Monocyte Cell Surface Research Articles

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

Heparin binding proteins on monocyte cell surfaces regulates pre-inflammatory responses in diabetes.

Many diabetic complications, such as renal and cardiovascular disease, share a common association with extensive and chronic inflammation due to infiltration by activated leukocytes that originate from the bone marrow (BM). Our previous study demonstrated that macrophage progenitor cells that divided in hyperglycemia induced intracellular synthesis of hyaluronan and became pro-inflammatory macrophages (Mpi), and that the presence of low concentrations of heparin (~50 nM) prevented the intracellular HA synthesis and promoted the formation of tissue repair macrophages (Mtr). However, the molecular mechanism underlying heparin's role is still unknown. This study showed that heparin can be internalized by dividing monocyte progenitor cells. Further, there are two most abundant heparin binding proteins, alpha-enolase (ENO-1) and cofilin-1, identified on monocyte cell surfaces. In addition to their conventional roles inside of cells, ENO-1 and cofilin-1 can be found on cell surfaces and are also involved in autoimmune diseases. Thus, this study provides new insight into heparin's role in regulating monocyte and macrophage function.

Melatonin Alters Innate Immune Function in Infants with Neonatal Encephalopathy

Introduction: Melatonin has been suggested an adjunctive therapy in neonatal encephalopathy (NE). Melatonin reduces oxidative stress and neutrophil activation; however, the immunological effects in NE have not been studied. Methods: Infants with NE and neonatal controls were prospectively recruited. Whole blood was sampled in the first week of life. Following endotoxin and or melatonin treatment, diurnal variation was measured by RT PCR for circadian rhythm genes (brain and Muscle Arnt-Like protein [BMAL1], circadian locomotor output cycles kaput [CLOCK], Nuclear Receptor Subfamily 1 Group D Member 2 [REV Erβ], and cryptochrome circadian clock [CRY]). Neutrophil and monocyte cell surface markers of activation CD11b, reactive oxygen intermediates (ROIs), and Toll-like receptor (TLR)-4 were also examined by flow cytometry in matching samples. Results: Serum and RNA samples from forty infants were included (controls n = 20; NE n = 20) over the first week of life. Melatonin reduced neutrophil CD11b and TLR-4 expression in response to LPS in infants with NE compared to controls. There were no differences in ROIs. BMAL1 and CLOCK baseline gene expression levels were similar. BMAL1 was significantly decreased with LPS stimulation in NE. There was no significant diurnal variation in melatonin, neutrophil, and monocyte function or circadian genes. Conclusions: Melatonin alters immune function ex vivo in infants with NE. Infants with NE have altered immune circadian responses following LPS stimulation, which have potential for modulation.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Surface translocation of ACE2 and TMPRSS2 upon TLR4/7/8 activation is required for SARS-CoV-2 infection in circulating monocytes

Infection of human peripheral blood cells by SARS-CoV-2 has been debated because immune cells lack mRNA expression of both angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease type 2 (TMPRSS2). Herein we demonstrate that resting primary monocytes harbor abundant cytoplasmic ACE2 and TMPRSS2 protein and that circulating exosomes contain significant ACE2 protein. Upon ex vivo TLR4/7/8 stimulation, cytoplasmic ACE2 was quickly translocated to the monocyte cell surface independently of ACE2 transcription, while TMPRSS2 surface translocation occurred in conjunction with elevated mRNA expression. The rapid translocation of ACE2 to the monocyte cell surface was blocked by the endosomal trafficking inhibitor endosidin 2, suggesting that endosomal ACE2 could be derived from circulating ACE2-containing exosomes. TLR-stimulated monocytes concurrently expressing ACE2 and TMPRSS2 on the cell surface were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir, TMPRSS2 inhibitor camostat, and anti-ACE2 antibody. Mass cytometry showed that ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with its hyperactivation and PD-L1 expression. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression makes circulating monocytes permissive to SARS-CoV-2 infection.

Galectins Differentially Regulate the Surface Glycosylation of Human Monocytes.

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14+CD16− monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes.

Decreased low-density lipoprotein receptor-related protein 1 expression in pro-inflammatory monocytes is associated with subclinical atherosclerosis.

