Mesenchymal stromal cells impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activate monocytes

Background aimsMesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulationMethodsTo understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approachesResultsMSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell–cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytesConclusionsOur results suggest that MSC impair the differentiation of CD14⁺⁺ CD16^? CD64⁺ classical monocytes into CD14⁺⁺ CD16⁺ CD64⁺⁺ activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.     

Tumor-induced STAT3 activation in monocytic myeloid-derived suppressor cells enhances stemness and mesenchymal properties in human pancreatic cancer

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1^Bright CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b⁺/Gr1⁺/Ly6G^?/Ly6C^hi) significantly increase the frequency of ALDH1^Bright CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14⁺ peripheral blood monocytes into Mo-MDSC (CD14⁺/HLA-DR^low/?) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1^Bright CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1^Bright CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1^Bright CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.     

miR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133⁺ side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.     

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC⁺CD31⁺CD34⁺CD14⁻KDR^high endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR⁺ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Selective activation of specific PKC isoforms dictating the fate of CD14+ monocytes towards differentiation or apoptosis

In this study, phorbol‐12‐myristate‐13‐acetate (PMA) at low concentrations (<10 nM; L‐PMA) induces the differentiation of CD14⁺ monocytes into monocyte‐derived macrophages (MDMs) while PMA at high concentrations (>100 nM; H‐PMA) causes the apoptosis of these cells. The pre‐treatment with Go6976 (a PKC‐α/β₁ selective inhibitor), not anilinemonoindolylmaleimide [a PKC‐β inhibitor (PKC‐β inh.)], significantly (P < 0.05) reduces the L‐PMA‐induced generation of MDMs in the cultured CD14⁺ monocytes. On the other hand, either of the above two PKC inhibitors is capable of suppressing the H‐PMA‐induced apoptosis of CD14⁺ monocytes. However, only the inclusion of PKC‐β inh., not Go6976, prevents the cells from serum deprivation‐induced cell apoptosis. Although the membrane translocation of conventional PKC‐α, β₁, and β₂ isoforms was observed in the H‐PMA‐treated CD14⁺ monocytes, only PKC‐β₂ exhibits a mitochondrial translocation activity among those PKCs responsive to H‐PMA treatment. Moreover, the activation of DEVD‐dependent caspases (DEVDase) was also detected in the H‐PMA‐treated CD14⁺ monocytes, indicating the involvement of a caspase‐dependent signaling pathway in the H‐PMA‐induced cell apoptosis of CD14⁺ monocytes. Together with our previous findings that the selective activation of PKC‐α or PKC‐β₁ induces the differentiation of CD14⁺ monocytes into MDMs or dendritic cells (MoDCs), respectively, the results in this study further demonstrate that PKC‐β₂ activation is responsible for relaying the apoptotic signal to intrinsic mitochondria‐dependent caspase signaling cascades in the CD14⁺ monocytes. It is likely that the selective activation of specific PKC isoforms provides a new strategy to manipulate the differential cell fate commitment of multipotent CD14⁺ monocytes towards apoptosis or differentiation into MDMs, MoDCs, and other cell types. J. Cell. Physiol. 226: 122–131, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1^GFP/+ mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C^low monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C^high and Ly6C^low monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C^high monocytes, but that these cells may transition through a Ly6C^low state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C^high monocytes via help from CD4⁺ T cells.     

Mechanisms of HIV Entry into the CNS: Increased Sensitivity of HIV Infected CD14+CD16+ Monocytes to CCL2 and Key Roles of CCR2, JAM-A,and ALCAM in Diapedesis

As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14⁺CD16⁺ monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14⁺CD16⁺ monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14⁺CD16⁺ monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14⁺ cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14⁺CD16⁺ monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14⁺CD16⁺ monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14⁺CD16⁺ monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis.     

Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages

Objective

The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14⁺CD16⁺ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation.

Methods

Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 μM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed.

Results

IS plasma concentration correlated positively with CD14⁺CD16⁺ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-β, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-β, PPARγ and TIMP-1 expression.

Conclusion

IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.     

Characterization of monocyte maturation/differentiation that facilitates their transmigration across the blood-brain barrier and infection by HIV: implications for NeuroAIDS

The prevalence of human immunodeficiency virus 1 (HIV) associated neurocognitive disorders resulting from infection of the central nervous system (CNS) by HIV continues to increase despite the success of combination antiretroviral therapy. Although monocytes are known to transport HIV across the blood–brain barrier (BBB) into the CNS, there are few specific markers that identify monocyte subpopulations susceptible to HIV infection and/or capable of infiltrating the CNS. We cultured human peripheral blood monocytes and characterized the expression of the phenotypic markers CD14, CD16, CD11b, Mac387, CD163, CD44v6 and CD166 during monocyte/macrophage (Mo/Mac) maturation/differentiation. We determined that a CD14⁺CD16⁺CD11b⁺Mac387⁺ Mo/Mac subpopulation preferentially transmigrates across our in vitro BBB model in response to CCL2. Genes associated with Mo/Mac subpopulations that transmigrate across the BBB and/or are infected by HIV were identified by cDNA microarray analyses. Our findings contribute to the understanding of monocyte maturation, infection and transmigration into the brain during the pathogenesis of NeuroAIDS.     

Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14⁺CD16⁺ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14⁺CD16⁺ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14⁺CD16⁺ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14⁺CD16⁺ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14⁺CD16⁺ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, increased CD14⁺CD16⁺ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14⁺CD16⁺ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14⁺CD16⁺ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14⁺CD16⁺ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14⁺CD16⁺ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14⁺CD16⁺ monocytes in dopamine rich brain regions.     

