Cristina Purificato

Suppressive Effect of 1α,25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functional Activities and Chemotaxis

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of vitamin D3, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)2D3 prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)2D3-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)2D3. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)2D3 is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)2D3-mediated immunomodulation.

Human Immunodeficiency Virus Type 1 gp120 Induces Abnormal Maturation and Functional Alterations of Dendritic Cells: a Novel Mechanism for AIDS Pathogenesis

ABSTRACT Dendritic cells (DCs) play a crucial role in bridging innate and acquired immune responses to pathogens. In human immunodeficiency virus type 1 (HIV-1) infection, immature DCs (iDCs) are also main targets for HIV-1 at the mucosal level. In this study, we evaluated the effects of HIV-1-DC interactions on the maturation and functional activity of these cells. Exposure of human monocyte-derived iDCs to either aldrithiol-2-inactivated HIV-1 or gp120 led to an upmodulation of activation markers indicative of functional maturation. Despite their phenotype, these cells retained antigen uptake capacity and showed an impaired ability to secrete cytokines or chemokines and to induce T-cell proliferation. Although gp120 did not interfere with DC differentiation, the capacity of these cells to produce interleukin-12 (IL-12) upon maturation was markedly reduced. Likewise, iDCs stimulated by classical maturation factors in the presence of gp120 lacked allostimulatory capacity and did not produce IL-12, in spite of their phenotype typical of activated DCs. Exogenous addition of IL-12 restores the allostimulatory capacity of gp120-exposed DCs. The finding that gp120 induces abnormal maturation of DCs linked to profound suppression of their activities unravels a novel mechanism by which HIV can lead to immune dysfunction in AIDS patients.

Phosphatidylcholine-specific phospholipase C activation is required for CCR5-dependent, NF-kB–driven CCL2 secretion elicited in response to HIV-1 gp120 in human primary macrophages

CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholine-specific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs). Using a combination of pharmacologic inhibition, confocal laser-scanner microscopy, and enzymatic activity assay, we demonstrate that R5 gp120 interaction with CCR5 activates PC-PLC, as assessed by a time-dependent modification of its subcellular distribution and a concentration-dependent increase of its enzymatic activity. Furthermore, PC-PLC is required for NF-kB-mediated CCL2 production triggered by R5 gp120. Notably, PC-PLC activation through CCR5 is specifically induced by gp120, since triggering CCR5 through its natural ligand CCL4 (MIP-1beta) does not affect PC-PLC cellular distribution and enzymatic activity, as well as CCL2 secretion, thus suggesting that different signaling pathways can be activated through CCR5 interaction with HIV-1 or chemokine ligands. The identification of PC-PLC as a critical mediator of well-defined gp120-mediated effects in MDMs unravels a novel mechanism involved in bystander activation and may contribute to define potential therapeutic targets to block Env-triggered pathologic responses.

IRF‐4 expression in the human myeloid lineage: up‐regulation during dendritic cell differentiation and inhibition by 1α,25‐dihydroxyvitamin D3

Interferon (IFN) regulatory factor (IRF)‐4 is a lymphoid‐ and myeloid‐restricted transcription factor of the IRF family. We analyzed its expression during differentiation of human monocytes along the macrophage or the dendritic cell (DC) pathway and in blood myeloid and plasmacytoid DC (M‐DC and P‐DC, respectively) subsets. Monocyte differentiation into DC, driven by granulocyte macrophage‐colony stimulating factor (GM‐CSF)/interleukin‐4 or GM‐CSF/IFN‐β, resulted in a strong up‐regulation of IRF‐4 mRNA and protein, which was further increased by lipopolysaccharide. It is interesting that 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3], a potent inhibitor of DC differentiation, completely abolished IRF‐4 up‐regulation. IRF‐4 was also detected in blood P‐DC and M‐DC. However, up‐regulation upon in vitro culture and down‐regulation by 1,25(OH)2D3 was observed in M‐DC but not in P‐DC. These results point to IRF‐4 as a potential player in human myeloid DC differentiation and as a novel target for the immunomodulatory activity of 1,25(OH)2D3.

