The role of LAIR-1 (CD305) in T cells and monocytes/macrophages in patients with rheumatoid arthritis

The LAIR-1 receptor is expressed on a majority of mononuclear leukocytes. It is used as a biomarker when testing synovial fluid for evidence of rheumatoid arthritis (RA). The primary objective of this study was to measure T cell- and monocyte/macrophage-specific LAIR-1 expression in RA patients and compare this to LAIR-1 expression in osteoarthritis (OA) patients and healthy individuals. LAIR-1 expression was significantly decreased in circulating CD4⁺ T cells in RA patients compared to both OA patients and healthy individuals. In contrast, LAIR-1 is high in CD14⁺ monocytes and local CD68⁺ macrophages in synovial tissues from RA patients. Upon stimulation with TNF-α, LAIR-1 expression decreased in T-helper (Th)1 and Th2 CD4⁺ T cells from healthy donors. These results indicate that LAIR-1 may exert different functions on T cells and monocytes/macrophages and suggest that LAIR-1 may be a novel therapeutic target for the treatment of RA.     

Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose siRNA depletion enhances HIV-1 Tat function

Background

The activation of mononuclear phagocytes (MPs), including monocytes, macrophages and dendritic cells, contributes to central nervous system inflammation in various neurological diseases. In HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), MPs are reservoirs of HTLV-I, and induce proinflammatory cytokines and excess T cell responses. The virus-infected or activated MPs may play a role in immuneregulation and disease progression in patients with HTLV-I-associated neurological diseases.

Results

Phenotypic analysis of CD14⁺ monocytes in HAM/TSP patients demonstrated high expression of CX₃CR1 and HLA-DR in CD14^lowCD16⁺ monocytes, compared to healthy normal donors (NDs) and asymptomatic carriers (ACs), and the production of TNF-?? and IL-1?? in cultured CD14⁺ cells of HAM/TSP patients. CD14⁺ cells of HAM/TSP patients also showed acceleration of HTLV-I Tax expression in CD4⁺ T cells. Minocycline, an inhibitor of activated MPs, decreased TNF-?? expression in CD14⁺ cells and IL-1?? release in PBMCs of HAM/TSP patients. Minocycline significantly inhibited spontaneous lymphoproliferation and degranulation/IFN-?? expression in CD8⁺ T cells of HAM/TSP patients. Treatment of minocycline also inhibited IFN-?? expression in CD8⁺ T cells of HAM/TSP patients after Tax11-19 stimulation and downregulated MHC class I expression in CD14⁺ cells.

Conclusion

These results demonstrate that minocycline directly inhibits the activated MPs and that the downregulation of MP function can modulate CD8⁺ T cells function in HAM/TSP patients. It is suggested that activated MPs may be a therapeutic target for clinical intervention in HAM/TSP.     

Functional Phenotype of Synovial Monocytes Modulating Inflammatory T-Cell Responses in Rheumatoid Arthritis (RA)

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14⁺CD16⁺, but not CD14^dimCD16⁺, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14⁺ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14⁺CD16⁺ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.     

Monocyte populations as markers of response to adalimumab plus MTX in rheumatoid arthritis

Introduction

The treatment of rheumatoid arthritis (RA) patients with anti-tumor necrosis factor alpha (TNFα) biological drugs has dramatically improved the prognosis of these patients. However, a third of the treated patients do not respond to this therapy. Thus, the search for biomarkers of clinical response to these agents is currently highly active. Our aim is to analyze the number and distribution of circulating monocytes, and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets in methotrexate (MTX) non-responder patients with RA, and to determine their value in predicting the clinical response to adalimumab plus MTX treatment.

Methods

This prospective work investigated the number of circulating monocytes, and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets, in 35 MTX non-responder patients with RA before and after three and six months of anti-TNFα treatment using multiparametric flow cytometry. The number of circulating monocytes in an age- and sex-matched healthy population was monitored as a control.

Results

Non-responder patients with RA show an increased number of monocytes and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets after three months of adalimumab plus MTX treatment that remained significantly increased at six months. In contrast, significant normalization of the numbers of circulating monocytes was found in responders at three months of adalimumab plus MTX treatment that lasts up to six months. CX3CR1 expression is increased in monocytes in non-responders. At three months of anti-TNFα treatment the number of circulating monocytes and their subsets was associated with at least 80% sensitivity, 84% specificity and an 86% positive predictive value (PPV) in terms of discriminating between eventual early responders and non-responders.