Cisplatin increases immune activity of monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b⁺ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-ɣ expression from CD8⁺ T-cells compared to CD11b⁺ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p < .0001). Splenocytes derived from tumor bearing mice released increase levels of IFN-ɣ after treatment with cisplatin when incubated with dendritic cells (DCs) and tumor antigen (62.0 v. 92.1 pg/mL; p = .03). Cisplatin induced an increase in T-cell and monocyte/macrophage activation markers (CD62L and CD301). Levels of IL-10, IL-6, and VEGF in the cell free ascites of mice treated with cisplatin decreased (p > .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.     

Cisplatin increases immune activity of monocytes and cytotoxic T-cells in a murine model of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is an immunologically active malignancy, but thus far immune therapy has had limited success in clinical trials. One barrier to implementation of efficacious immune therapies is a lack of knowledge of the effect of chemotherapy on the monocyte-derived component of the immune infiltrate within the tumor. We utilized the ID8 murine EOC model to investigate alterations within tumor ascites that occur following administration of platinum chemotherapy. Cisplatin treatment resulted in a significant increase in monocytes within the ascites of tumor bearing mice. We identified that CD11b⁺ cells from the ascites of mice that have been treated with cisplatin elicits an increase in IFN-ɣ expression from CD8⁺ T-cells compared to CD11b⁺ cells from a mouse treated with vehicle control (604.0 pg/mL v. 4328.0 pg/mL; p < .0001). Splenocytes derived from tumor bearing mice released increase levels of IFN-ɣ after treatment with cisplatin when incubated with dendritic cells (DCs) and tumor antigen (62.0 v. 92.1 pg/mL; p = .03). Cisplatin induced an increase in T-cell and monocyte/macrophage activation markers (CD62L and CD301). Levels of IL-10, IL-6, and VEGF in the cell free ascites of mice treated with cisplatin decreased (p > .05). These results indicate that treatment with cisplatin leads to an increase of anti-tumor activity within the ascites related to alterations in the ascites monocytes. Further investigation of these findings in humans is necessary to identify how these cells behave in different patient subgroups and if there is a role for monocyte directed therapy in conjunction with T-cell directed therapy and/or chemotherapy.     

Imbalance of Circulating Monocyte Subsets and PD-1 Dysregulation in Q Fever Endocarditis: The Role of IL-10 in PD-1 Modulation

Q fever endocarditis, a severe complication of Q fever, is associated with a defective immune response, the mechanisms of which are poorly understood. We hypothesized that Q fever immune deficiency is related to altered distribution and activation of circulating monocyte subsets. Monocyte subsets were analyzed by flow cytometry in peripheral blood mononuclear cells from patients with Q fever endocarditis and controls. The proportion of classical monocytes (CD14⁺CD16⁻ monocytes) was similar in patients and controls. In contrast, the patients with Q fever endocarditis exhibited a decrease in the non-classical and intermediate subsets of monocytes (CD16⁺ monocytes). The altered distribution of monocyte subsets in Q fever endocarditis was associated with changes in their activation profile. Indeed, the expression of HLA-DR, a canonical activation molecule, and PD-1, a co-inhibitory molecule, was increased in intermediate monocytes. This profile was not restricted to CD16⁺ monocytes because CD4⁺ T cells also overexpressed PD-1. The mechanism leading to the overexpression of PD-1 did not require the LPS from C. burnetii but involved interleukin-10, an immunosuppressive cytokine. Indeed, the incubation of control monocytes with interleukin-10 led to a higher expression of PD-1 and neutralizing interleukin-10 prevented C. burnetii-stimulated PD-1 expression. Taken together, these results show that the immune suppression of Q fever endocarditis involves a cross-talk between monocytes and CD4⁺ T cells expressing PD-1. The expression of PD-1 may be useful to assess chronic immune alterations in Q fever endocarditis.     

Circulating CD14brightCD16+ ‘Intermediate’ Monocytes Exhibit Enhanced Parasite Pattern Recognition in Human Helminth Infection

Circulating monocyte sub-sets have recently emerged as mediators of divergent immune functions during infectious disease but their role in helminth infection has not been investigated. In this study we evaluated whether ‘classical’ (CD14^brightCD16⁻), ‘intermediate’ (CD14^brightCD16⁺), and ‘non-classical’ (CD14^dimCD16⁺) monocyte sub-sets from peripheral blood mononuclear cells varied in both abundance and ability to bind antigenic material amongst individuals living in a region of Northern Senegal which is co-endemic for Schistosoma mansoni and S. haematobium. Monocyte recognition of excretory/secretory (E/S) products released by skin-invasive cercariae, or eggs, of S. mansoni was assessed by flow cytometry and compared between S. mansoni mono-infected, S. mansoni and S. haematobium co-infected, and uninfected participants. Each of the three monocyte sub-sets in the different infection groups bound schistosome E/S material. However, ‘intermediate’ CD14^brightCD16⁺ monocytes had a significantly enhanced ability to bind cercarial and egg E/S. Moreover, this elevation of ligand binding was particularly evident in co-infected participants. This is the first demonstration of modulated parasite pattern recognition in CD14^brightCD16⁺ intermediate monocytes during helminth infection, which may have functional consequences for the ability of infected individuals to respond immunologically to infection.     

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14⁺⁺CD16⁺ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14⁺⁺CD16⁺ monocyte subset.     

Phenotypic and Functional Heterogeneity of Bovine Blood Monocytes

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14⁺ CD16⁻, intermediate monocytes (intM) CD14⁺ CD16⁺ and nonclassical monocytes (ncM) CD14⁺ CD16⁺ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1β after LPS stimulation. Based on IL-1β secretion after LPS/ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNγ increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes.