Ángeles Domínguez-Soto

Folate Receptor β Is Expressed by Tumor-Associated Macrophages and Constitutes a Marker for M2 Anti-inflammatory/Regulatory Macrophages

Macrophage activation comprises a continuum of functional states critically determined by cytokine microenvironment. Activated macrophages have been functionally grouped according to their response to pro-Th1/proinflammatory stimuli [lipopolysaccharide, IFNgamma, granulocyte macrophage colony-stimulating factor (GM-CSF); M1] or pro-Th2/anti-inflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2]. We report that folate receptor beta (FRbeta), encoded by the FOLR2 gene, is a marker for macrophages generated in the presence of M-CSF (M2), but not GM-CSF (M1), and whose expression correlates with increased folate uptake ability. The acquisition of folate uptake ability by macrophages is promoted by M-CSF, maintained by IL-4, prevented by GM-CSF, and reduced by IFNgamma, indicating a link between FRbeta expression and M2 polarization. In agreement with in vitro data, FRbeta expression is detected in tumor-associated macrophages (TAM), which exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRbeta is expressed, and mediates folate uptake, by CD163(+) CD68(+) CD14(+) IL-10-producing TAM, and its expression is induced by tumor-derived ascitic fluid and conditioned medium from fibroblasts and tumor cell lines in an M-CSF-dependent manner. These results establish FRbeta as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of therapeutic drugs are a potential immunotherapy tool to target TAM.

Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin Mediates Binding and Internalization of Aspergillus fumigatus Conidia by Dendritic Cells and Macrophages

Aspergillus fumigatus is responsible for a large percentage of nosocomial opportunistic fungal infections in immunocompromised hosts, especially during cytotoxic chemotherapy and after bone marrow transplantation, and is currently a major direct cause of death in leukemia patients. Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and is used by viral and bacterial pathogens to gain access to human DC. We report that DC-SIGN specifically interacts with clinical isolates of A. fumigatus. DC-SIGN-dependent binding of A. fumigatus conidia can be demonstrated with stable transfectants and monocyte-derived DC and is inhibited by anti-DC-SIGN Abs. Binding and internalization of A. fumigatus conidia correlates with DC-SIGN cell surface expression levels and is abolished in the presence of A. funigatus-derived cell wall galactomannans. The clinical relevance of this interaction is emphasized by the presence of DC-SIGN in lung DC and alveolar macrophages, and further illustrated by the DC-SIGN-dependent attachment of A. fumigatus conidia to the cell membrane of IL-4-treated monocyte-derived macrophages. Our results suggest the involvement of DC-SIGN in the initial stages of pulmonary infection as well as in fungal spreading during invasive aspergillosis.

CCL2 Shapes Macrophage Polarization by GM-CSF and M-CSF: Identification of CCL2/CCR2-Dependent Gene Expression Profile

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF– and M-CSF–polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF–polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF–polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow–derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.

Dendritic Cell-Specific ICAM-3―Grabbing Nonintegrin Expression on M2-Polarized and Tumor-Associated Macrophages Is Macrophage-CSF Dependent and Enhanced by Tumor-Derived IL-6 and IL-10

Dendritic cell-specific ICAM-3–grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF–inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14+ CD163+ IL-10–producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewisx-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4–dependent) and regulatory (M-CSF–dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.

Serotonin Skews Human Macrophage Polarization through HTR2B and HTR7

Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT1–7) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization–associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT7 mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT2B and 5HT7 receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT2B was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT2B and 5HT7, whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.