Increased Circulating Levels of Vitamin D Binding Protein in MS Patients

Vitamin D (vitD) low status is currently considered a main environmental factor in multiple sclerosis (MS) etiology and pathogenesis. VitD and its metabolites are highly hydrophobic and circulate mostly bound to the vitamin D binding protein (DBP) and with lower affinity to albumin, while less than 1% are in a free form. The aim of this study was to investigate whether the circulating levels of either of the two vitD plasma carriers and/or their relationship are altered in MS. We measured DBP and albumin plasma levels in 28 MS patients and 24 healthy controls. MS patients were found to have higher DBP levels than healthy subjects. Concomitant interferon beta therapy did not influence DBP concentration, and the difference with the control group was significant in both females and males. No significant correlation between DBP and albumin levels was observed either in healthy controls or in patients. These observations suggest the involvement of DBP in the patho-physiology of MS.

Revisiting the Specificity of Small Molecule Inhibitors: The Example of Stattic in Dendritic Cells

Multiple lines of evidence place the signal transducer and activator of transcription (STAT)3 at a central node in the development, progression, and maintenance of many human tumors, pointing to STAT3 as a promising anticancer target (Aggarwal et al., 2009; Lee et al., 2011). This transcription factor is constitutively activated by aberrant upstream tyrosine kinase activity in a broad spectrum of human cancers. Of note, the persistent activation of STAT3 intrinsic to tumor cells is transmitted to stromal inflammatory and immune cells in the tumor microenvironment, establishing a feed-forward loop that promotes tumor growth and immunological tolerance.

Toll-like receptor cross-talk in human monocytes regulates CC-chemokine production, antigen uptake and immune cell recruitment

Chemokines production in monocytes/macrophages is crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbes. During microbial onset, multiple pathogen-associated structures can be present at infection sites, and simultaneously trigger different TLRs. We report here that TLR3, TLR4 and TLR8 engagement induce CCL1, CCL2 and CCL4 production in freshly isolated monocytes. While differentiating cells maintain the capacity to secrete CCL2 and CCL4, CCL1 is no longer induced at later differentiation stages. Although different pairs of TLR agonists have been described to synergistically induce cytokine production in different cell types, agonist combinations cooperate in reducing CCL1 and CCL2, but not CCL4 secretion in freshly isolated monocytes, and fail to rescue CCL1 production at later differentiation stages. The effects of single, but not combined, TLR engagement on chemokine expression mostly occur at transcriptional level, and are IL-10 independent. Conversely, inhibition of CCL1 secretion upon combined TLR engagement is partially rescued by blocking IL-23. A different chemotactic activity of monocyte-conditioned medium on blood mononuclear cells as well as antigen uptake capacity of TLR agonist activated monocytes parallel the regulated production of chemokines. Overall, these findings indicate that simultaneous engagement of TLRs may lead to different patterns of chemokine expression depending on cellular differentiation state, chemokine, and TLR agonist combination. These different responses may be relevant for the distinct but complementary functions of monocytes and macrophages in the immune response, and may have important implications for the therapeutic manipulation of the innate immune system.

Nuclear Phosphoinositide-Specific Phospholipase C β1 Controls Cytoplasmic CCL2 mRNA Levels in HIV-1 gp120-Stimulated Primary Human Macrophages

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Background: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. Results: CCL2 neutralization potently reduced the number of p24 Gag + cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. Conclusions: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

STAT3-silenced human dendritic cells have an enhanced ability to prime IFNγ production by both αβ and γδ T lymphocytes.

Dendritic cells (DC) are an attractive target for therapeutic manipulation of the immune system to enhance insufficient immune responses, such those occurring in cancer, or to dampen dangerous responses in allergic and autoimmune diseases. Main goal of this study was to manipulate human monocyte-derived DC (MDDC) function by silencing STAT3, since this transcription factor plays a key role as a negative regulator of immune surveillance, and is strongly involved in inflammation. STAT3 silencing did not affect the immunophenotype of both immature and toll-like receptor (TLR) ligand-matured DC. However, an altered cytokine secretion profile, characterized by lower IL10 and higher IL12 and TNFα levels, was observed in silenced DC with respect to control cells upon TLR triggering. Accordingly, STAT3 silenced MDDC promoted a higher IFNγ production by CD4(+) naïve T cells. Furthermore, STAT3 silencing in MDDC favored the activation of γδ T lymphocytes, an immune cell population with important antitumor effector activities. This effect was at least in part mediated by the increased IL12 production by silenced cells. STAT3 silencing also increased the levels of CCL4, a CCR5-binding chemokine known to be involved in T helper 1 (Th1) cell recruitment. Altogether these results strengthen the role of STAT3 as a critical check point of the suppression of Th1 responses, unraveling its potential to dampen DC capability to both induce and recruit different IFNγ producing T lymphocyte subsets.