LOX-1 unlocks human plasma cell potential.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is best known for promoting atherosclerosis. In this issue of Immunity, Joo et al. (2014) find that dendritic cells triggered through LOX-1 can directly support plasmablast production via the production of the cytokines APRIL and BAFF.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

CD66b+ monocytes represent a proinflammatory myeloid subpopulation in cancer.

Myeloid-derived suppressor cells (MDSC) populate the peripheral blood and contribute to immune regulation in cancer. However, there is limited knowledge on the myeloid cell types with proinflammatory capacities that may serve as opponents of MDSC. In the circulation of cancer patients, a monocyte subpopulation was identified with a specific immunophenotype and transcriptomic signature. They were predominantly CD14+CD33hiCD16-/+HLA-DR+/hi cells that typically expressed CD66b. In accordance with the transcriptomics data, NALP3, LOX-1 and PAI-1 levels were also significantly upregulated. The CD66b+ monocytes displayed high phagocytic activity, matrix adhesion and migration, and provided costimulation for T cell proliferation and IFN-γ secretion; thus, they did not suppress T cell responses. Irrespective of clinical stage, they were identified in various cancers. In conclusion, the CD66b+ monocytes represent a novel myeloid subpopulation which is devoid of immune regulatory influences of cancer and displays enhanced proinflammatory capacities.

Low-density neutrophils in chronic graft versus host disease (cGVHD) are primarily immature CD10- and enhance T cell activation.

Chronic graft-versus-host disease (cGVHD) is a frequent complication of allogeneic haematopoietic stem cell transplantation. Low density neutrophils (LDNs) in autoimmunity, which shares disease features with cGVHD, are proinflammatory, whereas those in cancer and sepsis suppress T cell immunity. Mature LDNs can be distinguished from immature LDNs on the basis of expression of CD10 and suppressive neutrophils can be identified using lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. The functionality of LDNs in cGVHD has not been specifically investigated. Here, we have determined the relative contribution of immature and mature neutrophils to LDNs in cGVHD and assessed whether these were suppressive or potentially proinflammatory. Peripheral blood LDNs and normal density neutrophils (NDNs) from 30 cGVHD patients and NDNs from 10 healthy controls (HCs) were immunophenotyped by flow cytometry. The ability of LDNs and NDNs to influence T cell proliferation and cytokine production in co-cultures was quantified. To further characterize LDNs, their propensity to undergo constitutive apoptosis and differentiate ex vivo was assessed. LDNs were elevated in cGVHD versus HCs, heterogeneous in phenotype, with a predominance of immature CD10- cells in most patients, but some mature CD10+ LOX-1+ LDNs were also detected. LDNs enhanced autologous T cell proliferation, interleukin (IL)-6 and interferon (IFN)-γ production. LDN, but not NDN, CD10 expression was inversely correlated with LOX-1, which correlated with IL-6 production. LDNs resisted apoptosis and differentiated into antigen-presenting/neutrophil-hybrid-like cells, which co-expressed major histocompatibility complex (MHC) class II HLA-DR and immuno-inhibitory programmed cell death ligand 1 (PD-L1), but did not suppress T cell proliferation. These data suggest LDNs in cGVHD are predominantly immature, proinflammatory and may have pathogenic potential.

Superiority in Rhesus Macaques of Targeting HIV-1 Env gp140 to CD40 versus LOX-1 in Combination with Replication-Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses.

We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly-ICLC adjuvant either alone or coadministered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly-ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to a P value of 0.01) than in a group without poly-ICLC. The responses were robust and cross-reactive and contained antibodies specific to multiple epitopes within gp140, including the C1, C2, V1, V2, and V3, C4, C5, and gp41 immunodominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to tier 1 viruses and antibody-dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines plus poly-ICLC. Together, these results indicate that prime-boost immunization via NYVAC-KC and either anti-CD40.Env gp140/poly-ICLC or anti-LOX-1.Env gp140/poly-ICLC induced balanced antibody and T cell responses against HIV-1 Env. Coadministration of NYVAC-KC with the DC-targeting vaccines increased T cell responses but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability than with LOX-1.IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. In this study, we have tested in nonhuman primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justify further development for future human clinical trials.

L5-LDL from ST-elevation myocardial infarction patients induces IL-1ß production via LOX-1 and NLRP3 inflammasome activation in macrophages.

