The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell-mediated resistance to metastasis.

The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the ß-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell-mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell-mediated resistance to metastasis.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

New insights into the multidimensional concept of macrophage ontogeny, activation and function.

Macrophages have protective roles in immunity to pathogens, tissue development, homeostasis and repair following damage. Maladaptive immunity and inflammation provoke changes in macrophage function that are causative of disease. Despite a historical wealth of knowledge about macrophages, recent advances have revealed unknown aspects of their development and function. Following development, macrophages are activated by diverse signals. Such tissue microenvironmental signals together with epigenetic changes influence macrophage development, activation and functional diversity, with consequences in disease and homeostasis. We discuss here how recent discoveries in these areas have led to a multidimensional concept of macrophage ontogeny, activation and function. In connection with this, we also discuss how technical advances facilitate a new roadmap for the isolation and analysis of macrophages at high resolution.

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.

Metabolic Reprogramming of Immune Cells in Cancer Progression.

Immune cells play a key role in host defense against infection and cancer. Upon encountering danger signals, these cells undergo activation leading to a modulation in their immune functions. However, recent studies reveal that immune cells upon activation also show distinct metabolic changes that impact their immune functions. Such metabolic reprogramming and its functional effects are well known for cancer cells. Given that immune cells have emerged as crucial players in cancer progression, it is important to understand whether immune cells also undergo metabolic reprogramming in tumors and how this might affect their contribution in cancer progression. This emerging aspect of tumor-associated immune cells is reviewed here, discussing metabolic reprogramming of different immune cell types, the key pathways involved, and its impact on tumor progression.

Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

An epithelial Nfkb2 pathway exacerbates intestinal inflammation by supplementing latent RelA dimers to the canonical NF-κB module.

Aberrant inflammation, such as that associated with inflammatory bowel disease (IBD), is fueled by the inordinate activity of RelA/NF-κB factors. As such, the canonical NF-κB module mediates controlled nuclear activation of RelA dimers from the latent cytoplasmic complexes. What provokes pathological RelA activity in the colitogenic gut remains unclear. The noncanonical NF-κB pathway typically promotes immune organogenesis involving Nfkb2 gene products. Because NF-κB pathways are intertwined, we asked whether noncanonical signaling aggravated inflammatory RelA activity. Our investigation revealed frequent engagement of the noncanonical pathway in human IBD. In a mouse model of experimental colitis, we established that Nfkb2-mediated regulations escalated the RelA-driven proinflammatory gene response in intestinal epithelial cells, exacerbating the infiltration of inflammatory cells and colon pathologies. Our mechanistic studies clarified that cell-autonomous Nfkb2 signaling supplemented latent NF-κB dimers, leading to a hyperactive canonical RelA response in the inflamed colon. In sum, the regulation of latent NF-κB dimers appears to link noncanonical Nfkb2 signaling to RelA-driven inflammatory pathologies and may provide for therapeutic targets.

Molecular profiling reveals a tumor-promoting phenotype of monocytes and macrophages in human cancer progression.

Monocytes and macrophages are major components of the tumor microenvironment, but their contributions to human cancer are poorly understood. We used molecular profiling combined with functional assays to investigate the role of these cells in human renal cell carcinoma (RCC). Blood monocytes from RCC patients displayed a tumor-promoting transcriptional profile that supported functions like angiogenesis and invasion. Induction of this protumor phenotype required an interleukin-1 receptor (IL-1R)-dependent mechanism. Indeed, targeting of IL-1-IL-1R axis in a human RCC xenograft model abrogated the protumor phenotype of tumor-associated macrophages (TAMs) and reduced tumor growth in vivo. Supporting this, meta-analysis of gene expression from human RCC tumors showed IL1B expression to correlate with myelomonocytic markers, protumor genes, and tumor staging. Analyzing RCC patient tumors confirmed the protumor phenotype of TAMs. These data provide direct evidence for a tumor-promoting role of monocytes and macrophages in human cancer and indicate IL-1-IL-1R as a possible therapeutic target.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm.

Plasticity is a hallmark of cells of the myelomonocytic lineage. In response to innate recognition or signals from lymphocyte subsets, mononuclear phagocytes undergo adaptive responses. Shaping of monocyte-macrophage function is an essential component of resistance to pathogens, tissue damage and repair. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. A better understanding of the molecular basis of myelomonocytic cell plasticity will open new vistas in immunopathology and therapeutic intervention.

Basophil-macrophage dialog in allergic inflammation.

Monocyte-macrophage differentiation under pathological conditions is poorly understood. In the present issue of Immunity, Egawa et al. (2013) report how basophils drive the differentiation of inflammatory monocytes into M2 macrophages, thereby regulating allergic skin inflammation.

Metabolic regulation of macrophage phenotype and function.

Studies in the last 20 years have given us a remarkable insight into the functional and phenotypic diversity of macrophages which reflects their integral role in host defence, homeostasis and pathogenesis. Mouse genetics, transcriptomic and epigenetic studies have provided an ontogenic and molecular perspective to the phenotypic diversity of these cells. Recently, metabolic studies have revealed the crucial role of metabolism and metabolites in shaping the phenotype and function of macrophages. Evidence pertaining to this aspect will be reviewed here.

Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors.

