Classical monocyte ontogeny dictates their functions and fates as tissue macrophages.

Classical monocytes (CMs) are ephemeral myeloid immune cells that circulate in the blood. Emerging evidence suggests that CMs can have distinct ontogeny and originate from either granulocyte-monocyte- or monocyte-dendritic-cell progenitors (GMPs or MDPs). Here, we report surface markers that allowed segregation of murine GMP- and MDP-derived CMs, i.e., GMP-Mo and MDP-Mo, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP-Mo yielded an equal increase in homeostatic CM progeny, such as blood-resident non-classical monocytes and gut macrophages; however, these cells differentially seeded various other selected tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo differentiated into distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse that differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.

Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

Type I Interferon Orchestrates Demand-Adapted Monopoiesis during Influenza A Virus Infection via STAT1-Mediated Upregulation of Macrophage Colony-Stimulating Factor Receptor Expression.

Whether and how a local virus infection affects the hematopoietic system in the bone marrow is largely unknown, unlike with systemic infection. In this study, we showed that influenza A virus (IAV) infection leads to demand-adapted monopoiesis in the bone marrow. The beta interferon (IFN-ß) promoter stimulator 1 (IPS-1)-type I IFN-IFN-α receptor 1 (IFNAR1) axis-mediated signaling was found to induce the emergency expansion of the granulocyte-monocyte progenitor (GMP) population and upregulate the expression of the macrophage colony-stimulating factor receptor (M-CSFR) on bipotent GMPs and monocyte progenitors via the signal transducer and activator of transcription 1 (STAT1), leading to a scaled-back proportion of granulocyte progenitors. To further address the influence of demand-adapted monopoiesis on IAV-induced secondary bacterial infection, IAV-infected wild-type (WT) and Stat1-/- mice were challenged with Streptococcus pneumoniae. Compared with WT mice, Stat1-/- mice did not demonstrate demand-adapted monopoiesis, had more infiltrating granulocytes, and were able to effectively eliminate the bacterial infection. IMPORTANCE Our findings show that influenza A virus infection induces type I interferon (IFN)-mediated emergency hematopoiesis to expand the GMP population in the bone marrow. The type I IFN-STAT1 axis was identified as being involved in mediating the viral-infection-driven demand-adapted monopoiesis by upregulating M-CSFR expression in the GMP population. As secondary bacterial infections often manifest during a viral infection and can lead to severe or even fatal clinical complications, we further assessed the impact of the observed monopoiesis on bacterial clearance. Our results suggest that the resulting decrease in the proportion of granulocytes may play a role in diminishing the IAV-infected host's ability to effectively clear secondary bacterial infection. Our findings not only provide a more complete picture of the modulatory functions of type I IFN but also highlight the need for a more comprehensive understanding of potential changes in hematopoiesis during local infections to better inform clinical interventions.

Maturation-associated gene expression profiles along normal human bone marrow monopoiesis.

Human monopoiesis is a tightly coordinated process which starts in the bone marrow (BM) haematopoietic stem cell (HSC) compartment and leads to the production of circulating blood mature monocytes. Although mature monocytes/macrophages have been extensively studied in both normal or inflammatory conditions, monopoiesis has only been assessed in vitro and in vivo animal models, due to low frequency of the monocytic precursors in the normal human BM. Here we investigated the transcriptional profile along normal human BM monopoiesis. Five distinct maturation-associated stages of monocytic precursors were identified and isolated from (fresh) normal human BM through fluorescence-activated cell sorting, and the gene expression profile (GEP) of each monocytic precursor subset was analysed by DNA-oligonucleotide microarrays. Overall, >6000 genes (18% of the genes investigated) were expressed in ≥1 stage of BM monopoiesis at stable or variable amounts, showing early decrease in cell proliferation with increased levels of expression of genes linked with cell differentiation. The here-defined GEP of normal human BM monopoiesis might contribute to better understand monocytic differentiation and the identification of novel monocytic candidate markers, while also providing a frame of reference for the study of monocytic maturation in both neoplastic and non-neoplastic disease conditions involving monocytic precursor cells.

Regulation of matrix metalloproteinase-9 during monopoiesis and zinc deficiency by chromatin remodeling.

