ABSTRACT
OBJECTIVE: In this study, we sought a comprehensive understanding of myeloid cell types driving fibrosis in diffuse cutaneous systemic sclerosis (dcSSc) skin. METHODS: We analyzed the transcriptomes of 2,465 myeloid cells from skin biopsy specimens from 12 dcSSc patients and 10 healthy control subjects using single-cell RNA sequencing. Monocyte-derived dendritic cells (mo-DCs) were assessed using immunohistochemical staining and immunofluorescence analyses targeting ficolin-1 (FCN-1). RESULTS: A t-distributed stochastic neighbor embedding analysis of single-cell transcriptome data revealed 12 myeloid cell clusters, 9 of which paralleled previously described healthy control macrophage/DC clusters, and 3 of which were dcSSc-specific myeloid cell clusters. One SSc-associated macrophage cluster, highly expressing Fcγ receptor IIIA, was suggested on pseudotime analysis to be derived from normal CCR1+ and MARCO+ macrophages. A second SSc-associated myeloid population highly expressed monocyte markers FCN-1, epiregulin, S100A8, and S100A9, but was closely related to type 2 conventional DCs on pseudotime analysis and identified as mo-DCs. Mo-DCs were associated with more severe skin disease. Proliferating macrophages and plasmacytoid DCs were detected almost exclusively in dcSSc skin, the latter clustering with B cells and apparently derived from lymphoid progenitors. CONCLUSION: Transcriptional signatures in these and other myeloid populations indicate innate immune system activation, possibly through Toll-like receptors and highly up-regulated chemokines. However, the appearance and activation of myeloid cells varies between patients, indicating potential differences in the underlying pathogenesis and/or temporal disease activity in dcSSc.
Subject(s)
Dendritic Cells/immunology , Lectins/immunology , Macrophages/immunology , Monocytes/immunology , Receptors, IgG/immunology , Scleroderma, Diffuse/immunology , Humans , Lectins/biosynthesis , Macrophages/metabolism , Monocytes/metabolism , Receptors, IgG/biosynthesis , Severity of Illness Index , FicolinsABSTRACT
Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.
Subject(s)
Antibodies/immunology , CD56 Antigen/biosynthesis , Hodgkin Disease/metabolism , Hodgkin Disease/therapy , Receptors, IgG/biosynthesis , Adult , Aged , Antineoplastic Agents/pharmacology , Epstein-Barr Virus Infections/complications , Female , GPI-Linked Proteins/biosynthesis , Herpesvirus 4, Human/metabolism , Hodgkin Disease/complications , Humans , Immunotherapy , In Vitro Techniques , Killer Cells, Natural/metabolism , Leukocytes, Mononuclear/cytology , Lymphocytes/metabolism , Lysosomal-Associated Membrane Protein 1/biosynthesis , Male , Middle Aged , Phenotype , Prospective Studies , Rituximab/pharmacologyABSTRACT
BACKGROUND: The preoperative diagnosis of periprosthetic joint infection (PJI) depends on a series of blood biomarkers. Previous studies have shown that CD64 expression on blood neutrophils and monocytes has a good diagnostic efficacy for diagnosing systemic and local infections. The purpose of the present study was to investigate the role of blood CD64 in the diagnosis of PJI. METHODS: On the basis of estimations made before the study was performed, 62 patients were recruited for joint revision surgery following the failure of primary hip or knee replacement. Venous blood was obtained within 24 hours after patient admission, and flow cytometry was performed to evaluate the CD64 expression of 3 groups of white blood cells (WBCs). CD64 expression was measured as CD64 mean fluorescence intensity (CD64MFI). The neutrophil CD64 index (nCD64 index; neutrophil CD64MFI [nCD64MFI]/lymphocyte CD64MFI [lCD64MFI]) and monocyte CD64 index (mCD64 index; monocyte CD64MFI [mCD64MFI]/lCD64MFI) were then calculated. The C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) at admission, synovial fluid indicators, leukocyte esterase test results, intraoperative histological results, and tissue or synovial fluid culture results were recorded. According to the modified Musculoskeletal Infection Society (MSIS) criteria, patients were divided into the PJI group and the non-PJI group. These blood indicators were then analyzed for the diagnosis of PJI. RESULTS: The PJI group included 18 patients, and the non-PJI group included 44 patients. The diagnostic value of the area under the receiver operating characteristic curve (AUC) was low for lCD64MFI, the nCD64 index, and the mCD64 index. The diagnostic value for nCD64MFI was moderate, with an AUC of 0.735 (95% confidence interval [CI], 0.595 to 0.874; p = 0.004). The diagnostic value for mCD64MFI was high, with an AUC of 0.898 (95% CI, 0.821 to 0.975; p < 0.001). The cutoff value for mCD64MFI was 28,968, with a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 1, 0.75, 0.62, and 1, respectively. This result was confirmed by internal validation with a different antibody. CONCLUSIONS: Flow cytometry can be used for patient screening before revision surgery, and blood mCD64MFI is a promising indicator for PJI. LEVEL OF EVIDENCE: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.
