Elena Sierra-Filardi

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.

Activin A skews macrophage polarization by promoting a proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers

M-CSF favors the generation of folate receptor β-positive (FRβ⁺), IL-10-producing, immunosuppressive, M2-polarized macrophages [M2 (M-CSF)], whereas GM-CSF promotes a proinflammatory, M1-polarized phenotype [M1 (GM-CSF)]. In the present study, we found that activin A was preferentially released by M1 (GM-CSF) macrophages, impaired the acquisition of FRβ and other M2 (M-CSF)-specific markers, down-modulated the LPS-induced release of IL-10, and mediated the tumor cell growth-inhibitory activity of M1 (GM-CSF) macrophages, in which Smad2/3 is constitutively phosphorylated. The contribution of activin A to M1 (GM-CSF) macrophage polarization was evidenced by the capacity of a blocking anti-activin A antibody to reduce M1 (GM-CSF) polarization markers expression while enhancing FRβ and other M2 (M-CSF) markers mRNA levels. Moreover, an inhibitor of activin receptor-like kinase 4/5/7 (ALK4/5/7 or SB431542) promoted M2 (M-CSF) marker expression but limited the acquisition of M1 (GM-CSF) polarization markers, suggesting a role for Smad2/3 activation in macrophage polarization. In agreement with these results, expression of activin A and M2 (M-CSF)-specific markers was oppositely regulated by tumor ascites. Therefore, activin A contributes to the proinflammatory macrophage polarization triggered by GM-CSF and limits the acquisition of the anti-inflammatory phenotype in a Smad2-dependent manner. Our results demonstrate that activin A-initiated Smad signaling skews macrophage polarization toward the acquisition of a proinflammatory phenotype.

Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development.

The generation of natural regulatory T cells (nTregs) is crucial for the establishment of immunologic self-tolerance and the prevention of autoimmunity. Still, the origin of nTregs and the mechanisms governing their differentiation within the thymus are poorly understood, particularly in humans. It was recently shown that conventional dendritic cells (cDCs) in human thymus were capable of inducing nTreg differentiation. However, the function of plasmacytoid DCs (pDCs), the other major subset of thymic DCs, remains unknown. Here we report that pDCs resident in the human thymus, when activated with CD40 ligand (CD40L) plus interleukin-3, efficiently promoted the generation of CD4(+)CD25(+)Foxp3(+) nTregs from autologous thymocytes. The progenitors of these nTregs were selectively found within CD4(+)CD8(+) thymocytes that had accomplished positive selection, as judged by their CD69(hi)TCR(hi) phenotype. Supporting the involvement of the CD40-CD40L pathway in pDC-induced nTreg generation, we show that positively selected CD4(+)CD8(+) progenitors specifically transcribed CD40L in vivo and up-regulated CD40L expression on T-cell receptor engagement, thereby promoting the activation of pDCs. Finally, evidence is provided that nTregs primed by pDCs displayed reciprocal interleukin-10/transforming growth factor-beta cytokine expression profiles compared with nTregs primed by cDCs. This functional diversity further supports a nonredundant tolerogenic role for thymic pDCs in the human thymus.

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-alpha and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNgamma) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163(+) tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.

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.

Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6

ABSTRACT Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the 2-substituted (1,3)-β-d-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages.

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.

Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods.

We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-beta-D-glucans which increased the viscosity of the growth media. The (1,3)-beta-D-glucan consisted of two main molecular species, with masses of approximately 10(7) and 10(4) Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foods.

Structural Requirements for Multimerization of the Pathogen Receptor Dendritic Cell-specific ICAM3-grabbing Non-integrin (CD209) on the Cell Surface

The myeloid C-type lectin dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) recognizes oligosaccharide ligands on clinically relevant pathogens (HIV, Mycobacterium, and Aspergillus). Alternative splicing and genomic polymorphism generate DC-SIGN mRNA variants, which have been detected at sites of pathogen entrance and transmission. We present evidence that DC-SIGN neck variants are expressed on dendritic and myeloid cells at the RNA and protein levels. Structural analysis revealed that multimerization of DC-SIGN within a cellular context depends on the lectin domain and the number and arrangement of the repeats within the neck region, whose glycosylation negatively affects oligomer formation. Naturally occurring DC-SIGN neck variants differ in multimerization competence in the cell membrane, exhibit altered sugar binding ability, and retain pathogen-interacting capacity, implying that pathogen-induced cluster formation predominates over the basal multimerization capability. Analysis of DC-SIGN neck polymorphisms indicated that the number of allelic variants is higher than previously thought and that multimerization of the prototypic molecule is modulated in the presence of allelic variants with a different neck structure. Our results demonstrate that the presence of allelic variants or a high level of expression of neck domain splicing isoforms might influence the presence and stability of DC-SIGN multimers on the cell surface, thus providing a molecular explanation for the correlation between DC-SIGN polymorphisms and altered susceptibility to HIV-1 and other pathogens.