INTRODUCTION: Matrix metalloproteinase-9 (MMP-9) cleaves various extracellular matrix proteins, hence significantly contributes to numerous physiological but also pathological processes. Monocytic differentiation is associated with increased MMP-9 gene expression. Interestingly, MMP-9 upregulation during monocytic differentiation is paralleled by a decline in intracellular zinc levels. Hence, an influence of zinc on the regulation of MMP-9 expression may exist. Although, previous studies suggest a vital role of zinc regarding MMP-9 activity, the possible relevance of zinc homeostasis during transcriptional regulation of MMP-9 for example via epigenetic mechanisms is rather unclear. AIM: This study aims to find a correlation between zinc deficiency and MMP-9 transcriptional regulation, focusing on epigenetics as the possible mechanism behind zinc deficiency-induced changes. METHODS: The effect of differentiation and zinc deficiency on MMP-9 expression and MMP9 promoter accessibility was investigated using the acute promyelocytic cell line NB4. Intracellular free zinc levels were detected by flow cytometry. MMP-9 gene expression was measured by real-time PCR and ELISA. Analysis of chromatin structures was done using chromatin accessibility by real-time PCR (CHART) assay. RESULTS: During monocytic differentiation of NB4 cells, the decrease in intracellular zinc levels was paralleled by an increased production of MMP-9. Assessment of chromatin structure revealed increased accessibility of certain regions within the MMP-9 promoter in differentiated cells. Interestingly, upregulated activation-induced MMP-9 gene expression as well as a more accessible MMP-9 promoter were in zinc-deficient NB4 cells whereas zinc resupplementation reversed the effects. CONCLUSION: These data demonstrate an important role of epigenetic mechanisms in regulating MMP-9 expression under zinc deficiency. This could provide an encouraging step to expand the research on using zinc for the treatment of various pathological conditions such as inflammatory, vascular and autoimmune diseases resulting from MMP-9 deregulation.

Mercury chloride activates the IFNγ-IRF1 signaling in myeloid progenitors and promotes monopoiesis in mice.

Inorganic mercury (Hg2+) is a highly toxic heavy metal in the environment. To date, the impacts of Hg2+ on the development of monocytes, or monopoiesis, have not been fully addressed. The aim of the present study was to investigate the impact of Hg2+ on monopoiesis. In this study, we treated B10.S mice and DBA/2 mice with 10 µM or 50 µM HgCl2 via drinking water for 4 wk, and we then evaluated the development of monocytes. Treatment with 50 µM HgCl2, but not 10 µM HgCl2, increased the number of monocytes in the blood, spleen and bone marrow (BM) of B10.S mice. Accordingly, treatment with 50 µM HgCl2, but not 10 µM HgCl2, increased the number of common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP) in the BM. Functional analyses indicated that treatment with 50 µM HgCl2 promoted the differentiation of CMP and GMP to monocytes in the BM of B10.S mice. Mechanistically, treatment with 50 µM HgCl2 induced the production of IFNγ, which activated the Jak1/3-STAT1/3-IRF1 signaling in CMP and GMP and enhanced their differentiation potential for monocytes in the BM, thus likely leading to increased number of mature monocytes in B10.S mice. Moreover, the increased monopoiesis by Hg2+ was associated with the increased inflammatory status in B10.S mice. In contrast, treatment with 50 µM HgCl2 did not impact the monopoiesis in DBA/2 mice. Our study reveals the impact of Hg on the development of monocytes.

Integrated single cell analysis shows chronic alcohol drinking disrupts monocyte differentiation in the bone marrow.

Chronic heavy alcohol drinking (CHD) rewires monocytes and macrophages toward heightened inflammatory states with compromised antimicrobial defenses that persist after 1-month abstinence. To determine whether these changes are mediated through alterations in the bone marrow niche, we profiled monocytes and hematopoietic stem cell progenitors (HSCPs) from CHD rhesus macaques using a combination of functional assays and single cell genomics. CHD resulted in transcriptional profiles consistent with increased activation and inflammation within bone marrow resident monocytes and macrophages. Furthermore, CHD resulted in transcriptional signatures associated with increased oxidative and cellular stress in HSCP. Differentiation of HSCP in vitro revealed skewing toward monocytes expressing "neutrophil-like" markers with greater inflammatory responses to bacterial agonists. Further analyses of HSCPs showed broad epigenetic changes that were in line with exacerbated inflammatory responses within monocytes and their progenitors. In summary, CHD alters HSCPs in the bone marrow leading to the production of monocytes poised to generate dysregulated hyper-inflammatory responses.