Subject(s)
Monocytes/metabolism , Prosthesis-Related Infections/blood , Prosthesis-Related Infections/diagnosis , Receptors, IgG/biosynthesis , Aged , Female , Humans , Male , Middle Aged , Pilot Projects , Prospective StudiesABSTRACT
Background: Immune checkpoint inhibitors (ICIs) frequently cause thyroid dysfunction but their underlying mechanism remains unclear. We have previously demonstrated increased circulating natural killer (NK) cells and human leukocyte antigen (HLA)-DR surface expression on inflammatory intermediate CD14+CD16+ monocytes in programmed cell death protein-1 (PD-1) inhibitor-treated patients. This study characterizes intrathyroidal and circulating immune cells and class II HLA in ICI-induced thyroiditis. Methods: This is a single-center prospective cohort study of 10 patients with ICI-induced thyroiditis by flow cytometry of thyroid fine needle aspirates (n = 9) and peripheral blood (n = 7) as compared with healthy thyroid samples (n = 5) and healthy volunteer blood samples (n = 44); HLA class II was tested in n = 9. Results: ICI-induced thyroiditis samples demonstrated overall increased T lymphocytes (61.3% vs. 20.1%, p = 0.00006), CD4-CD8- T lymphocytes (1.9% vs. 0.7%, p = 0.006), and, as a percent of T lymphocytes, increased CD8+T lymphocytes (38.6% vs. 25.7%; p = 0.0259) as compared with healthy thyroid samples. PD-1 inhibitor-induced thyroiditis had increased CD4+PD1+ T lymphocytes (40.4% vs. 0.8%; p = 0.021) and CD8+PD1+ T lymphocytes (28.8% vs. 1.5%; p = 0.038) in the thyroid compared with the blood. Circulating NK cells, certain T lymphocytes (CD4+CD8+, CD4-CD8- T, gamma-delta), and intermediate monocytes were increased in ICI-induced thyroiditis. Six patients typed as HLA-DR4-DR53 and three as HLA-DR15. Conclusions: ICI-induced thyroiditis is a T lymphocyte-mediated process with intra-thyroidal predominance of CD8+ and CD4-CD8- T lymphocytes. The HLA haplotypes may be involved but need further evaluation. These findings expand the limited understanding of ICI-induced thyroiditis, which could be further translated to guide immunomodulatory therapies for advanced thyroid cancer.