Subclinical atherosclerosis (SCA) occurs in asymptomatic individuals. Blood peripheral monocytes are involved in the development of atherosclerosis. Circulating monocytes acquire pro-inflammatory profiles, and they are involved in the early stages of atherosclerosis development. Low-density lipoprotein Receptor-related Protein 1 (LRP1) is expressed in monocytes, mainly in classical and intermediate subsets. Although LRP1 is highly expressed in macrophages and vascular smooth muscle cells (VSMCs) in atherosclerotic plaque formation, its expression in circulating monocytes has not been studied in SCA. The aim of this study was to characterize the LRP1 expression level in circulating monocytes of individuals with SCA and compared with individuals with low (LR) and intermediate (IR) risk of cardiovascular diseases, both without evidence of atherosclerotic lesions in carotid and coronary arteries. LRP1 and additional markers (CD11b, CD11c, and CD36) at cell surface of monocytes were analyzed by flow cytometry assays, whereas LRP1 and pro-inflammatory factors gene expressions were measured in isolated monocytes by quantitative RT-PCRs. Both LRP1 protein and LRP1 mRNA were significantly reduced in monocytes in SCA and IR respect to LR. Conversely, CD36, CD11b, and CD11c monocytic markers showed no significant changes between the different study groups. Finally, increased gene expressions of TNF-α and IL-1β were detected in monocytes of SCA, which were associated with decreased LRP1 expression at the cell surface in total monocytes. In summary, we propose that the decreased LRP1 expression at cell surface in total monocytes with pro-inflammatory profile is associated with the development of atherosclerosis in asymptomatic individuals.

The monocyte cell surface as a novel site of autoantigen generation in Rheumatoid Arthritis

Abstract Citrullination is recognized as a key pathogenic process in rheumatoid arthritis (RA), as evidenced by the formation of anti-citrullinated protein antibodies (APCAs) in the majority of patients; however, the mechanisms that result in citrullinated autoantigen generation are not fully understood. Although the citrullinating enzyme peptidylarginine deiminase IV (PAD4) is predominantly expressed by neutrophils and monocytes, the contribution of monocytes to the citrullinated autoantigen pool has been underexplored. In this study, we utilized multiple complementary methods including flow cytometry, immunofluorescence, and transmission electron microscopy, which revealed a predominantly extranuclear localization of PAD4 in monocytes with a fraction present on the cell surface. Surface PAD4 was enzymatically active and citrullinated both extracellular fibrinogen and endogenous surface proteins in a calcium dose–dependent manner. In addition, human monoclonal ACPAs cloned from patients with RA recognized fibrinogen citrullinated by monocyte-surface PAD4. Mass spectrometry analysis of citrullinated proteins from the cell surface fraction revealed CD11b to be a novel PAD4 substrate. Citrullinated CD11b was recognized by autoantibodies in 60% of ACPA+ RA patients compared to 6% of healthy controls (p=0.0021) and 0% of ACPA− RA patients (p≤0.001). Taken together, our study demonstrates that PAD4 is expressed on the surface of monocytes in an enzymatically active state that renders the monocyte surface a novel site of citrullinated autoantigen generation in RA. Supported by funds received from Bristol-Myers Squibb.

CD33 is downregulated by influenza virus H1N1pdm09 and induces ROS and the TNF-α, IL-1β, and IL-6 cytokines in human mononuclear cells.

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6h, and reactive oxygen species (ROS) production increased at 1.5h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection.

