A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets.

Human primary monocytes are heterogeneous in terms of phenotype and function, but are sub-divided only based on CD16 and CD14 expression. CD16 expression distinguishes a subset of monocytes with highly pro-inflammatory properties from non-CD16 expressing "classical" monocytes. CD14 expression further subdivides the CD16+ monocytes into non-classical CD14low and intermediate CD14high subsets. This long-standing CD16-CD14 classification system, however, has limitations as CD14 is expressed in a continuum, leading to subjectivity in delineating the non-classical and intermediate subsets; in addition, CD16 expression is unstable, making identification of the subsets impossible after in vitro culture or during inflammatory conditions in vivo. Hence, we aimed to identify the three monocyte subsets using an alternative combination of markers. Additionally, we wanted to address whether the monocyte subset perturbations observed during infection is real or an artifact of differential CD16 and/or CD14 regulation. Using cytometry by time-of-flight (CyTOF), we studied the simultaneous expression of 34 monocyte markers on total monocytes, and derived a combination of five markers (CD33, CD86, CD64, HLA-DR, and CCR2), that could objectively delineate the three subsets. Using these markers, we could also distinguish CD16+ monocytes from CD16- monocytes after in vitro stimulation. Finally, we found that the observed expansion of intermediate (CD14high) monocytes in dengue virus-infected patients was due to up-regulated CD16 expression on classical monocytes. With our new combination of markers, we can now identify monocyte subsets without CD16 and CD14, and accurately re-examine monocyte subset perturbations in diseases.

Monocyte Subsets Have Distinct Patterns of Tetraspanin Expression and Different Capacities to Form Multinucleate Giant Cells.

Monocytes are able to undergo homotypic fusion to produce different types of multinucleated giant cells, such as Langhans giant cells in response to M. tuberculosis infection or foreign body giant cells in response to implanted biomaterials. Monocyte fusion is highly coordinated and complex, with various soluble, intracellular, and cell-surface components mediating different stages of the process. Tetraspanins, such as CD9, CD63, and CD81, are known to be involved in cell:cell fusion and have been suggested to play a role in regulating homotypic monocyte fusion. However, peripheral human monocytes are not homogenous: they exist as a heterogeneous population consisting of three subsets, classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16+), at steady state. During infection with mycobacteria, the circulating populations of intermediate and non-classical monocytes increase, suggesting they may play a role in the disease outcome. Human monocytes were separated into subsets and then induced to fuse using concanavalin A. The intermediate monocytes were able to fuse faster and form significantly larger giant cells than the other subsets. When antibodies targeting tetraspanins were added, the intermediate monocytes responded to anti-CD63 by forming smaller giant cells, suggesting an involvement of tetraspanins in fusion for at least this subset. However, the expression of fusion-associated tetraspanins on monocyte subsets did not correlate with the extent of fusion or with the inhibition by tetraspanin antibody. We also identified a CD9High and a CD9Low monocyte population within the classical subset. The CD9High classical monocytes expressed higher levels of tetraspanin CD151 compared to CD9Low classical monocytes but the CD9High classical subset did not exhibit greater potential to fuse and the role of these cells in immunity remains unknown. With the exception of dendrocyte-expressed seven transmembrane protein, which was expressed at higher levels on the intermediate monocyte subset, the expression of fusion-related proteins between the subsets did not clearly correlate with their ability to fuse. We also did not observe any clear correlation between giant cell formation and the expression of pro-inflammatory or fusogenic cytokines. Although tetraspanin expression appears to be important for the fusion of intermediate monocytes, the control of multinucleate giant cell formation remains obscure.

Novel dual-targeting anti-proliferative dihydrotriazine-chalcone derivatives display suppression of cancer cell invasion and inflammation by inhibiting the NF-κB signaling pathway.

Chalcones present in edible plants possess anti-cancer and anti-inflammatory properties, with the Michael acceptor moiety reported to be responsible for their biological activities. In this study, two novel dihydrotriazine-chalcone compounds previously identified to exert anti-proliferative effects through dual-targeting of dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR), were evaluated for their anti-invasive and anti-inflammatory abilities. At non-lethal concentrations, the compounds suppressed in vitro migration of MDA-MB-231 breast carcinoma cells, which was correlated with a dose-dependent downregulation of phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) expression and secretion. At similar concentrations, these chalcone-based compounds suppressed expression of inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated murine macrophage-like RAW 264.7 cells, as well as tumor necrosis factor alpha (TNF-α) in LPS-stimulated human monocytes isolated from healthy donors. Mechanistically, inhibition of cancer cell invasion and inflammation by the compounds were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway, which corroborated with the reported mechanism of action of chalcones. Their abilities to target multiple biological mediators relevant to multi-step carcinogenesis and with bioactivities stronger than those of the parent chalcone scaffold have warranted dihydrotriazine-chalcone compounds as promising candidates for use in pharmacological intervention of aggressive cancers.