Subject(s)
Immune Checkpoint Inhibitors/pharmacology , Lymphocyte Subsets , T-Lymphocytes/cytology , Thyroid Gland/immunology , Thyroid Neoplasms/complications , Thyroiditis/complications , Adult , Aged , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Female , GPI-Linked Proteins/biosynthesis , HLA-DR Antigens/immunology , Haplotypes , Humans , Immunophenotyping , Inflammation , Killer Cells, Natural/cytology , Lipopolysaccharide Receptors/biosynthesis , Male , Middle Aged , Programmed Cell Death 1 Receptor/biosynthesis , Prospective Studies , Receptors, IgG/biosynthesis , Reference Values , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/immunology , Thyroiditis/blood , Thyroiditis/immunologyABSTRACT
Background: MicroRNAs (miRNAs) are a class of critical epigenetic regulators involved in several autoimmune diseases. Our previous study reported an miR-326-induced increase in T helper (Th) 17 cells in a mouse model of Hashimoto's thyroiditis (HT), but the pathogenic effect of miR-326 in HT patients has not been verified. The goal of the present study was to explore the pathogenic role of miR-326 and its underlying molecular mechanism in HT patients. Methods: A total of 58 HT patients and 55 normal controls were enrolled in this study. We examined whether Th17 cells and miR-326 were aberrantly altered in the peripheral blood mononuclear cells (PBMCs) of HT patients with flow cytometry and real-time polymerase chain reaction. Levels of membrane interleukin (IL)-23R (mIL-23R) were determined by flow cytometry and Western blot to explore the critical role of mIL-23R in the development of Th17 cells. Isolated CD3+ T cells were used to further investigate the ectodomain shedding of mIL-23R by a disintegrin and metalloprotease (ADAM17). Furthermore, miR-326 inhibitor and mimics were transfected into PBMCs derived from HT patients and healthy controls to verify the regulation of ADAM17 by miR-326. Results: We observed elevated miR-326 levels in the PBMCs of HT patients compared with those in the PBMCs of healthy controls. Consistent with IL-23-induced STAT3 overactivation, substantially more HT patient-derived PBMCs differentiated into Th17 cells under polarization culture conditions, which may, at least in part, have resulted from enhanced mIL-23R levels. Furthermore, ADAM17, an ectodomain sheddase of mIL-23R, was targeted and negatively regulated by miR-326. Inhibiting ADAM17 might attenuate the ectodomain shedding of mIL-23R. Conclusions: Our findings suggest that the effect of miR-326 on the IL-23/IL-23R/Th17 cell axis in HT patients might be partially due to the targeting of ADAM17.
Subject(s)
ADAM17 Protein/metabolism , Hashimoto Disease/immunology , Interleukin-23/metabolism , MicroRNAs/genetics , Receptors, Interleukin/metabolism , Th17 Cells/metabolism , Adult , Cell Differentiation , Electrochemistry , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Hashimoto Disease/metabolism , Healthy Volunteers , Humans , Leukocytes, Mononuclear/metabolism , Luminescence , Male , Middle Aged , Phosphorylation , Protein Domains , Receptors, IgG/biosynthesis , T-Lymphocytes/cytologySubject(s)
Appendicitis/metabolism , Granulocytes/metabolism , Monocytes/metabolism , Receptors, IgG/biosynthesis , Child , Child, Preschool , Critical Care , Female , Humans , Male , Pilot Projects , Prospective StudiesABSTRACT
OBJECTIVE: To investigate whether soluble growth stimulation expressed gene 2 (sST2), a prognostic marker in cardiovascular and inflammatory disorders, is associated with neurological injury after aneurysmal subarachnoid hemorrhage (SAH). METHODS: We studied SAH patients from 2 independent cohorts. Outcome assessments included functional status at 90 days using the modified Rankin Scale (mRS), mortality, and delayed cerebral ischemia (DCI). The relationships between sST2 plasma level and outcome measures were assessed in both cross-sectional and longitudinal analysis. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry. RESULTS: In the discovery cohort, sST2 predicted 90-day mRS 3-6 (C index = 0.724, p < 0.001) and mortality in Kaplan-Meier analysis (p < 0.001). The association with functional status was independent of age, sex, World Federation of Neurosurgical Societies score, modified Fisher score, treatment modality, and cardiac comorbidities (adjusted odds ratio = 2.28, 95% confidence interval = 1.04-5.00, p = 0.039). Higher sST2 concentration was observed in those patients with DCI (90.8 vs 53.7ng/ml, p = 0.003). These associations were confirmed in a replication cohort. In patients with high sST2, flow cytometry identified decreased expression of CD14 (4.27 × 105 ± 2,950 arbitrary unit (AU) vs 5.64 × 105 ± 1,290 AU, p < 0.001), and increased expression of CD16 (39,960 ± 272 AU vs 34,869 ± 183 AU, p < 0.001). INTERPRETATION: Plasma sST2 predicts DCI, functional outcome, and mortality after SAH, independent of clinical and radiographic markers. Elevated sST2 is also associated with changes in CD14+ CD16+ monocytes. ANN NEUROL 2019;86:384-394.
Subject(s)
Inflammation/genetics , Interleukin-1 Receptor-Like 1 Protein/metabolism , Subarachnoid Hemorrhage/genetics , Case-Control Studies , Cerebrospinal Fluid/metabolism , Cross-Sectional Studies , Female , Genetic Predisposition to Disease/genetics , Humans , Inflammation/complications , Inflammation Mediators/metabolism , Interleukin-1 Receptor-Like 1 Protein/blood , Interleukin-1 Receptor-Like 1 Protein/genetics , Lipopolysaccharide Receptors/biosynthesis , Longitudinal Studies , Male , Middle Aged , Monocytes/metabolism , Outcome Assessment, Health Care , Receptors, IgG/biosynthesis , Subarachnoid Hemorrhage/complicationsABSTRACT
The sinusoidal endothelial cells present in the liver (LSECs) are tipically characterized by the presence of pores (fenestrae). During some pathological conditions LSECs undergo "capillarization", a process characterized by loss of fenestrations and acquisition of a vascular phenotype. In chronic liver disease capillarization has been reported to precede the development of fibrosis. LSECs modification in the setting of HCV infection is currently poorly investigated. Considering that HCV accounts for important changes in hepatocytes and in view of the intimate connection between hepatocytes and LSECs, here we set out to study in great detail the LSECs modifications in individuals with HCV-dependent chronic hepatitis. Electron microscopy analysis, and evaluation of CD32, CD31 and caveolin-1 expression showed that in HCV infection LSECs display major morphological changes but maintain their phenotypical identity. Capillarization was observed only in cases at initial stages of fibrosis. Our findings showed that the severity of LSECs modifications appears to be correlated with hepatocytes damage and fibrosis stage providing novel insight in the pathogenesis of HCV-chronic hepatitis.
Subject(s)
Endothelial Cells , Hepacivirus/metabolism , Hepatitis C, Chronic , Liver , Adult , Aged , Caveolin 1/biosynthesis , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Female , Gene Expression Regulation , Hepatitis C, Chronic/metabolism , Hepatitis C, Chronic/pathology , Humans , Liver/metabolism , Liver/ultrastructure , Male , Middle Aged , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Receptors, IgG/biosynthesisABSTRACT
BACKGROUND AND AIMS: Circulating levels of oxidized lipoprotein (oxLDL) correlate with myocardial infarction risk and atherosclerosis severity. Our previous study demonstrates that oxLDL immune complexes (oxLDL-ICs) can signal through FcγRs on bone marrow-derived dendritic cells (BMDCs) and enhance their activation and inflammatory cytokine secretion. While global FcγR-/- studies have shown that activating FcγRs are proatherogenic, the role of the inhibitory FcγRIIb is unclear. We sought to determine the role of DC-specific FcγRIIb in atherosclerosis. METHODS: Bone marrow chimeras were generated by rescuing lethally irradiated Ldlr-/- mice with hematopoietic cells from littermate CD11c-Cre+ or CD11c-Cre-Fcgr2bfl/fl donors. Four weeks following transplant, recipients were placed on a Western diet for eight weeks. Various tissues and organs were analyzed for differences in inflammation. RESULTS: Quantitation of atherosclerosis in the proximal aorta demonstrated a 58% increase in female CD11c-Cre+Fcgr2bfl/fl recipients, but a surprising 44% decrease in male recipients. Hepatic cholesterol and triglycerides were increased in female CD11c-Cre+Fcgr2bfl/fl recipients. This was associated with an increase in CD36 and MHC Class II expression on hepatic CD11c+CD11b+ DCs in female livers. In contrast, male CD11c-Cre+Fcgr2bfl/fl recipients had decreased hepatic lipids with a corresponding decrease in CD36 and MHC Class II expression on CD11c+ cells. Interestingly, both sexes of CD11c-Cre+Fcgr2bfl/fl recipients had significant decreases in serum cholesterol and TGs with corresponding decreases in liver Fasn transcripts. CONCLUSIONS: The absence of FcγRIIb expression on CD11c+ cells results in sex-dependent alteration in liver inflammation influencing atherogenesis and sex-independent modulation of serum cholesterol and TGs.
Subject(s)
Atherosclerosis/blood , Cholesterol/blood , Receptors, IgG/physiology , Triglycerides/blood , Animals , CD11 Antigens/biosynthesis , Dendritic Cells/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Receptors, IgG/biosynthesis , Sex FactorsABSTRACT
Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1ß (IL-1ß) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1ß-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.
Subject(s)
Antibodies, Bacterial/immunology , Colitis, Ulcerative/immunology , Gastrointestinal Microbiome/immunology , Immunoglobulin G/immunology , Interleukin-1beta/immunology , Th17 Cells/immunology , Animals , Colitis/chemically induced , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Colitis, Ulcerative/microbiology , Colitis, Ulcerative/pathology , Dextran Sulfate/toxicity , Gene Expression Regulation , Genotype , Humans , Inflammation , Interleukin-8/biosynthesis , Interleukin-8/genetics , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Macrophages/immunology , Mice , Phagocytes/immunology , RNA, Messenger/biosynthesis , Reactive Oxygen Species , Receptors, IgG/biosynthesis , Receptors, IgG/genetics , Receptors, IgG/immunologyABSTRACT
Toll-like receptors (TLR) are critical mediators of the immune system with their activation linked to infection, inflammation and the pathogenesis of immune diseases including autoimmunity and cancer. For this reason, over the last 2 decades, TLR and their associated signalling pathways have been targeted therapeutically to enhance innate and adaptive immunity. Several TLR ligands, both endogenous and synthetic are at various phases of clinical testing, and new ligands are continually emerging. Agonists of TLR7 are known immune response modifiers, simultaneously stimulating several cell types, resulting in immune cell activation and cytokine and chemokine release. The immune stimulating properties of the TLR7 agonist Imiquimod has also been exploited for use in the treatment of malignant superficial tumours of the skin. Here, we investigated a novel TLR7 agonist UC-1V150 and demonstrate it activates both human and mouse myeloid cells in vitro and in vivo, to deliver potent FcγR-mediated engulfment of opsonized target cells.
Subject(s)
Benzaldehydes/chemistry , Benzaldehydes/pharmacology , Immunologic Factors/pharmacology , Macrophage Activation/drug effects , Macrophages/immunology , Membrane Glycoproteins/agonists , Phagocytosis/immunology , Purines/chemistry , Purines/pharmacology , Toll-Like Receptor 7/agonists , Aminoquinolines/pharmacology , Animals , Antibodies, Monoclonal/immunology , Cells, Cultured , Humans , Imiquimod , Macrophage Activation/immunology , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Phagocytosis/drug effects , Receptors, IgG/biosynthesisABSTRACT
Aryl hydrocarbon receptor (AhR), which was discovered as a receptor for environmental concomitants, plays an important role widely in the immune system. In this study, we assessed AhR involvement in immune-complex-mediated vascular injury by examining the reverse-passive Arthus reaction in AhR heterozygous knockout (AhR+/-) mice. In the cutaneous Arthus reaction, dermal edema was severer in AhR+/- mice than in wild-type mice. The number of infiltrating neutrophils and mRNA expression levels of CXC chemokine ligand 1 and IL-6 were also increased in AhR+/- mice. Similarly, in the peritoneal Arthus reaction, infiltration of neutrophils was increased in AhR+/- mice. Peritoneal macrophages from AhR+/- mice expressed higher levels of Fcγ receptor III and produced higher levels of CXC chemokine ligand 1 and IL-6 after immune complex treatment. In addition, AhR occupied the promoter regions of Fcγ receptor III gene in peritoneal macrophages in a ligand-dependent manner. Depletion of macrophages reduced the cutaneous Arthus reaction in AhR+/- mice, and adoptive transfer of AhR+/- mice macrophages into wild-type mice exacerbated the peritoneal Arthus reaction. Furthermore, AhR expression was decreased and Fcγ receptor III expression was increased in CD14+ monocytes in peripheral blood from patients with immune-complex-mediated vasculitis compared with those from healthy controls. These results suggest that downregulation of AhR is associated with the exacerbation of immune-complex-mediated vascular injury.
