Annalisa Del Prete

Cross-Linking of the Mannose Receptor on Monocyte-Derived Dendritic Cells Activates an Anti-Inflammatory Immunosuppressive Program

Immature monocyte-derived dendritic cells (DC) strongly express the endocytic mannose receptor (MR). Addition of a specific anti-MR mAb (clone PAM-1) for 24 h to cultures of immature DC induced phenotypical and functional maturation of the cells, assessed as up-regulation of costimulatory molecules and CD83, and chemotactic response to CCL19. A different isotype-matched anti-MR mAb (clone 19.2) had no significant effect. Engagement of MR with mAb PAM-1 induced the production of the anti-inflammatory cytokines IL-10, IL-1R antagonist, and of the nonsignaling IL-1R type II. In contrast IL-1β, TNF, and IL-12 were not produced. PAM-1-treated DC were unable to polarize Th1 effector cells and did not secrete the chemokines CXCL10 and CCL19; in turn, they produced large amounts of CCL22 and CCL17, thus favoring the amplification of Th2 circuits. T cells cocultured with PAM-1-matured DC initially proliferated but later became anergic and behaved as suppressor/regulatory cells. Natural ligands binding to MR had differential effects. MUC III (a partially purified mucin), biglycan (a purified complex proteoglycan), and mannosylated lipoarabinomannan from Mycobacterium tuberculosis affected cytokine production with high IL-10, IL-1R antagonist, IL-1R type II, and inhibition of IL-12. In contrast, mannan, dextran, and thyroglobulin had no significant effect. In conclusion, the appropriate engagement of the MR by mAb PAM-1 and selected natural ligands elicit a secretory program in mono-derived DC characterized by a distinct profile of cytokines/chemokines with the ability to dampen inflammation and to inhibit the generation of Th1-polarized immune responses.

Defective dendritic cell migration and activation of adaptive immunity in PI3Kγ‐deficient mice

Gene‐targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3‐kinase (PI3Kγ) in dendritic cell (DC) migration and induction of specific T‐cell‐mediated immune responses. DC obtained from PI3Kγ−/− mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kγ−/− mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8α− DC. Furthermore, PI3Kγ−/− mice showed a defective capacity to mount contact hypersensitivity and delayed‐type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen‐loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kγ plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kγ may be considered a new target to control exaggerated immune reactions.

Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A–deficient mice

Junctional adhesion molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. In the present work, we found that DCs also express JAM-A. To evaluate the biological relevance of this observation, Jam-A(-/-) mice were generated and the functional behavior of DCs in vitro and in vivo was studied. In vitro, Jam-A(-/-) DCs showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells. In vivo, Jam-A(-/-) mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium-restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity (CHS). Adoptive transfer experiments showed that JAM-A-deficient DCs elicited increased CHS in Jam-A(+/+) mice, further supporting the concept of a DC-specific effect. Thus, we identified here a novel, non-redundant role of JAM-A in controlling DC motility, trafficking to lymph nodes, and activation of specific immunity.

Trafficking properties of plasmacytoid dendritic cells in health and disease

Plasmacytoid dendritic cells (PDCs) represent a subset of circulating leukocytes characterized by the ability to release high levels of type I interferon (IFN). Under homeostatic conditions PDCs are confined to primary and secondary lymphoid organs. This is consistent with the restricted profile of functional chemotactic receptors expressed by circulating PDCs (i.e. CXCR4 and ChemR23). Accumulation of PDCs in non-lymphoid tissue is, however, observed in certain autoimmune diseases, allergic reactions and tumors. Indeed, PDCs are now considered to be involved in the pathogenesis of diseases characterized by a type I IFN-signature and are considered as a promising target for new intervention strategies. Here, current knowledge of the molecular mechanisms involved in the recruitment of PDCs under homeostatic and pathological conditions are summarized.

Targeted disruption of leukotriene B4 receptors BLT1 and BLT2 : A critical role for BLT1 in collagen-induced arthritis in mice

Leukotriene B4 mediates diverse inflammatory diseases through the G protein-coupled receptors BLT1 and BLT2. In this study, we developed mice deficient in BLT1 and BLT2 by simultaneous targeted disruption of these genes. The BLT1/BLT2 double-deficient mice developed normally and peritoneal exudate cells showed no detectable responses to leukotriene B4 confirming the deletion of the BLT1/BLT2 locus. In a model of collagen-induced arthritis on the C57BL/6 background, the BLT1/BLT2−/− as well as the previously described BLT1−/− animals showed complete protection from disease development. The disease severity correlated well with histopathology, including loss of joint architecture, inflammatory cell infiltration, fibrosis, pannus formation, and bone erosion in joints of BLT1/BLT2+/+ animals and a total absence of disease pathology in leukotriene receptor-deficient mice. Despite these differences, all immunized BLT1−/− and BLT1/BLT2−/− animals had similar serum levels of anti-collagen Abs relative to BLT1/BLT2+/+ animals. Thus, BLT1 may be a useful target for therapies directed at treating inflammation associated with arthritis.