Behind the monocyte's mystique: uncovering their developmental trajectories and fates.

Monocytes are circulating myeloid cells that are derived from dedicated progenitors in the bone marrow. Originally thought of as mere precursors for the replacement of tissue macrophages, it is increasingly clear that monocytes execute distinct effector functions and may give rise to monocyte-derived cells with unique properties from tissue-resident macrophages. Recently, the advent of novel experimental approaches such as single-cell analysis and fate-mapping tools has uncovered an astonishing display of monocyte plasticity and heterogeneity, which we believe has emerged as a key theme in the field of monocyte biology in the last decade. Monocyte heterogeneity is now recognized to develop as early as the progenitor stage through specific imprinting mechanisms, giving rise to specialized effector cells in the tissue. At the same time, monocytes must overcome their susceptibility towards cellular death to persist as monocyte-derived cells in the tissues. Environmental signals that preserve their heterogenic phenotypes and govern their eventual fates remain incompletely understood. In this review, we will summarize recent advances on the developmental trajectory of monocytes and discuss emerging concepts that contributes to the burgeoning field of monocyte plasticity and heterogeneity.

Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS.

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.

Type I interferon upregulation and deregulation of genes involved in monopoiesis in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is characterized by myelomonocytic bias and monocytic proliferation. Whether cell-intrinsic innate immune or inflammatory upregulation mediate disease pathogenesis and phenotype or whether the degree of aberrant monocytic differentiation influences outcomes remains unclear. We compared the transcriptomic features of bone marrow CD34+ cells from 19 patients with CMML and compared to healthy individuals. A total of 1495 genes had significantly differential expression in CMML (q<0.05, fold change>2), including 1271 genes that were significantly upregulated and 224 that were significantly downregulated in CMML. Top upregulated genes were associated with interferon (IFN) alpha and beta signaling, chemokine receptors, IFN gamma, G protein-coupled receptor ligand signaling, and genes involved in immunomodulatory interactions between lymphoid and non-lymphoid cells. Additionally, 6 gene sets were differentially upregulated and 139 were significantly downregulated in patients with myeloproliferative compared to myelodysplastic CMML. A total of 23 genes involved in regulation of monopoiesis were upregulated in CMML compared to healthy controls. We developed a prediction model using Cox regression including 3 of these genes, which differentiated patients into two prognostic subsets with distinct survival outcomes. This data warrants further evaluation of the roles and therapeutic potential of type I IFN signaling and monopoiesis in CMML.

Prenatal Development and Function of Human Mononuclear Phagocytes.

The human mononuclear phagocyte (MP) system, which includes dendritic cells, monocytes, and macrophages, is a critical regulator of innate and adaptive immune responses. During embryonic development, MPs derive sequentially in yolk sac progenitors, fetal liver, and bone marrow haematopoietic stem cells. MPs maintain tissue homeostasis and confer protective immunity in post-natal life. Recent evidence - primarily in animal models - highlight their critical role in coordinating the remodeling, maturation, and repair of target organs during embryonic and fetal development. However, the molecular regulation governing chemotaxis, homeostasis, and functional diversification of resident MP cells in their respective organ systems during development remains elusive. In this review, we summarize the current understanding of the development and functional contribution of tissue MPs during human organ development and morphogenesis and its relevance to regenerative medicine. We outline how single-cell multi-omic approaches and next-generation ex-vivo organ-on-chip models provide new experimental platforms to study the role of human MPs during development and disease.

Plasticity of monocyte development and monocyte fates.