Subject(s)
Macrophages/metabolism , Receptors, IgG/biosynthesis , Up-Regulation , Vascular System Injuries/immunology , Adult , Animals , Disease Models, Animal , Female , Humans , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Receptors, Aryl Hydrocarbon/deficiency , Vascular System Injuries/metabolism , Vascular System Injuries/pathologyABSTRACT
BACKGROUND: Exposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function. METHODS: FcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes. RESULTS: FcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG. CONCLUSIONS: Our data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.
Subject(s)
Antigen-Antibody Complex/metabolism , Brain/metabolism , Immunoglobulin G/metabolism , Receptors, IgG/biosynthesis , Signal Transduction/physiology , Animals , Animals, Newborn , Brain/cytology , Brain/growth & development , Cells, Cultured , Female , Gene Expression , Male , Pregnancy , Rats , Rats, Sprague-DawleyABSTRACT
IgG Fc receptors (FcγRs) and the C5a anaphylatoxin receptor (C5aR) were identified as key regulators of type II autoimmune injury in mice. However, and with respect to C5aR, the relative importance of C5a for IgG autoantibody-induced cellular destruction remained unclear. Using an experimental model of autoimmune hemolytic anemia (AIHA), we here report marked differences in the development of AIHA between mice lacking C5aR and C5-deficient (Hc0 ) strain, indicating a limited role of C5 in this type of C5aR-regulated disease. Ex-vivo-analyses of liver homogenates from anemic Hc0 mice demonstrate C5a-independent C5aR activation, upregulation of FcγR expression and amplification of erythrophagocytosis by macrophages. As assessed by pharmacological inhibition studies, targeting of C5aR, but not of C5, is effective in treating experimental AIHA. Collectively, these results define a previously unrecognized disease mechanism of C5aR activation in AIHA that does not necessarily involve C5 and C5a.
Subject(s)
Anemia, Hemolytic, Autoimmune/immunology , Autoimmunity/immunology , Complement C5a/deficiency , Kupffer Cells/immunology , Receptor, Anaphylatoxin C5a/metabolism , Receptors, IgG/immunology , Animals , Erythrocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis/immunology , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Receptor, Anaphylatoxin C5a/genetics , Receptors, IgG/biosynthesisABSTRACT
The synergistic effect of IL-18/IL-15/IL-12 stimulation potently activates NK cells, inducing high levels of IFN-γ production. As a result of this potent stimulatory effect, NK cell pre-activation with IL-18/IL-15/IL-12 is being developed as a cancer immunotherapy. Ex vivo expansion of NK cells enables the efficient generation of large numbers of NK cells for wide-scale and repeated therapeutic use, and is thus an important source of NK cells for clinical application. However, the effects of IL-18/IL-15/IL-12 stimulation on ex vivo expanded NK cells have not yet been assessed. Thus, the present study assessed the effects of IL-18/IL-15/IL-12 stimulation on NK cells expanded ex vivo using K562-based artificial antigen presenting cells expressing membrane-bound IL-21. We report that ex vivo expanded NK cells stimulated with IL-18/IL-15/IL-12 produce high levels of IFN-γ and TNFα, have potent cytotoxicity, and maintain prolonged IFN-γ production following removal of stimulation. IL-18/IL-15/IL-12 stimulation induces a phenotypically unique IFN-γ-producing population with reduced CD16 expression and greater CD25 expression as compared to stimulated IFN-γ- NK cells and unstimulated NK cells. We elucidate that the mechanism of synergy for induction and maintenance of IFN-γ production is not due to a further enhancement of STAT4 activation compared to stimulation with IL-12 alone. Furthermore, we demonstrate that the synergistic increase in IFN-γ is not solely under translational regulation, as elevated levels of IFN-γ mRNA contribute to the synergistic increase in IFN-γ. Overall, this study characterizes the response of ex vivo expanded NK cells to IL-18/IL-15/IL-12 stimulation and supports the use of ex vivo expanded NK cells as a feasible and efficient source of IL-18/IL-15/IL-12 pre-activated NK cells for adoptive transfer in cancer immunotherapies.