The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence.

Human primary monocytes comprise a heterogeneous population that can be classified into three subsets based on CD14 and CD16 expression: classical (CD14high/CD16-), intermediate (CD14high/CD16+), and non-classical (CD14low/CD16+). The non-classical monocytes are the most pro-inflammatory in response to TLR stimulation in vitro, yet they express a remarkably high basal level of miR-146a, a microRNA known to negatively regulate the TLR pathway. This concurrence of a pro-inflammatory status and a high miR-146a level has been associated with cellular senescence in other cell types. Hence, we assessed the three monocyte subsets for evidence of senescence, including proliferative status, telomere length, cellular ROS levels, and mitochondrial membrane potential. Indeed, the non-classical subset exhibited the clearest hallmarks of senescence, followed by the intermediate and then the classical subset. In addition, the non-classical subset secreted pro-inflammatory cytokines basally in vitro. The highly pro-inflammatory nature of the non-classical monocytes could be a manifestation of the senescence-associated secretory phenotype (SASP), likely induced by a high basal NF-κB activity and IL-1α production. Finally, we observed an accumulation of the non-classical monocytes, in conjunction with higher levels of plasma TNF-α and IL-8, in the elderly. These factors may contribute to inflamm-aging and age-related inflammatory conditions, such as atherosclerosis and osteoarthritis. With our new understanding that the non-classical monocyte subset is a senescent population, we can now re-examine the role of this subset in disease conditions where this subset expands.

Differential IL-1ß secretion by monocyte subsets is regulated by Hsp27 through modulating mRNA stability.

Monocytes play a central role in regulating inflammation in response to infection or injury, and during auto-inflammatory diseases. Human blood contains classical, intermediate and non-classical monocyte subsets that each express characteristic patterns of cell surface CD16 and CD14; each subset also has specific functional properties, but the mechanisms underlying many of their distinctive features are undefined. Of particular interest is how monocyte subsets regulate secretion of the apical pro-inflammatory cytokine IL-1ß, which is central to the initiation of immune responses but is also implicated in the pathology of various auto-immune/auto-inflammatory conditions. Here we show that primary human non-classical monocytes, exposed to LPS or LPS + BzATP (3'-O-(4-benzoyl)benzyl-ATP, a P2X7R agonist), produce approx. 80% less IL-1ß than intermediate or classical monocytes. Despite their low CD14 expression, LPS-sensing, caspase-1 activation and P2X7R activity were comparable in non-classical monocytes to other subsets: their diminished ability to produce IL-1ß instead arose from 50% increased IL-1ß mRNA decay rates, mediated by Hsp27. These findings identify the Hsp27 pathway as a novel therapeutic target for the management of conditions featuring dysregulated IL-1ß production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1ß.

MicroRNA-mediated immune modulation as a therapeutic strategy in host-implant integration.

The concept of implanting an artificial device into the human body was once the preserve of science fiction, yet this approach is now often used to replace lost or damaged biological structures in human patients. However, assimilation of medical devices into host tissues is a complex process, and successful implant integration into patients is far from certain. The body's immediate response to a foreign object is immune-mediated reaction, hence there has been extensive research into biomaterials that can reduce or even ablate anti-implant immune responses. There have also been attempts to embed or coat anti-inflammatory drugs and pro-regulatory molecules onto medical devices with the aim of preventing implant rejection by the host. In this review, we summarize the key immune mediators of medical implant reaction, and we evaluate the potential of microRNAs to regulate these processes to promote wound healing, and prolong host-implant integration.

MicroRNA expression profiling of human blood monocyte subsets highlights functional differences.

Within human blood there are two subsets of monocytes that can be identified by differential expression of CD16. Although numerous phenotypic and functional differences between the subsets have been described, little is known of the mechanisms underlying the distinctive properties of the two subsets. MicroRNAs (miRNAs) are small non-coding RNAs that can regulate gene expression through promoting mRNA degradation or repressing translation, leading to alterations in cellular processes. Their potential influence on the functions of monocyte subsets has not been investigated. In this study, we employed microarray analysis to define the miRNA expression profile of human monocyte subsets. We identified 66 miRNAs that were differentially expressed (DE) between CD16(+) and CD16(-) monocytes. Gene ontology analysis revealed that the predicted targets of the DE miRNAs were predominantly associated with cell death and cellular movement. We validated the functional impacts of selected DE miRNAs in CD16(-) monocytes, over-expression of miR-432 significantly increases apoptosis, and inhibiting miR-19a significantly reduces cell motility. Furthermore, we found that miR-345, another DE miRNA directly targets the transcription factor RelA in monocytes, which resulted in the differential expression of RelA in monocyte subsets. This implicates miR-345 indirect regulation of many genes downstream of RelA, including important inflammatory mediators. Together, our data show that DE miRNAs could contribute substantially to regulating the functions of human blood monocytes.