Monocytes are circulating myeloid immune precursor cells that are generated in the bone marrow. Mature monocytes are released into the circulation and, in case of need, recruited to peripheral sites of inflammation to differentiate into monocyte-derived effector cells. In absence of overt inflammation, monocytes also extravasate into selected tissues, where they complement tissue-resident macrophage compartments. Adjustment of these homeostatic monocyte infiltrates to local environment is critical to maintain health, as best established for the intestine. Defined gene expression changes that differ between gut segments presumably help strike the fine balance between the crucial function of these monocyte-derived macrophages as tissue rheostats and their detrimental hyperactivation. Environmental factors that dictate local monocyte differentiation remain incompletely understood. Definition of the latter could aid our general understanding of in vivo monocyte functions and their relation to inflammatory disorders. In this review, we summarize recent advances in our understanding of monocyte subsets, their differentiation into tissue macrophages, and selected contributions of monocyte-derived cells to steady-state physiology. Moreover, we will discuss emerging evidence for an intriguing bifurcation of monocyte development in the bone marrow and potential functional implications. Emphasis will be given to points of controversies, but we will largely focus on the healthy organism. For a discussion of monocyte and macrophage contributions to inflammatory conditions, we refer the reader to other dedicated reviews.

Systemic Reprogramming of Monocytes in Cancer.

Monocytes influence multiple aspects of tumor progression, including antitumor immunity, angiogenesis, and metastasis, primarily by infiltrating tumors, and differentiating into tumor-associated macrophages. Emerging evidence suggests that the tumor-induced systemic environment influences the development and phenotype of monocytes before their arrival to the tumor site. As a result, circulating monocytes show functional alterations in cancer, such as the acquisition of immunosuppressive activity and reduced responsiveness to inflammatory stimuli. In this review, we summarize available evidence about cancer-induced changes in monopoiesis and its impact on the abundance and function of monocytes in the periphery. In addition, we describe the phenotypical alterations observed in tumor-educated peripheral blood monocytes and highlight crucial gaps in our knowledge about additional cellular functions that may be affected based on transcriptomic studies. We also highlight emerging therapeutic strategies that aim to reverse cancer-induced changes in monopoiesis and peripheral monocytes to inhibit tumor progression and improve therapy responses. Overall, we suggest that an in-depth understanding of systemic monocyte reprogramming will have implications for cancer immunotherapy and the development of clinical biomarkers.

The Ontogeny of Monocyte Subsets.

Classical and non-classical monocytes, and the macrophages and monocyte-derived dendritic cells they produce, play key roles in host defense against pathogens, immune regulation, tissue repair and many other processes throughout the body. Recent studies have revealed previously unappreciated heterogeneity among monocytes that may explain this functional diversity, but our understanding of mechanisms controlling the functional programming of distinct monocyte subsets remains incomplete. Resolving monocyte heterogeneity and understanding how their functional identity is determined holds great promise for therapeutic immune modulation. In this review, we examine how monocyte origins and developmental influences shape the phenotypic and functional characteristics of monocyte subsets during homeostasis and in the context of infection, inflammation, and cancer. We consider how extrinsic signals and transcriptional regulators impact monocyte production and functional programming, as well as the influence of epigenetic and metabolic mechanisms. We also examine the evidence that functionally distinct monocyte subsets are produced via different developmental pathways during homeostasis and that inflammatory stimuli differentially target progenitors during an emergency response. We highlight the need for a more comprehensive understanding of the relationship between monocyte ontogeny and heterogeneity, including multiparametric single-cell profiling and functional analyses. Studies defining mechanisms of monocyte subset production and maintenance of unique monocyte identities have the potential to facilitate the design of therapeutic interventions to target specific monocyte subsets in a variety of disease contexts, including infectious and inflammatory diseases, cancer, and aging.

Mathematical Analysis of Cytokine-Induced Differentiation of Granulocyte-Monocyte Progenitor Cells.

Granulocyte-monocyte progenitor (GMP) cells play a vital role in the immune system by maturing into a variety of white blood cells, including neutrophils and macrophages, depending on exposure to cytokines such as various types of colony stimulating factors (CSF). Granulocyte-CSF (G-CSF) induces granulopoiesis and macrophage-CSF (M-CSF) induces monopoiesis, while granulocyte/macrophage-CSF (GM-CSF) favors monocytic and granulocytic differentiation at low and high concentrations, respectively. Although these differentiation pathways are well documented, the mechanisms behind the diverse behavioral responses of GMP cells to CSFs are not well understood. In this paper, we propose a mechanism of interacting CSF-receptors and transcription factors that control GMP differentiation, convert the mechanism into a set of differential equations, and explore the properties of this mathematical model using dynamical systems theory. Our model reproduces numerous experimental observations of GMP cell differentiation in response to varying dosages of G-CSF, M-CSF, and GM-CSF. In particular, we are able to reproduce the concentration-dependent behavior of GM-CSF induced differentiation, and propose a mechanism driving this behavior. In addition, we explore the differentiation of a fourth phenotype, monocytic myeloid-derived suppressor cells (M-MDSC), showing how they might fit into the classical pathways of GMP differentiation and how progenitor cells can be primed for M-MDSC differentiation. Finally, we use the model to make novel predictions that can be explored by future experimental studies.