Carbosilane dendrimers as gene delivery agents for the treatment of HIV infection

Despite the use of siRNA in the downregulation of HIV-1 replication which has been reported, CD4 T lymphocytes are difficult to transfect with non-viral vectors. We determined whether second generation carbosilane dendrimers (2G-NN16 and 2G-03NN24) may be efficient transfectants in CD4 T lymphocytes. Dendrimers were also tested on macrophages to determine whether they can modify macrophage phenotype and induce an inflammatory response. The nanoconjugate formed by 2G-03NN24/siRNA-Nef presents the highest inhibition of HIV-1 replication. Dendrimers presented safety properties because they did not induce proliferation on CD4 T lymphocytes and decrease the release of TNFα and IL-12p40 by macrophages. Both dendrimers also decrease the phagocytosis activity. Additionally, 2G-03NN24 dendrimer decreases the CCL2 and CCR2 expression in macrophages. Carbosilane dendrimers 2G-NN16 and 2G-03NN24 can be used as efficient non-viral vectors for gene therapy applications, mainly in the treatment of HIV infection.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells

Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)-directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK-regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

The pathogen receptor liver and lymph node sinusoidal endotelial cell C-type lectin is expressed in human Kupffer cells and regulated by PU.1†

Human LSECtin (liver and lymph node sinusoidal endothelial cell C‐type lectin, CLEC4G) is a C‐type lectin encoded within the L‐SIGN/DC‐SIGN/CD23 gene cluster. LSECtin acts as a pathogen attachment factor for Ebolavirus and the SARS coronavirus, and its expression can be induced by interleukin‐4 on monocytes and macrophages. Although reported as a liver and lymph node sinusoidal endothelial cell‐specific molecule, LSECtin could be detected in the MUTZ‐3 dendritic‐like cell line at the messenger RNA (mRNA) and protein level, and immunohistochemistry analysis on human liver revealed its presence in Kupffer cells coexpressing the myeloid marker CD68. The expression of LSECtin in myeloid cells was further corroborated through the analysis of the proximal regulatory region of the human LSECtin gene, whose activity was maximal in LSECtin+ myeloid cells, and which contains a highly conserved PU.1‐binding site. PU.1 transactivated the LSECtin regulatory region in collaboration with hematopoietic‐restricted transcription factors (Myb, RUNX3), and was found to bind constitutively to the LSECtin proximal promoter. Moreover, knockdown of PU.1 through the use of small interfering RNA led to a decrease in LSECtin mRNA levels in THP‐1 and monocyte‐derived dendritic cells, thus confirming the involvement of PU.1 in the myeloid expression of the lectin. Conclusion: LSECtin is expressed by liver myeloid cells, and its expression is dependent on the PU.1 transcription factor. (HEPATOLOGY 2009;49:287–296.)

RUNX3 negatively regulates CD36 expression in myeloid cell lines.

CD36 is a member of the scavenger receptor type B family implicated in the binding of lipoproteins, phosphatidylserine, thrombospondin-1, and the uptake of long-chain fatty acids. On mononuclear phagocytes, recognition of apoptotic cells by CD36 contributes to peripheral tolerance and prevention of autoimmunity by impairing dendritic cell (DC) maturation. Besides, CD36 acts as a coreceptor with TLR2/6 for sensing microbial diacylglycerides, and its deficiency leads to increased susceptibility to Staphylococcus aureus infections. The RUNX3 transcription factor participates in reprogramming DC transcription after pathogen recognition, and its defective expression leads to abnormally accelerated DC maturation. We present evidence that CD36 expression is negatively regulated by the RUNX3 transcription factor during myeloid cell differentiation and activation. In molecular terms, RUNX3 impairs the activity of the proximal regulatory region of the CD36 gene in myeloid cells through in vitro recognition of two functional RUNX-binding elements. Moreover, RUNX3 occupies the CD36 gene proximal regulatory region in vivo, and its overexpression in myeloid cells results in drastically diminished CD36 expression. The down-regulation of CD36 expression by RUNX3 implies that this transcription factor could impair harmful autoimmune responses by contributing to the loss of pathogen- and apoptotic cell-recognition capabilities by mature DCs.

Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages

Human CD14++CD16− and CD14+/loCD16+ monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14++ CD16− and CD16+ monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF–driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF–driven differentiation of CD16+ monocytes, or addition of IL-10–containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory–associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14++CD16ˉ monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF–treated CD16+ monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.