Subject(s)
Interferon-gamma/biosynthesis , Interleukin-12/pharmacology , Interleukin-15/pharmacology , Interleukin-18/pharmacology , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , STAT4 Transcription Factor/metabolism , Adoptive Transfer/methods , Cells, Cultured , Humans , Immunotherapy/methods , Interleukin-2 Receptor alpha Subunit/biosynthesis , Interleukins/metabolism , Neoplasms/therapy , Receptors, IgG/biosynthesis , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: Three different subsets of circulating human monocytes, CD14brightCD16- (classical), CD14brightCD16+ (intermediate), and CD14dimCD16+ (non-classical) have been recently identified. It has been reported that CD14brightCD16+ monocytes are increased in rheumatoid arthritis (RA). However, the role of each monocyte subset in the pathogenesis of RA is still unclear. The purpose of this study was to investigate the association of CD14brightCD16+ monocytes with RA. METHODS: The study enrolled 35 patients with RA and 14 healthy volunteers. The three subsets of peripheral blood monocytes were analyzed by flow cytometry. Serum cytokines were measured at baseline in patients with RA and in healthy volunteers. CD14brightCD16- monocytes were isolated and cultured in vitro with different cytokines for 14 hours, and CD16 induction was assessed. RESULTS: The proportion of CD14brightCD16+ monocytes, and serum interleukin (IL)-6, IL-8, and IL-10 were increased in patients with RA compared to healthy controls. The proportion of CD14brightCD16+ monocytes correlated with the disease activity of RA positively, whereas the proportion of CD14brightCD16- monocytes correlated negatively. When isolated CD14brightCD16- monocytes were stimulated with IL-6, IL-8, and IL-10, the only cytokine that significantly induced CD16 expression on the cells was IL-10. CONCLUSIONS: The proportion of CD16brightCD14+ monocytes was positively correlated with RA disease activity. The expression of CD16 in monocytes was induced by IL-10 but not IL-6, and IL-8 was enhanced in the sera of patients with RA. Our results suggest that CD16brightCD14+ monocytes are involved in the pathogenesis of RA and that IL-10 is a key cytokine that regulates CD16 expression in monocytes.
Subject(s)
Arthritis, Rheumatoid/immunology , Interleukin-10/immunology , Monocytes/immunology , Aged , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , GPI-Linked Proteins/biosynthesis , GPI-Linked Proteins/immunology , Humans , Lipopolysaccharide Receptors/biosynthesis , Lipopolysaccharide Receptors/immunology , Male , Middle Aged , Receptors, IgG/biosynthesis , Receptors, IgG/immunologyABSTRACT
INTRODUCTION: Neutrophilic granulocytes express cluster of differentiation 64 (CD64) antigen upon activation. CD64 can be used as a marker of bacterial infection and sepsis. The goal of this study was to determine whether CD64 is a useful biomarker for critically ill patients and analyze longitudinal measurements with regard to outcome and sepsis severity. METHODS: In this prospective observational study, CD64 analysis was performed daily until discharge from ICU or death. Demographics, clinical, laboratory data, and outcome defined as 28-day survival were recorded. Patients were included when admitted to the ICU with sepsis, severe sepsis, or septic shock and within 24 h from start of antibiotic treatment. RESULTS: Hundred and fifty-five consecutive patients were enrolled. At baseline, a difference in CD64 of 2.26 (1.33-4.47) vs. 1.49 (0.89-2.24) (P = 0.004) was seen between patients with a positive culture and negative culture. CD64 at day 1 was higher with patients with septic shock when compared with sepsis (P = 0.012). No difference of CD64 between survivors and nonsurvivors was seen. CONCLUSION: This study demonstrated that CD64 discriminates between critically ill patients with culture positive and negative sepsis and correlates with severity of disease. However, CD64 index is not a good predictor for 28-day mortality in the critically ill patient.