Conclusions

The absolute number of circulating monocytes and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets at three months of adalimumab plus MTX treatment, have a predictive value (with high specificity and sensitivity) in terms of the clinical response after six months of anti-TNFα treatment in patients with RA.     

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14⁺ peripheral blood mononuclear cells (CD14⁺ PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14⁺ PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14⁺ PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.     

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL₁₇₇ and PRL₁₈₈; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6 h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na⁺/Cl^? cotransporter and a Na⁺/H⁺ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na⁺/K⁺/2Cl^? cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6 h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6 h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.     

Chemokine receptors expression on peripheral CD4-lymphocytes in rheumatoid arthritis: Coexpression of CCR7 and CD95 is associated with disease activity

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation triggered by infiltrating CD4 lymphocytes. The positioning and activation of lymphocyte in inflamed synovial tissues are dependent on a number of factors including their chemokine receptor expression profile. We aimed to investigate which chemokine receptors pattern correlate with serum cytokine levels and with disease activity. Forty patients with RA (34 female and 6 male) with age range from 21 to 68 years were included. Twenty healthy volunteers (16 female and 4 male) with matched age (range 21–48 years) were served as healthy controls (HCs). Expression of chemokine receptors (CCR5, CX3CR1 and CCR7) together with the apoptosis-related marker (CD95) was analyzed using three-color flow cytometry analysis after gating on CD4⁺ peripheral blood lymphocytes. Plasma levels of IL-6, IL-10, IL-12 and TNF-α cytokines were measured in all participants using ELISA. Disease activity score (DAS28-CRP) system was assessed and active disease was defined as DAS28 ⩾3.2. Twenty-five (62.4%) patients were classified as active RA (ARA) and 15 (37.5%) patients with inactive RA (IRA). Percentages of CD4⁺ lymphocytes expressing CD95 with either of CCR7 or CCR5 were significantly higher in ARA compared to IRA and HCs groups, while the expression of CX3CR1 on T-cells was found significantly lower in both CD95⁻ and CD95⁺ T-cells in RA groups than HC. Percentages of CD4⁺CD95⁺CCR7⁺ cells correlated positively with IL-6 (r = 0.390). Whereas CD4⁺CD95⁺CX3CR1⁺ were negatively correlated with TNF-α (r = −0.261). Correlation of CD4⁺CD95⁺CCR7⁺ T cell subset with disease activity and inflammatory cytokines suggests a role for this cell subset in the pathogenesis of RA. Further investigation will be required to fully characterize this cell subset and its role in disease progression.     

CD14 monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells

Here, the effect of CD14⁺ monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-γ (IFN-γ) secretion capacities of CD4⁺ and CD8⁺ T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E₂ (PGE₂) as an important soluble mediator. CD14⁺ monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1β, either exogenously added or produced by CD14⁺ monocytes in culture, could trigger expression of high levels of PGE₂ by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE₂ expression, but also reversed the promotional effect of CD14⁺ monocytes and partially restored CD4⁺ and CD8⁺ T cell proliferation and IFN-γ secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs.     

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2 %. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310 % increase in TNF-α release from monocytes at 16 h, respectively. PAR-1 agonist peptide, SFLLR-NH₂, and PAR-2 agonist peptide tc-LIGRLO-NH₂ provoked up to 210 and 240 % increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH₂-induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH₂, an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH₂-induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroach major allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.     

Trypanosoma cruzi Disrupts Thymic Homeostasis by Altering Intrathymic and Systemic Stress-Related Endocrine Circuitries

We have previously shown that experimental infection caused by Trypanosoma cruzi is associated with changes in the hypothalamus-pituitary-adrenal axis. Increased glucocorticoid (GC) levels are believed to be protective against the effects of acute stress during infection but result in depletion of CD4⁺CD8⁺ thymocytes by apoptosis, driving to thymic atrophy. However, very few data are available concerning prolactin (PRL), another stress-related hormone, which seems to be decreased during T. cruzi infection. Considering the immunomodulatory role of PRL upon the effects caused by GC, we investigated if intrathymic cross-talk between GC and PRL receptors (GR and PRLR, respectively) might influence T. cruzi-induced thymic atrophy. Using an acute experimental model, we observed changes in GR/PRLR cross-activation related with the survival of CD4⁺CD8⁺ thymocytes during infection. These alterations were closely related with systemic changes, characterized by a stress hormone imbalance, with progressive GC augmentation simultaneously to PRL reduction. The intrathymic hormone circuitry exhibited an inverse modulation that seemed to counteract the GC-related systemic deleterious effects. During infection, adrenalectomy protected the thymus from the increase in apoptosis ratio without changing PRL levels, whereas an additional inhibition of circulating PRL accelerated the thymic atrophy and led to an increase in corticosterone systemic levels. These results demonstrate that the PRL impairment during infection is not caused by the increase of corticosterone levels, but the opposite seems to occur. Accordingly, metoclopramide (MET)-induced enhancement of PRL secretion protected thymic atrophy in acutely infected animals as well as the abnormal export of immature and potentially autoreactive CD4⁺CD8⁺ thymocytes to the periphery. In conclusion, our findings clearly show that Trypanosoma cruzi subverts mouse thymus homeostasis by altering intrathymic and systemic stress-related endocrine circuitries with major consequences upon the normal process of intrathymic T cell development.     

