Fine-tuning nucleophosmin in macrophage differentiation and activation.

M-CSF-driven differentiation of peripheral blood monocytes is one of the sources of tissue macrophages. In humans and mice, the differentiation process involves the activation of caspases that cleave a limited number of proteins. One of these proteins is nucleophosmin (NPM1), a multifunctional and ubiquitous protein. Here, we show that caspases activated in monocytes exposed to M-CSF cleave NPM1 at D213 to generate a 30-kDa N-terminal fragment. The protein is further cleaved into a 20-kDa fragment, which involves cathepsin B. NPM1 fragments contribute to the limited motility, migration, and phagocytosis capabilities of resting macrophages. Their activation with lipopolysaccharides inhibits proteolytic processes and restores expression of the full-length protein that negatively regulates the transcription of genes encoding inflammatory cytokines (eg, NPM1 is recruited with NF-κB on the MCP1 gene promoter to decrease its transcription). In mice with heterozygous npm gene deletion, cytokine production in response to lipopolysaccharides, including CXCL1 (KC), MCP1, and MIP2, is dramatically enhanced. These results indicate a dual function of NPM1 in M-CSF-differentiated macrophages. Proteolysis of the protein participates in the establishment of a mature macrophage phenotype. In response to inflammatory stimuli, the full-length protein negatively regulates inflammatory cytokine production.

Alpha-defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14(-)/CD24(+) phenotype. The proteome profile of these cells is dramatically distinct from that of CD14(+)/CD24(-) monocytes from CMML patients or healthy donors. More specifically, CD14(-)/CD24(+) CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit macrophage colony-stimulating factor (M-CSF)-driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors, and competitive experiments with uridine diphosphate (UDP)/uridine triphosphate (UTP), we demonstrate that HNP1-3 secreted by CD14(-)/CD24(+) cells inhibit M-CSF-induced differentiation of CD14(+)/CD24(-) cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.

Colony-stimulating factor-1-induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages.

The differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8-activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R-dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.

A role for caspases in the differentiation of erythroid cells and macrophages.

Several cysteine proteases of the caspase family play a central role in many forms of cell death by apoptosis. Other enzymes of the family are involved in cytokine maturation along inflammatory response. In recent years, several caspases involved in cell death were shown to play a role in other cellular processes such as proliferation and differentiation. In the present review, we summarize the current knowledge of the role of caspases in the differentiation of erythroid cells and macrophages. Based on these two examples, we show that the nature of involved enzymes, the pathways leading to their activation in response to specific growth factors, and the specificity of the target proteins that are cleaved by the activated enzymes strongly differ from one cell type to another. Deregulation of these pathways is thought to play a role in the pathophysiology of low-grade myelodysplastic syndromes, characterized by excessive activation of caspases and erythroid precursor apoptosis, and that of chronic myelomonocytic leukemia, characterized by a defective activation of caspases in monocytes exposed to M-CSF, which blocks their differentiation.

Intratumoral CpG-B Promotes Antitumoral Neutrophil, cDC, and T-cell Cooperation without Reprograming Tolerogenic pDC.

Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunologic outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here, we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells, subsequent increased antitumor T-cell priming in draining lymph nodes, and enhanced effector T-cell infiltration in the tumor microenvironment. These results reinforce the concept that i.t. delivery of TLR9 agonists alters the tumor microenvironment by improving the antitumor activity of both innate and adaptive immune cells.Significance: Intratumoral delivery of CpG-B disrupts the tolerogenic tumor microenvironment and inhibits tumor growth. Cancer Res; 78(12); 3280-92. ©2018 AACR.

Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes.

Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2(-/-) mice exhibited enhanced effector CD4(+) and CD8(+) T cell responses, impaired CD4(+) regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell-mediated immunity.

Th17 Cell Plasticity and Functions in Cancer Immunity.

Th17 cells represent a particular subset of T helper lymphocytes characterized by high production of IL-17 and other inflammatory cytokines. Th17 cells participate in antimicrobial immunity at mucosal and epithelial barriers and particularly fight against extracellular bacteria and fungi. While a role for Th17 cells in promoting inflammation and autoimmune disorders has been extensively and elegantly demonstrated, it is still controversial whether and how Th17 cells influence tumor immunity. Although Th17 cells specifically accumulate in many different types of tumors compared to healthy tissues, the outcome might however differ from a tumor type to another. Th17 cells were consequently associated with both good and bad prognoses. The high plasticity of those cells toward cells exhibiting either anti-inflammatory or in contrast pathogenic functions might contribute to Th17 versatile functions in the tumor context. On one hand, Th17 cells promote tumor growth by inducing angiogenesis (via IL-17) and by exerting themselves immunosuppressive functions. On the other hand, Th17 cells drive antitumor immune responses by recruiting immune cells into tumors, activating effector CD8(+) T cells, or even directly by converting toward Th1 phenotype and producing IFN-γ. In this review, we are discussing the impact of the tumor microenvironment on Th17 cell plasticity and function and its implications in cancer immunity.

New role for antigen-presenting activated pDCs in promoting Th17 cells and impacting antitumor immunity.

Plasmacytoid dendritic cells (pDCs) are not only potent inflammatory cytokine producers but also function as antigen-presenting cells (APCs). We have shown that vaccination using CpG-B activated tumor antigen (Ag) presenting pDCs induce Th17 cells that promote intratumoral immune cell recruitment, including antitumor cytotoxic T lymphocytes CTLs. Therefore, strategies targeting both innate and adaptive pDC functions may improve antitumor T-cell immunity.

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regression.

Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity.

Tolerogenic and activatory plasmacytoid dendritic cells in autoimmunity.