Sulforaphane and its methylcarbonyl analogs inhibit the LPS-stimulated inflammatory response in human monocytes through modulating cytokine production, suppressing chemotactic migration and phagocytosis in a NF-κB- and MAPK-dependent manner.

Sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)-butane], an aliphatic isothiocyanate (ITC) naturally derived from cruciferous vegetables and largely known for its chemopreventive potential also appears to possess anti-inflammatory potential. In this study, structural analogs of SF {compound 1 [1-isothiocyanato-4-(methylcarbonyl)-butane] and 2 [1-isothiocyanato-3-(methylcarbonyl)-propane]} containing a carbonyl group in place of the sulfinyl group in SF, were evaluated for their anti-inflammatory activities. In RAW 264.7 cells, the ITCs at non-toxic concentrations caused an inhibition of NO and prostaglandin E2 (PGE2) release through suppressing expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as a reduction in matrix metalloproteinase-9 (MMP-9) expression, secretion and gelatinolytic activity. Further work performed on human monocytes isolated from blood of healthy donors revealed that the ITCs not only suppressed the expression and release of pro-inflammatory mediators IL-1ß, IL-6, TNF-α and MMP-9, but also suppressed their antibody-independent phagocytic and chemotactic migratory abilities. These anti-inflammatory activities were mediated through suppression of the NF-κB and MAPK signaling pathways. In addition, the ITCs were revealed to interact with the cysteines in inhibitor of nuclear factor-κB kinase ß subunit (IKKß), which could contribute at least partly to the suppression of NF-κB signaling. In conclusion, results obtained in this study provide deeper insights into the anti-inflammatory properties of SF and its methylcarbonyl analogs and the underlying mechanisms. These compounds thus serve as promising candidates for clinical applications in controlling inflammatory conditions.

Increased myeloid-derived suppressor cells in gastric cancer correlate with cancer stage and plasma S100A8/A9 proinflammatory proteins.

Immune dysfunction may contribute to tumor progression in gastric cancer (GC) patients. One mechanism of immune dysfunction is the suppression of T cell activation and impairment of the efficacy of cancer immunotherapy by myeloid-derived suppressor cells (MDSCs). We assessed the phenotype and immunosuppressive function of MDSCs in GC patients. We further investigated the role of S100A8/A9 in GC and the relationship between S100A8/A9 and MDSC function. Lastly, the effect of MDSCs on survival rates and its potential as a prognostic factor in GC patients were investigated. MDSCs from PBMCs of GC patients were identified by comparing the expression of specific surface markers with PBMCs from healthy individuals. The ability of MDSCs to suppress T lymphocyte response and the effect of S100A8/A9 and RAGE blocking were tested in vitro by (autologous) MLR. GC patients had significantly more MDSCs than healthy individuals. These MDSCs suppressed both T lymphocyte proliferation and IFN-γ production and had high arginase-I expression. Levels of S100A8/A9 in plasma were higher in GC patients compared with healthy individuals, and they correlated with MDSC levels in the blood. Blocking of S100A8/A9 itself and the S100A8/A9 receptor RAGE on MDSCs from GC patients abrogated T cell effector function. We found that high levels of MDSCs correlated with more advanced cancer stage and with reduced survival (p = 0.006). S100A8/A9 has been identified as a potential target to modulate antitumor immunity by reversing MDSC-mediated immunosuppression.

The three human monocyte subsets: implications for health and disease.

Human blood monocytes are heterogeneous and conventionally subdivided into two subsets based on CD16 expression. Recently, the official nomenclature subdivides monocytes into three subsets, the additional subset arising from the segregation of the CD16+ monocytes into two based on relative expression of CD14. Recent whole genome analysis reveal that specialized functions and phenotypes can be attributed to these newly defined monocyte subsets. In this review, we discuss these recent results, and also the description and utility of this new segregation in several disease conditions. We also discuss alternative markers for segregating the monocyte subsets, for example using Tie-2 and slan, which do not necessarily follow the official method of segregating monocyte subsets based on relative CD14 and CD16 expressions.

Macrophages in human colorectal cancer are pro-inflammatory and prime T cells towards an anti-tumour type-1 inflammatory response.

