MicroRNAs as Molecular Switches in Macrophage Activation

The efficacy of macrophage- mediated inflammatory response relies on the coordinated expression of key factors, which expression is finely regulated at both transcriptional and post-transcriptional level. Several studies have provided compelling evidence that microRNAs play pivotal roles in modulating macrophage activation, polarization, tissue infiltration, and resolution of inflammation. In this review, we highlight the essential molecular mechanisms underlying the different phases of inflammation that are targeted by microRNAs to inhibit or accelerate restoration to tissue integrity and homeostasis. We further review the impact of microRNA-dependent regulation of tumor-associated macrophages and the relative implication for tumor biology

Rapid induction of arachidonic acid release by monocyte chemotactic protein-1 and related chemokines. Role of Ca2+ influx, synergism with platelet-activating factor and significance for chemotaxis.

Monocyte Chemotactic Protein-1 (MCP-1), a member of the Cys-Cys branch of the chemokine superfamily, induced a mepacrine- and manoalide-sensitive increase in the release of [3H]arachidonic acid from prelabeled human monocytes and monocytic THP-1 leukemic cells. The effect was rapid (<30 s), reached maximum at optimal chemotactic concentrations, and was completely blocked by pretreatment of monocytes with Bordetella pertussis toxin. A specific antiserum and heat inactivation blocked the induction of arachidonic release by MCP-1. No [3H]arachidonic acid release was observed in the absence of Ca2+ influx (5 mM EGTA or 5 mM Ni2+) or in monocytes loaded with a Ca(2+)-buffering agent. However, using ionophore-permeabilized monocytes and controlled intracellular Ca2+ concentration it was possible to dissociate MCP-1-induced Ca2+ influx from [3H]arachidonic acid release. Thus, the MCP-1-induced increase in [Ca2+]i is necessary but not sufficient for arachidonic acid accumulation. Phospholipase A2 inhibitors (mepacrine, p-bromophenacyl bromide, and manoalide) blocked monocyte polarization and chemotaxis induced by MCP-1. The related Cys-Cys chemokines RANTES and LD78/MIP1 alpha also induced a rapid release of [3H]arachidonic acid, and their chemotactic activity was blocked by phospholipase A2 inhibitors. Brief (5 min) pretreatment of monocytes with platelet-activating factor amplified MCP-1-induced arachidonic acid release and, at MCP-1 suboptimal concentrations, synergized in inducing monocyte migration. Since MCP-1 and platelet-activating factor are induced concomitantly by inflammatory cytokines in monocytes and endothelial cells, we speculate that the observed synergism may have in vivo relevance. The results presented here show that the Cys-Cys chemokines MCP-1, LD78/MIP1 alpha, and RANTES cause rapid release of arachidonic acid in monocytes and that this may be important in inducing monocyte chemotaxis

HIV-1 Coreceptor Activity of CCR5 and Its Inhibition by Chemokines: Independence from G Protein Signaling and Importance of Coreceptor Downmodulation

AbstractHIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of Gi-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection

New Insights on the Emerging Genomic Landscape of CXCR4 in Cancer: A Lesson from WHIM

Deciphering the molecular alterations leading to disease initiation and progression is currently crucial to identify the most relevant targets for precision therapy in cancer patients. Cancers express a complex chemokine network influencing leucocyte infiltration and angiogenesis. Moreover, malignant cells also express a selective repertoire of chemokine receptors that sustain their growth and spread. At present, different cancer types have been shown to overexpress C-X-C chemokine receptor type 4 (CXCR4) and to respond to its ligand C-X-C motif chemokine 12 (CXCL12). The CXCL12/CXCR4 axis influences cancer biology, promoting survival, proliferation, and angiogenesis, and plays a pivotal role in directing migration of cancer cells to sites of metastases, making it a prognostic marker and a therapeutic target. More recently, mutations in the C-terminus of CXCR4 have been identified in the genomic landscape of patients affected by Waldenstrom's macroglobulinemia, a rare B cell neoplasm. These mutations closely resemble those occurring in Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis (WHIM) syndrome, an immunodeficiency associated with CXCR4 aberrant expression and activity and with chemotherapy resistance in clinical trials. In this review, we summarize the current knowledge on the relevance of CXCR4 mutations in cancer biology, focusing on its importance as predictors of clinical presentation and response to therapy

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Serum micrornas as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Role of the chemokine decoy receptor D6 in balancing inflammation, immune activation, and antimicrobial resistance in Mycobacterium tuberculosis infection

D6 is a decoy and scavenger receptor for inflammatory CC chemokines. D6-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis. The death of D6−/− mice was associated with a dramatic local and systemic inflammatory response with levels of M. tuberculosis colony-forming units similar to control D6-proficient mice. D6-deficient mice showed an increased numbers of mononuclear cells (macrophages, dendritic cells, and CD4 and CD8 T lymphocytes) infiltrating inflamed tissues and lymph nodes, as well as abnormal increased concentrations of CC chemokines (CCL2, CCL3, CCL4, and CCL5) and proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, and interferon γ) in bronchoalveolar lavage and serum. High levels of inflammatory cytokines in D6−/− infected mice were associated with liver and kidney damage, resulting in both liver and renal failure. Blocking inflammatory CC chemokines with a cocktail of antibodies reversed the inflammatory phenotype of D6−/− mice but led to less controlled growth of M. tuberculosis. Thus, the D6 decoy receptor plays a key role in setting the balance between antimicrobial resistance, immune activation, and inflammation in M. tuberculosis infection