Antithrombin III,but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27 ± 7 years) were pre-incubated with clinically relevant concentrations of AT-III (n = 11) and C1-INH (n = 12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3 h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n = 6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.     

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

Background

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods

Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4⁺CD45RO⁺, CCR7^-, CD49d^high population, and that these cells are derived from the effector memory CD4⁺ T cells in resting blood.

Results

After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4⁺CD45RO⁺ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.

Conclusion

Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.     

Acceleration of Bone Repair in NOD/SCID Mice by Human Monoosteophils,Novel LL-37-Activated Monocytes

Background

An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function.

Methods and Findings

Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries.

Conclusion

Human derived monoosteophils, characterized as CD45⁺α3⁺α3β⁺CD34⁻CD14⁻BAP (bone alkaline phosphatase)⁻ cells, can function in an animal model of bone injury.     

Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34⁺ stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34⁺ haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14⁺ monocytes were found. They consisted of two subpopulations: CD14⁺⁺CD16⁺ and CD14⁺CD16^?, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14⁺ monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3⁺ T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu_369–377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8⁺ T lymphocytes (CTL). The CD14⁺⁺CD16⁺ subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34⁺ stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

Skewed ratios between CD3(+) T cells and monocytes are associated with poor prognosis in patients with HBV-related acute-on-chronic liver failure

Tempering of the innate immune response by T lymphocytes has been demonstrated to play a critical role in protecting animals from inflammation-induced death; however, its role in humans remains unknown. Patients with HBV-related acute-on-chronic liver failure (ACLF) share a striking similarity to the inflammatory response in septic shock where a hyperactive innate response is observed. The present study attempted to characterize the features of CD3⁺ T cells and monocytes and evaluate their clinical implications in 55 patients with HBV-related ACLF, 30 patients with chronic hepatitis B (CHB) and 30 healthy controls (HC). We found that the ratio between circulating CD3⁺ T cells and monocytes (T/M) was decreased in ACLF patients, due to decreased CD3 counts and increased monocyte counts compared with CHB and HC subjects. We also found that the T/M ratios were decreased from the early to the intermediate stage and reached the lowest value at the late stage in ACLF patients. Analyses with clinical parameters revealed that T/M ratios were negatively correlated with the Model for End-Stage Liver Disease Score and direct bilirubin, and positively correlated with prothrombin activity. Moreover, increased T/M ratios were observed in patients with good prognosis, but not in patients with a poor outcome; and ACLF patients who received liver transplantation exhibited an increased T/M ratio. Importantly, we found that programmed death-1 receptor (PD-1) was drastically upregulated on both CD4⁺ T and CD8⁺ T cells in ACLF, which at least in part contributed to the T-cell loss in these patients. Mechanically, the in vitro co-culture assay revealed that both CD4⁺ T and CD8⁺ T cells, as well as regulatory T cells, could inhibit TNF-α secretion by monocytes. In addition, the TNF-α levels in ACLF serum were negatively correlated with T/M ratios. In conclusion, our study identified the novel potential role of T/M ratio in predicting disease progression and provided novel evidences for further studies of the immunopathogenesis in ACLF.     

The CD14+CD16+ Inflammatory Monocyte Subset Displays Increased Mitochondrial Activity and Effector Function During Acute Plasmodium vivax Malaria

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14⁺CD16⁻ (classical), CD14⁺CD16⁺ (inflammatory), and CD14^loCD16⁺ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16⁺ cells, in particular the CD14⁺CD16⁺ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14⁺ were distinguished from CD14^lo monocytes by displaying larger and more active mitochondria. CD14⁺CD16⁺ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14⁺CD16⁺ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.