Citral modulates human monocyte responses to Staphylococcus aureus infection

Staphylococcus aureus is a Gram-positive bacterium that is considered an important human pathogen. Due to its virulence and ability to acquire mechanisms of resistance to antibiotics, the clinical severity of S. aureus infection is driven by inflammatory responses to the bacteria. Thus, the present study aimed to investigate the modulating role of citral in inflammation caused by S. aureus infection. For this, we used an isolate obtained from a nasal swab sample of a healthy child attending a day-care centre in Vitória da Conquista, Bahia, Brazil. The role of citral in modulating immunological factors against S. aureus infection was evaluated by isolating and cultivating human peripheral blood mononuclear cells. The monocytes were treated with 4%, 2%, and 1% citral before and after inoculation with S. aureus. The cells were analysed by immunophenotyping of monocyte cell surface molecules (CD54, CD282, CD80, HLA-DR, and CD86) and cytokine dosage (IL-1β, IL-6, IL-10, IL-12p70, IL-23, IFN-γ, TGF-β, and TNF-α), and evaluated for the expression of 84 genes related to innate and adaptive immune system responses. GraphPad Prism software and variables with P values < 0.05, were used for statistical analysis. Our data demonstrated citral’s action on the expression of surface markers involved in recognition, presentation, and migration, such as CD14, CD54, and CD80, in global negative regulation of inflammation with inhibitory effects on NF-κB, JNK/p38, and IFN pathways. Consequently, IL-1β, IL-6, IL-12p70, IL-23, IFN-γ, and TNF-α cytokine expression was reduced in groups treated with citral and groups treated with citral at 4%, 2%, and 1% and infected, and levels of anti-inflammatory cytokines such as IL-10 were increased. Furthermore, citral could be used as a supporting anti-inflammatory agent against infections caused by S. aureus. There are no data correlating citral, S. aureus, and the markers analysed here; thus, our study addresses this gap in the literature.

Serglycin induces osteoclastogenesis and promotes tumor growth in giant cell tumor of bone

Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex–mediated thrombotic disorders.

Tissue Factor Expression As a Prognostic Marker in Patients with B-Cell Malignant Lymphoma

Background. Studies in tumor cell lines have shown an increased expression of TF. Expression of TF in adenocarcinoma of the pancreas is associated with tumor progression. It has been described that between 5% and 10% of patients with malignant lymphoma (ML) may develop VTD. The mechanisms of ML-associated thrombosis are unknown, however, expression of functional TF may increase as much as 75-fold in peripheral blood monocytes, particularly during relapse. As expected, thrombosis was found in patients with the highest TF levels. The role of TF in the biology of ML has not yet been determined. Considering indirect evidence, it seems that, besides its procoagulant effects, TF may have a role in tumor progression.Objective. The purpose of the present study was to elucidate whether the interaction of tissue factor and TF/VIIa could induce relapsed in non Hodgkin Lymphoma patients.Patients and Methods. We therefore performed a prospective study (cohort) to evaluate the risk of TF in non Hodgkin Lymphoma patients without a previous episode of venous thromboembolism. 59 patients 30 women and 29 men were enrolled in this study. The median age was 43.3 ± 14.5 (range 20 to 59). The TF and FT/VIIa activity was performed on fresh plasma (American Diagnostics, USA), and other tests (PC, PS, AT and activated protein C resistance) (STAGO, Asnieres, Francia).Results.59 patients with B-cell ML, 30 women and 29 men with a mean age of 43.3 years (ranges 20 to 59). According to the Ann Arbor Staging system, 44% percent of patients (n=26) had a stage IV and only 14% at stage I. ML was mostly limited to lymph nodes (74%).Results of hemostatic markers were between the normal ranges. However, concentrations of factors involved in the initiation phase of fluid phase of blood coagulation (TF, FT/VIIa complex, and FVII) had an abnormal distribution. None case with APCR was found.Comparison between complete remission and relapsing patients. Thirty-one patients (52.5%) achieved complete remission at the end of follow-up and 28 (47.5%) relapsed. Mean follow up for patients with complete remission was 48 months vs. 18 months for relapsing patients (p <0.001). After comparing TF concentrations between the groups, relapsing patients had higher levels of TF (p=0.005). TF/FVIIa complex levels were also significantly higher in patients with relapsing disease (p=0.019). As expected LDH levels were significantly lower in patients with complete remission (p=0.0001).TF levels and response to treatment. Twenty-nine patients had normal TF levels. Relapses were more frequently found in patients with elevated TF concentrations (n=19) as compared with patients with normal TF levels (n=9) (p=0.013). Elevated TF/FVIIa complexes levels were also more frequently seen among patients with relapsing ML (n=32) as compared with patients with complete remission (n=27) (p=0.046).As expected, other factors associated with a poor prognosis were DHL levels (p=0.001) and IPI (p=0.001).Discussion. TF and TF/FVIIIa concentrations were significantly different in patients who achieved complete remission as compared with those with relapse after treatment. As expected for the staging of patients included LDH serum levels as well as IPI score were also significantly different between these two groups of patients. Moreover, estimation of the relative risks associated with relapsing disease showed that also these four variables were associated with relapsing disease. However, linear regression analysis showed that TF, LDH, and IPI but not TF/FVIIa complexes remained as a risk factor for relapse in patients with ML. To our knowledge, we have demonstrated for the first time that high TF concentration is a prognosis factor for relapse in patients with B-cell ML.Our results seem consistent with previous reports regarding an association between the increased TF expression and advanced staging of the malignant disease. It has been demonstrated the presence of high plasma levels of TF and FVIII in patients with solid tumors suggesting that it may be an important mechanism to activate blood coagulation system in cancer patients. Tumor cells may also induce TF expression on the cell surface of peripheral blood monocytes and macrophages. DisclosuresNo relevant conflicts of interest to declare.