L5-LDL, the most electronegative LDL associated with major cardiovascular risks, significantly rises in patients with ST-segment elevation myocardial infarction (STEMI). The inflammatory nature of atherosclerotic vascular diseases has prompted us to investigate whether L5-LDL induces the production of inflammatory cytokines, especially vascular ischemia-related interleukin (IL)-1ß, in the pathogenesis of STEMI. Clinical data showed that plasma levels of L5-LDL and IL-1ß were higher in the STEMI patients than in the controls (P < 0.05). In THP-1-derived human macrophages, L5-LDL significantly increased the levels of both IL-1ß and cleaved caspase-1, indicating the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes by L5-LDL. Knockdown of NLRP3 and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) resulted in decreased L5-LDL-induced IL-1ß. Furthermore, knock down of the lectin-type oxidized LDL receptor (LOX-1) in THP-1 cells attenuated L5-LDL-induced activation of NF-κB and caspase-1, leading to subsequent inhibition of IL-1ß in macrophages. Furthermore, blockade LOX-1 with neutralizing antibody also inhibited L5-LDL-induced IL-1ß in human peripheral blood mononuclear cell-derived macrophages. In conclusion, L5-LDL induces IL-1ß production in macrophages by activation of NF-κB and caspase-1 through the LOX-1-dependent pathway. This study represents the evidence linking L5-LDL and the inflammatory cytokine IL-1ß in STEMI, and identifies L5-LDL as a novel therapeutic target in acute myocardial infarction. NEW & NOTEWORTHY: This study represents the evidence linking L5-LDL and the inflammatory cytokine IL-1ß in ST-segment elevation myocardial infarction (STEMI). We elucidate the molecular mechanism underlying L5-LDL-induced production of IL-1ß in macrophages. The results showed that L5-LDL induced activation of caspase-1 and NF-κB through the lectin-type oxidized LDL receptor (LOX-1)-dependent pathway, leading to the production of IL-1ß.

The role of the lectin-like oxLDL receptor (LOX-1) in traffic-generated air pollution exposure-mediated alteration of the brain microvasculature in Apolipoprotein (Apo) E knockout mice.

Recent studies have shown a strong correlation between air pollution-exposure and detrimental outcomes in the central nervous system, including alterations in blood brain barrier (BBB) integrity, neuroinflammation, and neurodegeneration. However, the mechanisms mediating these pathologies have not yet been fully elucidated. We have previously reported that exposure to traffic-generated air pollution results in increased circulating oxidized low-density lipoprotein (oxLDL), associated with alterations in BBB integrity, in atherosclerotic Apolipoprotein E null (ApoE-/-) mice. Thus, we investigated the role of the lectin-like oxLDL receptor (LOX)-1 in mediating these deleterious effects in ApoE-/- mice exposed to a mixture of gasoline and diesel engine exhaust (MVE: 100 PM µg/m3) for 6 h/d, 7d/week, for 30 d by inhalation. Concurrent with exposures, a subset of mice were treated with neutralizing antibodies to LOX-1 (LOX-1 Ab) i.p., or IgG (control) i.p., every other day during exposures. Resulting brain microvascular integrity, tight junction (TJ) protein expression, matrix metalloproteinase (MMP)-9/-2 activity, ROS, and markers of cellular adhesion and monocyte/macrophage sequestration were assessed. MVE-exposure resulted in decreased BBB integrity and alterations in microvascular TJ protein expression, associated with increased LOX-1 expression, MMP-9/-2 activities, and lipid peroxidation, each of which was attenuated with LOX-1 Ab treatment. Furthermore, MVE-exposure induced cerebral microvascular ROS and adhesion molecules, expression of which was not normalized through LOX-1 Ab-treatment. Such findings suggest that alterations in brain microvascular structure and integrity observed with MVE-exposure may be mediated, at least in part, via LOX-1 signaling.

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1ß as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells.

Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 µg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 µg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 µg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.

Lectin-like oxidized LDL receptor-1 expresses in mouse bone marrow-derived mesenchymal stem cells and stimulates their proliferation.