High macrophage infiltration into tumours often correlates with poor prognoses; in colorectal, stomach and skin cancers, however, the opposite is observed but the mechanisms behind this phenomenon remain unclear. Here, we sought to understand how tumour-associated macrophages (TAMs) in colorectal cancer execute tumour-suppressive roles. We found that TAMs in a colorectal cancer model were pro-inflammatory and inhibited the proliferation of tumour cells. TAMs also produced chemokines that attract T cells, stimulated proliferation of allogeneic T cells and activated type-1 T cells associated with anti-tumour immune responses. Using colorectal tumour tissues, we verified that TAMs in vivo were indeed pro-inflammatory. Furthermore, the number of tumour-infiltrating T cells correlated with the number of TAMs, suggesting that TAMs could attract T cells; and indeed, type-1 T cells were present in the tumour tissues. Patient clinical data suggested that TAMs exerted tumour-suppressive effects with the help of T cells. Hence, the tumour-suppressive mechanisms of TAMs in colorectal cancer involve the inhibition of tumour cell proliferation alongside the production of pro-inflammatory cytokines, chemokines and promoting type-1 T-cell responses. These new findings would contribute to the development of future cancer immunotherapies based on enhancing the tumour-suppressive properties of TAMs to boost anti-tumour immune responses.

Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies.

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 microm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.

Identification of novel functional differences in monocyte subsets using proteomic and transcriptomic methods.

Human blood monocytes can be broadly divided into two distinct subsets: CD14+CD16- and CD14+/lowCD16+ subsets. Perturbation in their proportions in the blood has been observed in several disease conditions. Although numerous phenotypic and functional differences between the two subsets have already been described, the roles contributed by each subset during homeostasis or disease conditions are still largely unclear. To uncover novel differences to aid in elucidating their functions, we perform a global analysis of the two subsets utilizing both proteomics and transcriptomics approaches. From the proteomics and transcriptomics data, the expression of 613 genes by the two subsets is detected at both the protein and mRNA levels. These 613 genes are assessed for up-regulation in each subset at the protein and mRNA levels using a cutoff fold change of > or =|1.5| between subsets. Proteins and mRNAs up-regulated in each subset are then mapped in silico into biological functions. This mapping reveals copious functional differences between the subsets, many of which are seen at both protein and mRNA levels. For instance, expression of genes involved in F(CY) receptor-mediated phagocytosis are up-regulated in the CD14+/lowCD16+ subset, while those involved in antimicrobial function are up-regulated in the CD14+CD16- subset. We uncover novel functional differences between the monocyte subsets from differences in gene expression at the protein and mRNA levels. These functional differences would provide new insights into the different roles of the two monocyte subsets in regulating innate and adaptive immune responses.

The controlled presentation of TGF-beta1 to hepatocytes in a 3D-microfluidic cell culture system.

3D-microfluidic cell culture systems (3D-microFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100-200 pg/ml TGF-beta1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-beta1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-microFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-beta1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-beta1 or without TGF-beta1. The controlled presentation of TGF-beta1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.

Stem cells in microfluidics.

With the introduction of microtechnology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and their influence on cell fate studied. Microfluidic devices can be made transparent and the cells monitored real time by imaging, using fluorescence markers to probe cell functions and cell fate. This article gives a perspective on the yet untapped utility of microfluidic devices for stem cell research. It will guide the biologists through some basic microtechnology and the application of microfluidics to cell research, as well as highlight to the engineers the cell culture capabilities of microfluidics.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture.

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

Dendrimer hydrazides as multivalent transient inter-cellular linkers.

Three-dimensional (3D) multi-cellular aggregates (MCAs), as a model scaffold-free tissue construct, are useful for engineering cell-dense and matrix-poor tissues for repair and regeneration applications. To facilitate rapid MCA formation with high degrees of linker consistency and performance, we synthesized a class of dendrimer hydrazides with 8, 16 and 32 arms that can react with the aldehyde on the modified cell surfaces to form MCAs. DAB-AM-16 hydrazide with 32 arms demonstrated the best cell aggregation ability as compared to the dendrimer hydrazides with fewer arms, facilitating MCA formation at lower linker concentrations, minimizing cytotoxicity. Characterization of the MCAs formed with 2 microm of DAB-AM-16 hydrazide indicated that the cells proliferated well, maintained 3D cell-cell interaction and 3D cell morphology even as the inter-cellular linker gradually disappeared from the cell surfaces. Cells cultured as MCAs also demonstrated improved cell functions than the cells cultured in 2D monolayer. The dendrimer hydrazides would be a class of consistent, economical, and high performance multivalent transient inter-cellular linkers useful in forming scaffold-free 3D tissue constructs for soft-tissue engineering and regenerative medicine.

A gel-free 3D microfluidic cell culture system.

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Transient inter-cellular polymeric linker.

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.