Diminished Non-Classical Monocytes in the Blood Associate with Disease Severity in Alcoholic Hepatitis.

ObjectiveAlcoholic hepatitis (AH) holds a high mortality, and vast macrophage infiltration of the liver is involved in the progressive liver injury. No efficient medical treatment exists, and macrophages may be a future treatment target. Here, we examine associations between non-classical monocyte subsets and cell surface markers of migration with disease activity in patients with severe AH.MethodsWe analyzed samples from two cohorts of patients with AH. Cohort 1 included 15 AH patients, followed for 30 days, and 8 healthy controls (HCs). Cohort 2 included 23 AH patients, followed for 90 days, and 9 HCs. Peripheral blood mononuclear cells (PBMCs) from both cohorts were analyzed by flow cytometry. Liver biopsies from cohort 2 were analyzed by RNA sequencing.ResultsCirculating non-classical monocytes in all but absent in patients with AH compared to HC in both cohorts (both p<0.0001). The frequency of non-classical monocytes was significantly associated with Maddrey’s discriminant function (mDF) (r=−0.79, p=0.0008, cohort 1), Child–Pugh score (CP) (r=−0.56, p=0.03, cohort 1), Model for End-Stage Liver Disease (MELD) (r=−0.54, p=0.02, cohort 2) and C-reactive protein (CRP) (r=−0.51, p=0.027, cohort 2). The surface expression of CD11b was increased on non-classical monocytes in patients with AH compared to HC (p<0.0001) (cohort 1). The mRNA expression of CD11b was increased in liver biopsies in patients with AH compared to HC (cohort 2) (p<0.0001).ConclusionIn this study, we describe an almost complete depletion of circulating non-classical monocytes in the blood in two independent cohorts of patients with AH, which may be associated with a possible harmful recruitment of these cells to the liver. These results contribute to a better understanding of the disease, which hopefully can lead to therapies that target the acute inflammatory response leading to severe AH.

Classical complement and inflammasome activation converge in CD14highCD16- monocytes in HIV associated TB-immune reconstitution inflammatory syndrome.

Inflammasome-derived cytokines, IL-1β and IL-18, and complement cascade have been independently implicated in the pathogenesis of tuberculosis (TB)-immune reconstitution inflammatory syndrome (TB-IRIS), a complication affecting HIV+ individuals starting antiretroviral therapy (ART). Although sublytic deposition of the membrane attack complex (MAC) has been shown to promote NLRP3 inflammasome activation, it is unknown whether these pathways may cooperatively contribute to TB-IRIS. To evaluate the activation of inflammasome, peripheral blood mononuclear cells (PBMCs) from HIV-TB co-infected patients prior to ART and at the IRIS or equivalent timepoint were incubated with a probe used to assess active caspase-1/4/5 followed by screening of ASC (apoptosis-associated speck-like protein containing a CARD domain) specks as a readout of inflammasome activation by imaging flow cytometry. We found higher numbers of monocytes showing spontaneous caspase-1/4/5+ASC-speck formation in TB-IRIS compared to TB non-IRIS patients. Moreover, numbers of caspase-1/4/5+ASC-speck+ monocytes positively correlated with IL-1β/IL-18 plasma levels. Besides increased systemic levels of C1q and C5a, TB-IRIS patients also showed elevated C1q and C3 deposition on monocyte cell surface, suggesting aberrant classical complement activation. A clustering tSNE analysis revealed TB-IRIS patients are enriched in a CD14highCD16- monocyte population that undergoes MAC deposition and caspase-1/4/5 activation compared to TB non-IRIS patients, suggesting complement-associated inflammasome activation during IRIS events. Accordingly, PBMCs from patients were more sensitive to ex-vivo complement-mediated IL-1β secretion than healthy control cells in a NLRP3-dependent manner. Therefore, our data suggest complement-associated inflammasome activation may fuel the dysregulated TB-IRIS systemic inflammatory cascade and targeting this pathway may represent a novel therapeutic approach for IRIS or related inflammatory syndromes.