The bone marrow-derived mesenchymal stem cells (bmMSCs) have been widely used in cell transplant therapy, and the proliferative ability of bmMSCs is one of the determinants of the therapy efficiency. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) as a transmembrane protein is responsible for binding, internalizing and degrading oxidized low density lipoprotein (ox-LDL). It has been identified that LOX-1 is expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts and monocytes. In these cells, low concentration of ox-LDL (<40 µg/mL) stimulates their proliferation via LOX-1 activation. However, it is poor understood that whether LOX-1 is expressed in bmMSCs and which role it plays. In this study, we investigated the status of LOX-1 expression in bmMSCs and its function on bmMSC proliferation. Our results showed that primary bmMSCs exhibiting a typical fibroblast-like morphology are positive for CD44 and CD90, but negative for CD34 and CD45. LOX-1 in both mRNA and protein levels is highly expressed in bmMSCs. Meanwhile, bmMSCs exhibit a strong potential to take up ox-LDL. Moreover, LOX-1 expression in bmMSCs is upregulated by ox-LDL with a dose- and time-dependent manner. Presence of ox-LDL also enhances the proliferation of bmMSCs. Knockdown of LOX-1 expression significantly inhibits ox-LDL-induced bmMSC proliferation. These findings indicate that LOX-1 plays a role in bmMSC proliferation.

LOX-1 as a natural IFN-alpha-mediated signal for apoptotic cell uptake and antigen presentation in dendritic cells.

The identification of molecules responsible for apoptotic cell (AC) uptake by dendritic cells (DCs) and induction of T-cell immunity against AC-associated antigens is a challenge in immunology. DCs differentiated in the presence of interferon-alpha (IFN-alpha-conditioned DCs) exhibit a marked phagocytic activity and a special attitude in inducing CD8(+) T-cell response. In this study, we found marked overexpression of the scavenger receptor oxidized low-density lipoprotein receptor 1 (LOX-1) in IFN-alpha-conditioned DCs, which was associated with increased levels of genes belonging to immune response families and high competence in inducing T-cell immunity against antigens derived from allogeneic apoptotic lymphocytes. In particular, the capture of ACs by IFN-alpha DCs led to a substantial subcellular rearrangement of major histocompatibility complex class I and class II molecules, along with enhanced cross-priming of autologous CD8(+) T cells and CD4(+) T-cell activation. Remarkably, AC uptake, CD8(+) T-cell cross-priming, and, to a lesser extent, priming of CD4(+) T lymphocytes were inhibited by a neutralizing antibody to the scavenger receptor LOX-1 protein. These results unravel a novel LOX-1-dependent pathway by which IFN-alpha can, under both physiologic and pathologic conditions, render DCs fully competent for presenting AC-associated antigens for cross-priming CD8(+) effector T cells, concomitantly with CD4(+) T helper cell activation.

Lectin-like oxidized low-density lipoprotein receptor-1 delivers heat shock protein 60-fused antigen into the MHC class I presentation pathway.

Heat shock protein (Hsp) 60 elicits a potent proinflammatory response in the innate immune system and has been proposed as a danger signal of stressed or damaged cells to the immune system. Previous studies reported CD14, TLR2, and TLR4 as mediators of signaling but probably not of binding. Although the receptor for Hsp60 was proposed to be saturable and specific on macrophages, it is not well defined. In the current study, we found that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), as a receptor for Hsp60, could bind and internalize Hsp60 via the C terminus of Hsp60. Yeast two-hybrid assay revealed that the second beta-sheet containing the long-loop region of LOX-1 played an important role in this interaction. Furthermore, LOX-1 might be engaged as a common receptor for different Hsp60 species. Bone marrow-derived dendritic cells could cross-present Hsp60-fused OVA Ag on MHC class I molecules via LOX-1. Inhibition of the recognition of Hsp60 by LOX-1 decreases Hsp60-mediated cross-presentation of OVA and specific CTL response and protective tumor immunity in vivo. Taken together, these results demonstrate that LOX-1 functions as a receptor for Hsp60 and is involved in the delivery of Hsp60-fused Ag into the MHC class I presentation pathway.

The oxidized low-density lipoprotein receptor mediates vascular effects of inhaled vehicle emissions.