Monocytes are effectors of the inflammatory response to microbes. Human CD14(+) monocytes specialize in phagocytosis and production of reactive oxygen species and secrete inflammatory cytokines in response to a broad range of microbial cues. Here, we have characterized the functions of human monocytes that lack CD14 (CD14(dim)) and express CD16. CD14(dim) monocytes were genetically distinct from natural killer cells. Gene expression analyses indicated similarities with murine patrolling Gr1(dim) monocytes, and they patrolled the endothelium of blood vessels after adoptive transfer, in a lymphocyte function-associated antigen-1-dependent manner. CD14(dim) monocytes were weak phagocytes and did not produce ROS or cytokines in response to cell-surface Toll-like receptors. Instead, they selectively produced TNF-α, IL-1ß, and CCL3 in response to viruses and immune complexes containing nucleic acids, via a proinflammatory TLR7-TLR 8-MyD88-MEK pathway. Thus, CD14(dim) cells are bona fide monocytes involved in the innate local surveillance of tissues and the pathogenesis of autoimmune diseases.

MeSsAGe risk score: tool for renal biopsy decision in steroid-dependent nephrotic syndrome.

BACKGROUND: A lack of consensus exists as to the timing of kidney biopsy in children with steroid-dependent nephrotic syndrome (SDNS) where minimal change disease (MCD) predominates. This study aimed at examining the applicability of a biomarker-assisted risk score model to select SDNS patients at high risk of focal segmental glomerulosclerosis (FSGS) for biopsy. METHODS: Fifty-five patients with SDNS and biopsy-proven MCD (n = 40) or FSGS (n = 15) were studied. A risk score model was developed with variables consisting of age, sex, eGFR, suPAR levels and percentage of CD8+ memory T cells. Following multivariate regression analysis, total risk score was calculated as sum of the products of odds ratios and corresponding variables. Predictive cut-off point was determined using receiver operator characteristics (ROC) curve analysis. RESULTS: Plasma suPAR levels in FSGS patients were significantly higher, while percentage of CD45RO+CD8+CD3+ was significantly lower than in MCD patients and controls. ROC analysis suggests the risk score model with threshold score of 16.7 (AUC 0.84, 95% CI 0.72-0.96) was a good predictor of FSGS on biopsy. The 100% PPV cut-off was >24.0, while the 100% NPV was <13.3. CONCLUSION: A suPAR and CD8+ memory T cell percentage-based risk score model was developed to stratify SDNS patients for biopsy and for predicting FSGS.

TLR7 and TLR9 ligands regulate antigen presentation by macrophages.

The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state.

Ubiquitin-conjugating enzyme Ubc13 controls breast cancer metastasis through a TAK1-p38 MAP kinase cascade.

Metastatic spread is the leading cause of cancer mortality. Breast cancer (BCa) metastatic recurrence can happen years after removal of the primary tumor. Here we show that Ubc13, an E2 enzyme that catalyzes K63-linked protein polyubiquitination, is largely dispensable for primary mammary tumor growth but is required for metastatic spread and lung colonization by BCa cells. Loss of Ubc13 inhibited BCa growth and survival only at metastatic sites. Ubc13 was dispensable for transforming growth factor ß (TGFß)-induced SMAD activation but was required for activation of non-SMAD signaling via TGFß-activating kinase 1 (TAK1) and p38, whose activity controls expression of numerous metastasis promoting genes. p38 activation restored metastatic activity to Ubc13-deficient cells, and its pharmacological inhibition attenuated BCa metastasis in mice, suggesting it is a therapeutic option for metastatic BCa.

Annexin-A1 regulates TLR-mediated IFN-ß production through an interaction with TANK-binding kinase 1.

TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-ß and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-ß and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-ß and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-ß and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.

Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

Protumoral role of monocytes in human B-cell precursor acute lymphoblastic leukemia: involvement of the chemokine CXCL10.

Myelomonocytic cells play a key role in the progression of many solid tumors. However, very little is known about their contribution to the progression of hematopoietic cancers. We investigated the role of monocytes in the progression of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We demonstrated that coculturing human monocytes in vitro with CD19+ BCP-ALL blasts from patients "conditioned" them to an inflammatory phenotype characterized by significant up-regulation of the chemokine, CXCL10. This phenotype was also observable ex vivo in monocytes isolated from BCP-ALL patients, which show elevated CXCL10 production compared with monocytes from healthy donors. Functionally, the "conditioned" monocytes promoted migration and invasive capacity of BCP-ALL cells. Increased invasion was mediated by matrix metalloproteinase 9 expression and activity in the BCP-ALL cells induced by the monocyte-derived CXCL10. However, neither the "conditioned" monocytes nor the CXCL10 produced by these cells had any effect on the proliferation/viability of BCP-ALL cells and angiogenesis. Collectively, our results strongly suggest a protumoral role for human monocytes in BCP-ALL, orchestrated by CXCL10 and its effect on tumor cell migration and invasion. These observations highlight the importance of the CXCL10/CXCR3 chemokine circuit in BCP-ALL progression.

Macrophage plasticity and polarization in tissue repair and remodelling.

Mononuclear phagocyte plasticity includes the expression of functions related to the resolution of inflammation, tissue repair and remodelling, particularly when these cells are set in an M2 or an M2-like activation mode. Macrophages are credited with an essential role in remodelling during ontogenesis. In extraembryonic life, under homeostatic conditions, the macrophage trophic and remodelling functions are recapitulated in tissues such as bone, mammary gland, decidua and placenta. In pathology, macrophages are key components of tissue repair and remodelling that occur during wound healing, allergy, parasite infection and cancer. Interaction with cells bearing stem or progenitor cell properties is likely an important component of the role of macrophages in repair and remodelling. These properties of cells of the monocyte-macrophage lineage may represent a tool and a target for therapeutic exploitation.