Regulation of Human Monocyte Surface Antigen Expression. I. Up-Modulation of Mo3e Antigen Expression on U-937 and HL-60 Cells Stimulated by Pharmacologic Activators of Protein Kinase C

Mo3e is a protein (p 50,80) that is expressed on the surface of human monocytic cells after exposure in vitro to soluble activating factors that include bacterial lipopolysaccharide, muramyl dipeptide, and phorbol myristate acetate (PMA). The surface expression of Mo3e may represent a cellular event that occurs in response to the formation of "secondary messengers" that include diacylglycerol, inositol trisphosphate, and calcium ions. This postulate is based on the stimulatory effect of agents that can mimic the activity of endogenous diacylglycerol (PMA and other biologically active phorbol compounds, mezerein, and L-alpha-1,2 dioctanoylglycerol) and inositol trisphosphate (ionomycin) on Mo3e expression by U-937 and HL-60 cells. The inhibitory effect of phospholipid-active calmodulin inhibitors (trifluoperazine, chlorpromazine, and dibucaine), calcium antagonists (nicardipine and TMB-8), and EGTA further support the involvement of phospholipid- and calcium-dependent protein kinase (protein kinase C) and calcium ions in the up-modulation of Mo3e surface expression.

Apolipoprotein M promotes the anti-inflammatory effect of high-density lipoprotein by binding to scavenger receptor BI.

BackgroundInflammation participates pivotally in the pathogenesis of atherosclerosis. Apolipoprotein M (apoM) is a high-density lipoprotein (HDL)-associated plasma protein that affects HDL metabolism and shows various anti-inflammatory functions in atherosclerosis. In this study, we aim to determine whether apoM is expressed in peripheral blood mononuclear cells (PBMCs) and promoted the anti-inflammatory effect of HDL by combing with scavenger receptor BI (SR-BI).MethodsThe expression of apoM in PBMCs is detected by a confocal fluorescence microscope and flow cytometry. The interactions between apoM and SR-BI are detected with co-immunoprecipitation. The multiplexed Luminex xMAP assay detects the inflammatory factors induced by apoM+ HDL and apoM– HDL in inflammatory cell model.ResultsApoM is expressed on CD14+ monocytes, CD3+ T cells, and CD19+ B cells, CD16+ and CD56+ NK cells. CD14+ monocytes have the highest ratio of apoM+ cells. ApoM+ HDL, apoM– HDL, and recombinant apoM protein could be co-precipitated with SR-BI on the surface of human THP-1 monocytic leukemia cells. In vitro, apoM+ HDL induces significantly less expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β than apoM– HDL.ConclusionsApoM was expressed on all PBMCs. ApoM interacted with SR-BI on THP-1. ApoM+ HDL has a more significant anti-inflammatory effect than apoM– HDL.

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype.

Simple SummaryWhen activated by tumour antigen-specific IgEs, monocytes may contribute to the restriction of cancer growth in animal models of cancer. In this study, we investigated the effects of IgE stimulation on the activation state of human monocytes from healthy subjects and from patients with cancer. Cross-linking of cognate Fc receptors by IgE on human monocytes potentiated: (a) upregulation of activatory and down regulation of regulatory monocyte cell surface markers; (b) phosphorylation of intracellular protein kinases in monocytes previously described to be downstream of the mast cell and basophil FcεRI signalling pathway; (c) ovarian cancer patient monocyte-mediated cytotoxic killing of tumour cells and release of pro-inflammatory mediators, potentially associated with favourable patient survival. IgE can therefore activate human monocytes to acquire a pro-inflammatory phenotype capable of mediating effector functions against tumour cells. This may contribute to the mechanism of cancer immunotherapy using IgE antibodies.IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

Phenotypic and Proliferative Analysis of T Cell and Monocyte Populations in Patients with Intermediate/High/Very High-Risk MDS Compared to Age-Matched Healthy Donors