RATIONALE: To determine vascular signaling pathways involved in inhaled air pollution (vehicular engine emission) exposure-induced exacerbation of atherosclerosis that are associated with onset of clinical cardiovascular events. OBJECTIVES: To elucidate the role of oxidized low-density lipoprotein (oxLDL) and its primary receptor on endothelial cells, the lectin-like oxLDL receptor (LOX-1), in regulation of endothelin-1 expression and matrix metalloproteinase activity associated with inhalational exposure to vehicular engine emissions. METHODS: Atherosclerotic apolipoprotein E knockout mice were exposed by inhalation to filtered air or mixed whole engine emissions (250 µg particulate matter [PM]/m(3) diesel + 50 µg PM/m(3) gasoline exhausts) 6 h/d for 7 days. Concurrently, mice were treated with either mouse IgG or neutralizing antibodies to LOX-1 every other day. Vascular and plasma markers of oxidative stress and expression proatherogenic factors were assessed. In a parallel study, healthy human subjects were exposed to either 100 µg PM/m(3) diesel whole exhaust or high-efficiency particulate air and charcoal-filtered "clean" air (control subjects) for 2 hours, on separate occasions. MEASUREMENTS AND MAIN RESULTS: Mixed emissions exposure increased oxLDL and vascular reactive oxygen species, as well as LOX-1, matrix metalloproteinase-9, and endothelin-1 mRNA expression and also monocyte/macrophage infiltration, each of which was attenuated with LOX-1 antibody treatment. In a parallel study, diesel exhaust exposure in volunteer human subjects induced significant increases in plasma-soluble LOX-1. CONCLUSIONS: These findings demonstrate that acute exposure to vehicular source pollutants results in up-regulation of vascular factors associated with progression of atherosclerosis, endothelin-1, and matrix metalloproteinase-9, mediated through oxLDL-LOX-1 receptor signaling, which may serve as a novel target for future therapy.

LOX-1 deletion improves neutrophil responses, enhances bacterial clearance, and reduces lung injury in a murine polymicrobial sepsis model.

Inflammatory tissue injury and immunosuppression are the major causes of death in sepsis. Novel therapeutic targets that can prevent excessive inflammation and improve immune responses during sepsis could be critical for treatment of this devastating disease. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a membrane protein expressed in endothelial cells, has been known to mediate vascular inflammation. In the present study, we demonstrated that LOX-1 deletion markedly improved the survival rate in a murine model of polymicrobial sepsis. Wild-type (LOX-1(+/+)) and LOX-1 knockout (LOX-1(-/-)) mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. LOX-1 deletion significantly reduced systemic inflammation and inflammatory lung injury during sepsis, together with decreased production of proinflammatory cytokines and reduced lung edema formation. Furthermore, LOX-1 deletion improved host immune responses after the induction of sepsis, as indicated by enhanced bacterial clearance. Interestingly, we were able to demonstrate that LOX-1 is expressed in neutrophils. LOX-1 deletion prevented neutrophil overreaction and increased neutrophil recruitment to infection sites after sepsis induction, contributing at least partly to increased immune responses in LOX-1 knockout mice. Our study results indicate that LOX-1 is an important mediator of inflammation and neutrophil dysfunction in sepsis.

Involvement of CD91 and scavenger receptors in Hsp70-facilitated activation of human antigen-specific CD4+ memory T cells.

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress-inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen-specific CD4(+) T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen-specific cells were detected combining HLA-DRB1*0401 or HLA-DRB1*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen-specific CD4(+) T cell responses.

Prior exposure to oxidized low-density lipoprotein limits apoptosis in subsequent generations of endothelial cells by altering promoter methylation.

Oxidized LDL (ox-LDL) plays a critical role in atherogenesis, including apoptosis. As hypercholesterolemia causes epigenetic changes resulting in long-term phenotypic consequences, we hypothesized that repeated and continuous exposure to ox-LDL may alter the pattern of apoptosis in human umbilical vein endothelial cells (HUVECs). We also analyzed global and promoter-specific methylation of apoptosis-related genes. As expected, ox-LDL evoked a dose-dependent increase in apoptosis in the first passage HUVECs that was completely abrogated by lectin-like ox-LDL receptor (LOX-1)-neutralizing antibody. Ox-LDL-induced apoptosis was associated with upregulation of proapoptotic LOX-1, ANXA5, BAX, and CASP3 and inhibition of antiapoptotic BCL2 and cIAP-1 genes accompanied with reciprocal changes in the methylation of promoter regions of these genes. Subsequent passages of cells displayed attenuated apoptotic response to repeat ox-LDL challenge with blunted gene expression and exaggerated methylation of LOX-1, BAX, ANXA5, and CASP3 genes (all P < 0.05 vs. first exposure to ox-LDL). Treatment of cells with LOX-1 antibody before initial ox-LDL treatment prevented both gene-specific promoter methylation and expression changes and reduction of apoptotic response to repeat ox-LDL challenge. Based on these data, we conclude that exposure of HUVECs to ox-LDL induces epigenetic changes leading to resistance to apoptosis in subsequent generations and that this effect may be related to the LOX-1-mediated increase in DNA methylation.