Plasmacytoid dendritic cells (pDCs) are a particular subset of DCs that link innate and adaptive immunity. They are responsible for the substantial production of type 1 interferon (IFN-I) in response to viral RNA or DNA through activation of TLR7 and 9. Furthermore, pDCs present antigens (Ag) and induce naïve T cell differentiation. It has been demonstrated that pDCs can induce immunogenic T cell responses through differentiation of cytotoxic CD8(+) T cells and effector CD4(+) T cells. Conversely, pDCs exhibit strong tolerogenic functions by inducing CD8(+) T cell deletion, CD4(+) T cell anergy, and Treg differentiation. However, since IFN-I produced by pDCs efficiently activates and recruits conventional DCs, B cells, T cells, and NK cells, pDCs also indirectly affect the nature and the amplitude of adaptive immune responses. As a consequence, the precise role of Ag-presenting functions of pDCs in adaptive immunity has been difficult to dissect in vivo. Additionally, different experimental procedures led to conflicting results regarding the outcome of T cell responses induced by pDCs. During the development of autoimmunity, pDCs have been shown to play both immunogenic and tolerogenic functions depending on disease, disease progression, and the experimental conditions. In this review, we will discuss the relative contribution of innate and adaptive pDC functions in modulating T cell responses, particularly during the development of autoimmunity.

Targeting apoptosis proteins in hematological malignancies.

The apoptotic machinery plays a key role in hematopoietic cell homeostasis. Terminally differentiated cells are eliminated, at least in part, by apoptosis, whereas part of the apoptotic machinery, including one or several caspases, is required to go through very specific steps of the differentiation pathways. A number of hematological diseases involve a deregulation of this machinery, which in most cases is a decrease in cell sensitivity to pro-apoptotic signals through over-expression of anti-apoptotic molecules. In some situations however, e.g. in the erythroid lineage of low grade myelodysplastic syndromes, cell sensitivity to apoptosis is increased in a death receptor-dependent manner and cell death pathways are inhibited only when these diseases progress into high grade and acute leukemia. Therapeutic strategies targeting the apoptotic machinery specifically block cell death inhibitors that are over-expressed in transformed cells, mainly Bcl-2-related proteins and Inhibitor of Apoptosis Proteins (IAPs). Another strategy is the activation of the extrinsic pathway to apoptosis, mainly through the death receptor agonist Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) or agonistic antibodies targeting TRAIL receptors. The use of inhibitors of death receptors could make sense when these receptors are involved in excessive cell death or activation of survival pathways. Most of the drugs targeting apoptotic pathways introduced in clinics have demonstrated their tolerability. Their efficacy, either alone or in combination with other drugs such as demethylating agents and histone deacetylase inhibitors, is currently tested in both myeloid and lymphoid hematological diseases.

Various functions of caspases in hematopoiesis.

The role of cysteine proteases of the caspase family in apoptosis is well defined. Some caspases were initially shown to be involved in cytokine maturation along inflammatory response. In the recent years, several other non apoptotic functions of caspases were identified. In hematopoietic cells, caspases play a role in specific pathways of differentiation (erythropoiesis, differentiation of monocytes into macrophages, formation of proplatelets by megakaryocytes). These enzymes also play a non-apoptotic and complex role in regulating the maturation and proliferation of specific lymphocytes. Lastly, the apoptotic functions of caspases regulate the life span of several but not all blood cell types. The present review summarizes the current knowledge in these different functions. We show that the nature of involved enzymes, the pathways leading to their activation and the specificity of their cellular target proteins varies strongly from a cell type to another. We indicate also that, in most situations, specific Bcl-2-related proteins are involved in the control of caspase activation. Lastly, we discuss the deregulation of these pathways in hematopoietic diseases, including those in which an excess in caspase activation leads to cell death and those in which a default in caspase activation could block cell differentiation.

Trefoil factor TFF1-induced protection of conjunctival cells from apoptosis at premitochondrial and postmitochondrial levels.

PURPOSE: Goblet cells of the conjunctival epithelium synthesize and secrete TFF1 (Trefoil factor 1), a small protease-resistant peptide that, together with mucins, is responsible for the rheologic properties of the tear film. This study aimed to determine whether TFF1, whose synthesis increases in inflammatory conditions such as pterygium, could protect conjunctival cells from apoptosis. METHODS: Chang conjunctival cells, either wild-type or expressing TFF1 through stable transfection, were exposed to benzalkonium chloride (BAK) and ultraviolet (UV) irradiation to trigger apoptosis. The authors used cell fractionation to detect lipid raft-associated proteins, coimmunoprecipitation to explore the formation of a death-inducing signaling complex (DISC), and a combination of immunofluorescence, immunoblotting, flow cytometry, siRNA-mediated decrease in gene expression, and electrophoretic mobility shift assay to explore the mechanisms of TFF1-protective effects. RESULTS: TFF1 protects Chang conjunctival cells from apoptosis induced by UV irradiation and BAK at two levels. First, TFF1 prevents caspase-8 activation at the level of the DISC that involves Fas receptor in plasma membrane rafts, which in turn decreases the mitochondrial release of cytochrome c. Second, TFF1 interferes with caspase-9 and caspase-3 activation through an NF-kappaB-induced increase in the expression of XIAP (X-linked inhibitor of apoptosis protein). CONCLUSIONS: TFF1 upregulation on inflammatory conditions may be a protective mechanism that limits conjunctival cell loss by inhibiting apoptosis upstream and downstream of the mitochondrial events. These observations suggest a potential interest of TFF1 or related peptides to prevent cell death in ocular surface disorders.