Subject(s)
Gene Expression Regulation , Neutrophils/metabolism , Receptors, IgG/biosynthesis , Severity of Illness Index , Shock, Septic/blood , Shock, Septic/mortality , Acute Disease , Biomarkers/blood , Critical Illness , Disease-Free Survival , Female , Humans , Male , Predictive Value of Tests , Survival RateABSTRACT
The phase III Thai RV144 vaccine trial showed an estimated vaccine efficacy (VE) to prevent HIV-1 infection of 31.2%, which has motivated the search for immune correlates of vaccine protection. In a recent report, several single nucleotide polymorphisms (SNPs) in FCGR2C were identified to associate with the level of VE in the RV144 trial. To investigate the functional significance of these SNPs, we utilized a large scale B cell RNA sequencing database of 462 individuals from the 1000 Genomes Project to examine associations between FCGR2C SNPs and gene expression. We found that the FCGR2C SNPs that associated with vaccine efficacy in RV144 also strongly associated with the expression of FCGR2A/C and one of them also associated with the expression of Fc receptor-like A (FCRLA), another Fc-γ receptor (FcγR) gene that was not examined in the previous report. These results suggest that the expression of FcγR genes is influenced by these SNPs either directly or in linkage with other causal variants. More importantly, these results motivate further investigations into the potential for a causal association of expression and alternative splicing of FCGR2C and other FcγR genes with the HIV-1 vaccine protection in the RV144 trial and other similar studies.
Subject(s)
AIDS Vaccines/therapeutic use , B-Lymphocytes/metabolism , B-Lymphocytes/virology , HIV Infections/genetics , HIV Infections/prevention & control , Polymorphism, Genetic , Receptors, IgG/biosynthesis , Receptors, IgG/genetics , Alleles , Alternative Splicing , B-Lymphocytes/cytology , Europe , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , HIV-1 , Homozygote , Humans , Polymorphism, Single Nucleotide , Quantitative Trait Loci , Receptors, Fc , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Sequence Analysis, RNAABSTRACT
AIMS: Our objective was to compare the protective efficacy of ischemic preconditioning (IPC) and remote ischemic preconditioning (RIPC) against liver ischemia/reperfusion injury (IRI) and to evaluate their combined protective effect in mouse liver transplantation (MLT). MATERIALS AND METHODS: Mice were randomly allocated to sham, IPC, RIPC, or IPC+RIPC groups. The animals were sacrificed at 2h, 24h, and 3 days after reperfusion. Blood samples were collected to evaluate alanine aminotransferase, TNF-α, and innate immune response. Liver tissue samples were obtained for histological evaluation, terminal deoxynucleotidyltransferased UTP nick end labeling, malondialdehyde (MDA) assay. KEY FINDINGS: Mice given preconditioning measures had significantly lower increase in transaminase, TNF-α expression, MDA formation, liver injury scores, and apoptosis index at 2h, 24h and 3 days after liver transplantation. The percentages of CD11b(+), CD11b(+)CD16/32(+) and CD11b(+) CD16/32(high) in white blood cells at 3 days after MLT were significantly lower than in the sham group. The results of factorial analysis demonstrated no synergistic effect for IPC and RIPC, except for MDA formation 2h after reperfusion (p=0.038). SIGNIFICANCE: Based on the synergistic and addictive effect on liver IRI induced by MLT between IPC and RIPC, the study suggested ways in which combined preconditionings could be elicited in patients undergoing planned procedures complicated by IRI to support better outcomes.