molecular Docking of Phosphatidylserine Receptor were studied to observe protein – protein interaction against Surface L Protein of Hepatitis B Virus by using online protein interaction software. It was found from the in-silico studies that Phosphatidylserine Receptors i.e. TIM3 (PDB: 5F71), AXL (PDB: 5U6B), MERTK (PDB: 2POC) and Gas6 (Growth Arrest Specific protein 6) (PDB: 2C5D) have shown effective binding efficacy against Surface L Protein of Hepatitis B Virus, whereas TIM3 (PDB: F71) and Gas6 (PDB: 2C5D) has shown maximum binding energy with respect to both the software used to analyse the protein-protein docking. This interaction study can form the basis of the experimental attempt in understanding the viral-host protein interaction pattern during hepatitis b viral infection.

Coreceptor AXL Facilitates African Swine Fever Virus Entry via Apoptotic Mimicry.

African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.

Dipalmitoyl-phosphatidylserine-filled cationic maltodextrin nanoparticles exhibit enhanced efficacy for cell entry and intracellular protein delivery in phagocytic THP-1 cells.

Vaccination through the upper respiratory tract is a promising strategy, and particulate antigens, such as antigens associated with nanoparticles, triggered a stronger immune response than the sole antigens. Cationic maltodextrin-based nanoparticles loaded with phosphatidylglycerol (NPPG) are efficient for intranasal vaccination but non-specific to trigger immune cells. Here we focused on phosphatidylserine (PS) receptors, specifically expressed by immune cells including macrophages, to improve nanoparticle targeting through an efferocytosis-like mechanism. Consequently, the lipids associated with NPPG have been substituted by PS to generate cationic maltodextrin-based nanoparticles with dipalmitoyl-phosphatidylserine (NPPS). Both NPPS and NPPG exhibited similar physical characteristics and intracellular distribution in THP-1 macrophages. NPPS cell entry was faster and higher (two times more) than NPPG. Surprisingly, competition of PS receptors with phospho-L-serine did not alter NPPS cell entry and annexin V did not preferentially interact with NPPS. Although the protein association is similar, NPPS delivered more proteins than NPPG in cells. On the contrary, the proportion of mobile nanoparticles (50%), the movement speed of nanoparticles (3 µm/5 min), and protein degradation kinetics in THP-1 were not affected by lipid substitution. Together, the results indicate that NPPS enter cells and deliver protein better than NPPG, suggesting that modification of the lipids of cationic maltodextrin-based nanoparticles may be a useful strategy to enhance nanoparticle efficacy for mucosal vaccination.

The transcription factor XBP1 in dendritic cells promotes the TH2 cell response in airway allergy.

Dendritic cells (DCs) that express T cell immunoglobulin domain molecule-4 (TIM4), a cell surface receptor for phosphatidylserine, induce T helper 2 (TH2) cell responses and allergic reactions. We elucidated the role of the transcription factor X-box-binding protein-1 (XBP1) in the induction of the TH2 cell response through its role in generating TIM4+ DCs. We found that XBP1 was required for TIM4 mRNA and protein expression in airway DCs in response to the cytokine interleukin-2 (IL-2) and that this pathway was required for TIM4 expression on DCs in response to the allergens PM2.5 and Derf1. The IL-2-XBP1-TIM4 axis in DCs contributed to Derf1/PM2.5-induced, aberrant TH2 cell responses in vivo. An interaction between the guanine nucleotide exchange factor Son of sevenless-1 (SOS1) and the GTPase RAS promoted XBP1 and TIM4 production in DCs. Targeting the XBP1-TIM4 pathway in DCs prevented or alleviated experimental airway allergy. Together, these data suggest that XBP1 is required for TH2 cell responses by inducing the development of TIM4+ DCs, which depends on the IL-2-XBP1-SOS1 axis. This signaling pathway provides potential therapeutic targets for the treatment of TH2 cell-dependent inflammation or allergic diseases.

Abstract 1272: Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) has risen to become the 3rd leading cause of cancer related deaths in the United States with a 5-year survival rate of 11% and a median survival of just 4-6 months. PDA is notoriously chemo-resistant and has shown little to no sensitivity to the current immune checkpoint blockade therapy. A novel marker for the tumor microenvironment is phosphatidylserine, first characterized as a selective marker of tumor vasculature. Phosphatidylserine (PS) is a phospholipid exposed on the outer leaflet of the plasma membrane of tumor associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions to suppress the immune response. Exposed PS drives immunosuppression by binding to PS receptors expressed on tumor-infiltrating macrophages and dendritic cells. To this point, PS has been targeted with antibodies, such as Bavituximab, that have shown excellent specificity for tumor vasculature and an immune stimulatory environment evidence by polarization of macrophages to a pro-inflammatory M1-like phenotype, a reduction in myeloid-derived suppressor cells, enhanced maturation of dendritic cells and increased primed T cell activity. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) that exploits the PS-binding domain of β2-glycoprotein 1 (β2GP1), an abundant serum protein that binds PS. Betabodies were constructed by fusing domain V of β2GP1 to the Fc of an IgG2a. Betabodies are smaller than full length antibodies (~85 kDa vs 150 kDa), have good affinity for PS (~1 nM) and bind directly to PS. We have demonstrated that betabodies bind specifically to externalized PS through ELISA, ICC and flow cytometry. We established that betabodies localize robustly and specifically to the tumor microenvironment through in vivo localization experiments in tumor-bearing mice. Further studies will exploit the specificity of betabodies to deliver novel microtubule-disrupting agents to exposed PS in the microenvironment of PDA. Because betabodies are internalized inefficiently, therapeutic payloads will be linked to betabodies via enzyme cleavable linkers. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors. In addition, similarly to PS targeting antibodies, preliminary data in vitro has shown that treatment of both bone marrow derived macrophages and Raw 264.7 with the unconjugated betabodies repolarize M2 macrophages to a M1 phenotype. The mechanism of action has not yet been fully elucidated; however, it may involve the interaction between the β2GP1 domains and endogenous LPS. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors via targeted drug delivery as well as modulation of the immuno-microenvironment like its PS targeting predecessors. Citation Format: Natalie Zara Phinney, Xianming Huang, Mary Lynn Trawick, Kevin Pinney, Rolf Brekken. Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1272.