Amphibian macrophage development and antiviral defenses.

Macrophage lineage cells represent the cornerstone of vertebrate physiology and immune defenses. In turn, comparative studies using non-mammalian animal models have revealed that evolutionarily distinct species have adopted diverse molecular and physiological strategies for controlling macrophage development and functions. Notably, amphibian species present a rich array of physiological and environmental adaptations, not to mention the peculiarity of metamorphosis from larval to adult stages of development, involving drastic transformation and differentiation of multiple new tissues. Thus it is not surprising that different amphibian species and their respective tadpole and adult stages have adopted unique hematopoietic strategies. Accordingly and in order to establish a more comprehensive view of these processes, here we review the hematopoietic and monopoietic strategies observed across amphibians, describe the present understanding of the molecular mechanisms driving amphibian, an in particular Xenopus laevis macrophage development and functional polarization, and discuss the roles of macrophage-lineage cells during ranavirus infections.

Serum amyloid A regulates monopoiesis in hyperlipidemic Ldlr(-/-) mice.

We previously showed that feeding a Western-type diet (WTD) to Ldlr(-/-) mice lacking serum amyloid A (SAA) (Saa(-/-) Ldlr(-/-) mice), the level of total blood monocytes was higher than in Ldlr(-/-) mice. In this investigation we demonstrate that higher levels of bone marrow monocytes and macrophage-dendritic cell progenitor (MDP) cells were found in WTD-fed Saa(-/-) Ldlr(-/-) mice compared to Ldlr(-/-) mice and lower levels of GMP cells and CMP cells in Ldlr(-/-) mice. These data indicate that SAA regulates the level of bone marrow monocytes and their myeloid progenitors in hyperlipidemic Ldlr(-/-) mice.

Regulation of the Interleukin-6 gene expression during monocytic differentiation of HL-60 cells by chromatin remodeling and methylation.

The pro-inflammatory cytokine Interleukin (IL)-6 is involved in the proliferation and differentiation of leukocytes and non-immune cells, but its overproduction is associated with inflammatory and autoimmune disorders. The main producers of IL-6 are mature monocytes, whereas progenitor cells and the promyeloid cell line HL-60 do not synthesize IL-6. In contrast, HL-60 cells differentiated into monocytic cells were able to express IL-6 after lipopolysaccharide (LPS) stimulation. This study investigated the chromatin structure of the IL-6 promoter and the effect of methylation on IL-6 gene regulation during monopoiesis. The results show that the proximal IL-6 promoter regions I to III (+13/-329) were inaccessible in undifferentiated HL-60 cells but became significantly accessible in differentiated HL-60 cells stimulated with LPS. Region IL-6 VI (-1099/-1142) remained closed, but the upstream region IL-6 VII (-2564/-2877) relaxed after differentiation and LPS treatment. The opening of IL-6 IV (-309/-521) and IL-6V (-500/-722), containing DNA and histone methylation sites, was differentiation-dependent only. Demethylation experiments using 5-aza-2'-deoxycytidine (AZA) followed by LPS stimulation revealed a significant enhanced IL-6 mRNA expression and protein release by HL-60 cells. AZA treatment resulted in significant increased IL-6 promoter accessibilities, identifying methylation as an important repressor of IL-6 gene regulation in promyeloid cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) had no effect on IL-6 promoter accessibility. Our data indicate that during monopoiesis the proximal IL-6 promoter is reorganized into an accessible conformation allowing transcription of IL-6 after LPS stimulation. DNA methylation appears to be the essential epigenetic mechanism in IL-6 gene expression of mature monocytes and their progenitors by controlling the chromatin structure.