Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma.

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma.

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

Resveratrol attenuates TLR-4 mediated inflammation and elicits therapeutic potential in models of sepsis.

Sepsis is a potentially fatal condition triggered by systemic inflammatory response to infection. Due to the heightened immune reactivity and multi-organ pathology, treatment options are limited and several clinical trials have not produced the desired outcome, hence the interest in the discovery of novel therapeutic strategies. The polyphenol resveratrol (RSV) has shown promise against several pathological states, including acute and chronic inflammation. In this study, we evaluated its therapeutic potential in a murine model of sepsis and in patients undergoing transrectal ultrasound biopsy. RSV was able to inhibit lipopolysaccharide (LPS) stimulated inflammatory responses through blocking Phospholipase D (PLD) and its downstream signaling molecules SphK1, ERK1/2 and NF-κB. In addition, RSV treatment resulted in the downregulation of MyD88, an adaptor molecule in the TLR4 signaling pathway, and this effect at least in part, involved RSV-induced autophagy. Notably, RSV protected mice against polymicrobial septic shock induced upon cecal ligation and puncture, and inhibited pro-inflammatory cytokine production by human monocytes from transrectal ultrasound (TRUS) biopsy patients. Together, these findings demonstrate the immune regulatory activity of RSV and highlight its therapeutic potential in the management of sepsis.

Increased monocyte-platelet aggregates and monocyte-endothelial adhesion in healthy individuals with vitamin D deficiency.

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n = 7) and without vitamin D deficiency (n = 16). Vitamin D deficient (VitD-Def) subjects (25(OH)D3 level < 26 ng/mL) expressed significant downregulation of vitamin D receptor (VDR) on monocytes as compared to controls (P < .0001), and VDR expression was well-associated with serum 25(OH)D3 levels. Increased monocyte-platelet aggregates (MPA), a marker for platelet activation, were also observed in VitD-Def subjects (P < .05) which suggests a pro-inflammatory monocyte phenotype. Monocyte adhesion to endothelial cells, an early-stage atherosclerosis event, was also higher in VitD-Def individuals, and inversely correlated to serum 25(OH)D3 level (P < .05). Taken together, these results indicate the pro-inflammatory state and atherogenic potential of monocytes in VitD-Def healthy subjects, and propound the use of vitamin D supplementation as a prospective immunomodulatory and anti-inflammatory therapy in atherosclerosis.

Macrophage polarisation associated with atherosclerosis differentially affects their capacity to handle lipids.

BACKGROUND AND AIMS: Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes macrophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro-inflammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation. METHODS: We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFNγ+LPS, IL-4, IL-10, oxPAPC and CXCL4. RESULTS: RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL-4 and IL-10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity. CONCLUSIONS: Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, informing how the plaque environment influences atherosclerosis progression by influencing macrophage phenotypes.

Integrated in silico and 3D in vitro model of macrophage migration in response to physical and chemical factors in the tumor microenvironment.

Macrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions; however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. In our model design, we propose IL-8, CCL2, and ß-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2 or to the co-addition of a sub-saturating concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.

Modulating Macrophage Phenotype by Sustained MicroRNA Delivery Improves Host-Implant Integration.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host-implant integration. Here, electrospun poly(caprolactone-co-ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2-inducing miRs, Let-7c and miR-124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1-inducing miR, Anti-Let-7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR-124 treatment group, followed by Anti-Let-7c and Let-7c treatment groups. Based on immunohistochemical quantifications, these miR-encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host-implant integration at longer time points.

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.

Targeting immune cells for cancer therapy.

Recent years have seen a renaissance in the research linking inflammation and cancer with immune cells playing a central role in smouldering inflammation in the tumor microenvironment. Diverse immune cell types infiltrate the tumor microenvironment, and the dynamic tumor-immune cell interplay gives rise to a rich milieu of cytokines and growth factors. Fundamentally, this intricate cross-talk creates the conducive condition for tumor cell proliferation, survival and metastasis. Interestingly, the prominent impact of immune cells is expounded in their contrary pro-tumoral role, as well as their potential anti-cancer cellular weaponry. The latter is known as immunotherapy, a concept born out of evidence that tumors are susceptible to immune defence and that by manipulating the immune system, tumor growth can be successfully restrained. Naturally, a deeper understanding of the multifaceted roles of various immune cell types thus contributes toward developing innovative anti-cancer strategies. Therefore, in this review we first outline the roles played by the major immune cell types, such as macrophages, neutrophils, natural killer cells, T cells and B cells. We then explain the recently-explored strategies of immunomodulation and discuss some important approaches via an immunology perspective.

Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.

The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8-PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC.

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.

BACKGROUND: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. METHOD: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. RESULTS: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. CONCLUSIONS: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.

Multiplexed Label-Free Fractionation of Peripheral Blood Mononuclear Cells for Identification of Monocyte-Platelet Aggregates.