Background: Myelodysplastic syndromes (MDS) have been associated with alterations in the bone marrow microenvironment and include abnormal innate and adaptive immune responses. The extent to which these changes contribute to the pathogenesis of MDS is unclear. The heterogeneous nature of MDS makes it difficult to distinguish changes in intrinsic cell function for microenvironmental alterations in the mutated myeloid compartment. T cells, which are in the lymphoid compartment and less frequently contain MDS mutations, can potentially be used to evaluate immune dysfunction in the presence of malignant myeloid cells. Here we examine the immunophenotypes and proliferative function of T lymphocyte and monocyte populations from higher-risk MDS patients. Methods: Peripheral blood mononuclear cells (PBMC) from patients with IPSS-R Intermediate/High/Very High-risk MDS (herein referred to collectively as higher-risk MDS) and age-matched healthy donors (n = 15 and n = 12, respectively) were polyclonally stimulated with an antibody against CD3 (OKT3) in the presence of IL-2 (50 U/mL). Phenotypic analysis was performed at baseline and 46 hours after the addition of OKT3 and IL-2. T cell proliferation was measured by staining PBMC with proliferation dye (CellTrace Violet) and measuring fold proliferation after eight days in culture. Proliferation statistical comparisons were made by unpaired t-test. Phenotypic parameters were measured by flow cytometry and statistical analysis performed with the Kruskal-Wallis one-way analysis. Results: As previously reported, higher-risk MDS patient T cells had decreased capacity for proliferation when compared to healthy donor counterparts (Figure 1A p = 0.0096). This difference in proliferative capacity was not associated with either the level of PD-1 on the surface of T cells (r = 0.2956) or the level of PD-L1 on monocytes (r = 0.3859) in stimulated PBMCs. Patients with higher-risk MDS tended to have lower PD-L1 surface expression on monocytes compared to healthy donor counterparts, albeit not significantly (Figure 1C p = 0.1). However, PD-1 expression on T cells was comparable between groups both before and after stimulation (Figure 1B CD4 cells: p = 0.58; CD8 cells p = 0.58). This suggests that T cell proliferation from patients with higher-risk MDS might not be inhibited through the PD-1/PD-L1 axis. Of note, higher-risk MDS patient monocytes expressed less CD80 and HLA-DR both pre- and post-stimulation when compared to healthy donors (Figure 1C CD80: p = 0.0001 and 0.037; DR: p = 0.012 and 0.019, respectively), implying a potential dysfunction in the monocytic stimulatory and antigen presentation capacity. PD-L1 surface expression is low at baseline (an average of &amp;lt;5% for both donors and higher-risk MDS); upon stimulation, there is a high correlation between HLA-DR and PD-L1 in healthy donors (p = 0.0003, r = 0.859). Additionally, there is less upregulation of PD-L1 on the surface of monocytic cells in the higher-risk MDS group and subsequently less correlation between PD-L1/HLA-DR (p = 0.03) in stimulated PBMC samples. Taken together, these results show a decreased baseline expression and/or ability to upregulate monocyte activation markers after stimulation. We hypothesize that these changes might contribute to the observation that higher-risk MDS patient T cells are less capable of proliferation. Conclusion: There was no significant difference in PD-1 and PD-L1 expression between higher-risk MDS and healthy donor patients, despite reduced T cell proliferation among patients with MDS. Additionally, decreased levels of CD80 and HLA-DR on intermediate/high-risk MDS patient monocytes suggests a suppressed ability to upregulate activation markers, possibly leading to decreased T cell proliferation. Targeting the PD-1/PD-L1 axis alone may not be therapeutic in patients with higher-risk MDS, and we hypothesize that the immune evasion observed in higher-risk MDS patients may not be due exclusively to increases in inhibitory interactions like PD-L1 and PD-1, but may result from a decrease in stimulatory receptors, such as CD80, and antigen presentation molecules, such as HLA-DR. Disclosures Fields: PersImmune, Inc.: Ended employment in the past 24 months. Ferrari:PersImmune, Inc.: Ended employment in the past 24 months. Tarke:PersImmune, Inc.: Ended employment in the past 24 months. Luger:Arcturus Therapeutics: Current Employment. Lane:PersImmune, Inc.: Current Employment. Vitiello:PersImmune, Inc.: Current equity holder in private company, Ended employment in the past 24 months. Bejar:Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company.