Lectin-like oxidized LDL receptor 1 is involved in CRP-mediated complement activation.

BACKGROUND: C-reactive protein (CRP) is purported to be a risk factor that acts independently of LDL cholesterol in predicting all-cause mortality in patients with ischemic heart disease. Lectin-like oxidized LDL receptor 1 (LOX-1) impairs endothelial function and exacerbates myocardial injury. We recently demonstrated that CRP increased vascular permeability through direct binding to LOX-1. Here we examined, using a hypertensive rat model, whether LOX-1 is involved in CRP-induced complement activation. METHODS AND RESULTS: In the cultured LOX-1-expressing cell line hLOX-1-CHO, CRP increased complement activation, but did not do so in native CHO cells. Depleting C1q from serum abolished CRP-induced complement activation. Incubation of CRP with serum on immobilized recombinant LOX-1 similarly showed that CRP activated C1q-requiring classical complement pathway in a LOX-1-dependent manner. Interestingly, the interaction between CRP and LOX-1 was dependent on Ca²âº ion and competed with phosphocholine, suggesting that LOX-1 bound to the B-face of CRP with a phosphocholine-binding domain. This was in contrast to Fcγ receptors, to which CRP bound in A-face with complement-binding domain. In vivo, intradermal injection of CRP to hypertensive SHRSP rats induced complement activation detected by C3d deposition and leukocyte infiltration around the injected area. Anti-LOX-1 antibody reduced the extent of complement activation and leukocyte infiltration. CONCLUSIONS: LOX-1 appears to be involved in CRP-induced complement activation, and thus may serve to locate the site of CRP-induced complement activation and inflammation.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

High efficiency CD91- and LOX-1-mediated re-presentation of gp96-chaperoned peptides by MHC II molecules.

Exogenous antigens enter antigen-presenting cells through non-specific mechanisms and are presented by the MHC II molecules. We show here that antigens chaperoned by the heat shock protein gp96 enter dendritic cells and B cells through a specific, CD91- and LOX-1-mediated mechanism, and are presented by MHC II molecules, in addition to MHC I molecules as previously demonstrated. Receptor utilization results in high efficiency uptake such that antigen concentrations as low as 10(-9) M, if chaperoned by gp96, lead to productive antigen presentation. Chaperoning by gp96 increases the efficiency of uptake over un-chaperoned peptides by up to two orders of magnitude. Consistent with these studies in vitro, immunization of mice with gp96-peptide complexes (containing 5 ng peptide) results in generation of a peptide-specific CD4+ T cell response. The high efficiency suggests a mechanism in which dendritic cells, exposed in vivo to heat shock protein-chaperoned peptides liberated by virus-infected host cells or by the lysis of infecting bacteria, may prime and expand specific CD4+ responses.

LOX index, a novel predictive biochemical marker for coronary heart disease and stroke.

BACKGROUND: Lectin-like oxidized LDL receptor 1 (LOX-1) is implicated in atherothrombotic diseases. Activation of LOX-1 in humans can be evaluated by use of the LOX index, obtained by multiplying the circulating concentration of LOX-1 ligands containing apolipoprotein B (LAB) times that of the soluble form of LOX-1 (sLOX-1) [LOX index = LAB x sLOX-1]. This study aimed to establish the prognostic value of the LOX index for coronary heart disease (CHD) and stroke in a community-based cohort. METHODS: An 11-year cohort study of 2437 residents age 30-79 years was performed in an urban area located in Japan. Of these, we included in the analysis 1094 men and 1201 women without history of stroke and CHD. We measured LAB and sLOX-1 using ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody and with 2 monoclonal antibodies against LOX-1, respectively. RESULTS: During the follow-up period, there were 68 incident cases of CHD and 91 cases of stroke (with 60 ischemic strokes). Compared with the bottom quartile, the hazard ratio (HR) of the top quartile of LOX index was 1.74 (95% CI 0.92-3.30) for stroke and 2.09 (1.00-4.35) for CHD after adjusting for sex, age, body mass index, drinking, smoking, hypertension, diabetes, non-HDL cholesterol, and use of lipid-lowering agents. Compared with the bottom quartile of LOX index, the fully adjusted HRs for ischemic stroke were consistently high from the second to the top quartile: 3.39 (95% CI 1.34-8.53), 3.15 (1.22-8.13) and 3.23 (1.24-8.37), respectively. CONCLUSIONS: Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.