Monocytes and platelets play key roles in atherosclerosis and thrombosis, and circulating monocyte-platelet aggregates (MPA) in blood have been widely proposed as surrogate biomarkers for cardiovascular risk stratification and monitoring antiplatelet therapies. However, conventional MPA characterization is based on whole-blood fixation and flow cytometry analysis which adversely affect cell viability and detection accuracy due to significant leukocyte and platelet contaminations. Herein, we introduce a rapid and label-free microfluidic approach for complete size-based fractionation of peripheral blood mononuclear cells (PBMCs) into monocytes, lymphocytes, and platelets. The developed biochip enables gentle sorting of intact MPA in the enriched monocytes with efficient depletion of lymphocytes and platelets for accurate MPA quantification. We first compared the developed microfluidic technology (dean flow fractionation, DFF) with standard magnetic negative isolation (MACS) and observed that DFF-sorted monocytes had similar viability, purity, and key immune functions (phagocytosis, macrophage differentiation) as MACS-sorted monocytes. As proof of concept for diabetes testing, we isolated and characterized monocytes/MPA from a cohort of healthy ( n = 5) and type 2 diabetes mellitus (T2DM) subjects ( n = 8) in PBMCs and DFF-sorted monocytes. High-speed imaging, immunofluorescence, and flow cytometry analysis clearly indicated higher levels of MPA in T2DM patients ( P < 0.05) with enhanced MPA detection sensitivity in DFF-sorted monocytes ( P < 0.05). Taken together, the developed DFF technology greatly facilitates high-throughput (∼130 µL min-1) label-free isolation of monocyte/MPA from PBMCs and can be further developed into a clinical tool for point-of-care cardiovascular risk stratification in metabolic disorders including T2DM.

Corrigendum: Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

[This corrects the article DOI: 10.3389/fimmu.2018.00416.].

Label-free leukocyte sorting and impedance-based profiling for diabetes testing.

Circulating leukocytes comprise of approximately 1% of all blood cells and efficient enrichment of these cells from whole blood is critical for understanding cellular heterogeneity and biological significance in health and diseases. In this work, we report a novel microfluidic strategy for rapid (< 1 h) label-free leukocyte sorting and impedance-based profiling to determine cell activation in type 2 diabetes mellitus (T2DM) using whole blood. Leukocytes were first size-fractionated into different subtypes (neutrophils, monocytes, lymphocytes) using an inertial spiral sorter prior to single-cell impedance measurement in a microfluidic device with coplanar electrode design. Significant changes in membrane dielectric properties (size and opacity) were detected between healthy and activated leukocytes (TNF-α/LPS stimulated), during monocyte differentiation and among different monocyte subsets (classical, intermediate, non-classical). As proof-of-concept for diabetes testing, neutrophil/monocyte dielectric properties in T2DM subjects (n = 8) were quantified which were associated with cardiovascular risk factors including lipid levels, C-reactive protein (CRP) and vascular functions (LnRHI) (P < 0.05) were observed. Overall, these results clearly showed that T2DM subjects have pro-inflammatory leukocyte phenotypes and suggest leukocyte impedance signature as a novel surrogate biomarker for inflammation.

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.

Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model.

In the hepatitis B virus (HBV)-related hepatocellular carcinoma tumor microenvironment (TME), monocytes reportedly impede natural T cell functions via PD-L1/PD-1 signaling. However, it remains unclear if T cell receptor-redirected T cells (TCR T cells) are similarly inhibited. Hence, we developed a 3D intrahepatic TME microfluidic model to investigate the immunosuppressive potential of monocytes toward HBV-specific TCR T cells and the role of PD-L1/PD-1 signaling. Interestingly, in our 3D static microfluidic model, we observed that monocytes suppressed only retrovirally transduced (Tdx) TCR T cell cytotoxicity toward cancer cells via PD-L1/PD-1, while mRNA electroporated (EP) TCR T cell cytotoxicity was not affected by the presence of monocytes. Importantly, when co-cultured in 2D, both Tdx and EP TCR T cell cytotoxicity toward cancer cells were not suppressed by monocytes, suggesting our 3D model as a superior tool compared to standard 2D assays for predicting TCR T cell efficacy in a preclinical setting, which can thus be used to improve current immunotherapy strategies.

An automated staining protocol for seven-colour immunofluorescence of human tissue sections for diagnostic and prognostic use.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection and quantification of the expression of up to six markers, plus a nuclear counterstain, on a single tissue section. Recent studies have shown the potential for mIF to advance our understanding of complex disease processes, including cancer. It is important that the technique be standardised and validated to facilitate its transition into clinical use. Traditional approaches to mIF rely on manual processing of sections, which is time-consuming and a source of significant variation between samples/individuals. Here we determined if an automated diagnostic tissue stainer could be used for mIF incorporating tyramide signal amplification (TSA), and how the final image quality compared with sections stained semi-automatically or manually. Using tissue microarrays of fixed human breast tumour sections, we observed comparable antibody labelling between the diagnostic autostainer and manual technique. The diagnostic autostainer produced higher signal intensity with similar spectral unmixing efficiency. We also found that microwave treatment for antibody stripping during TSA labelling could be replaced by the heating option incorporated within the diagnostic-use autostainer. These data show that diagnostic autostainers used for traditional immunohistochemistry protocols can be readily adapted to achieve rapid preparation of high-quality sections using a TSA method for clinical mIF.

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.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.