Molecular pathways in cancer-related inflammation.

Accumulating evidence shows that chronic inflammation is associated to increased risk of cancer. An inflammatory component is present also in the microenvironment of tumours epidemiologically unrelated to inflammation. Extensive investigations over the past decade have uncovered many of the important mechanistic pathways underlying cancer-related inflammation. Pathways linking inflammation and cancer have been identified: an intrinsic one (driven by genetic events that cause neoplasia) and an extrinsic one (driven by inflammatory conditions which predispose to cancer). Smouldering inflammation is a component of the tumour microenvironment and is a recognized hallmark of cancer. Key orchestrators at the intersection of the intrinsic and extrinsic pathways include transcription factors (e.g. Nuclear Factor kappa-B, NFKB) that modulate the inflammatory response through soluble mediators (cytokines, chemokines) and cellular components (e.g. tumor-associated macrophages), promoting tumorigenesis. NFKB aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immunity, and alters responses to hormones and chemotherapeutic agents. Emerging evidence also suggests that persistent inflammation promotes genetic instability. Thus, cancer-related inflammation represents a target for innovative diagnostic and therapeutic strategies.

Role of mitochondria and reactive oxygen species in dendritic cell differentiation and functions

Dendritic cells (DC) are potent antigen-presenting cells capable of inducing T and B responses and immune tolerance. We have characterized some aspects of energy metabolism accompanying the differentiation process of human monocytes into DC. Compared to precursor monocytes, DC exhibited a much larger number of mitochondria and consistently (i) a higher endogenous respiratory activity and (ii) a more than sixfold increase in ATP content and an even larger increase in the activity of the mitochondrial marker enzyme citrate synthase. The presence in the culture medium of rotenone, an inhibitor of the respiratory chain Complex I, prevented the increase in mitochondrial number and ATP level, without affecting cell viability. Rotenone inhibited DC differentiation, as revealed by the observation that the expression of CD1a, which is a specific surface marker of DC differentiation, was strongly reduced. Cells cultured in the presence of rotenone displayed a lower content of growth factor-induced, mitochondrially generated, hydrogen peroxide. A similar drop in ROS was observed upon addition of catalase, which caused functional effects similar to those produced by rotenone treatment. These results suggest that ROS play a crucial role in DC differentiation and that mitochondria are an important source of ROS in this process.

Blocking TH17-polarizing cytokines by histone deacetylase inhibitors in vitro and in vivo

Histone deacetylase (HDAC) inhibitors are small molecules inducing cell‐cycle arrest, differentiation, and apoptosis, currently undergoing clinical trials as anticancer drugs. In addition, emerging evidence suggests HDAC inhibitors may have anti‐inflammatory and immunomodulatory properties as well, although the molecular mechanisms remain poorly defined. Given the central role of dendritic cells (DC) in the induction and maintenance of the inflammatory and immune response, we investigated the effects of HDAC inhibitors on the maturation and activation of human monocyte‐derived DC in the presence of LPS and IFN‐γ. Our results show that the production of TH1‐ and TH17‐inducing cytokines, namely IL‐12 and IL‐23, was inhibited by trichostatin A (72% and 52%, respectively) and suberoylanilide hydroxamic acid (86% and 83%). Strikingly, HDAC inhibitors were effective if added simultaneously as well as after the proinflammatory challenge, and their effect was not associated to a reduction of expression or function of LPS/IFN‐γ receptors. These findings were confirmed in two different murine models. In addition, HDAC inhibitors selectively blocked the production of TH1‐attracting chemokines CXCL9, CXCL10, and CXCL11. The reduction of TH1‐ and TH17‐inducing cytokines as well as TH1‐attracting chemokines may represent relevant mechanisms through which HDAC inhibitors at nonproapoptotic doses exert their immunomodulatory properties.

Signaling events involved in cytokine and chemokine production induced by secretory phospholipase A2 in human lung macrophages

Secretory phospholipases A2 (sPLA2) are enzymes released during inflammatory reactions. These molecules activate immune cells by mechanisms either related or unrelated to their enzymatic activity. We examined the signaling events activated by group IA (GIA) and group IB (GIB) sPLA2 in human lung macrophages leading to cytokine/chemokine production. sPLA2 induced the production of cytokines (TNF‐α, IL‐6 and IL‐10) and chemokines (CCL2, CCL3, CCL4 and CXCL8), whereas no effect was observed on IL‐12, CCL1, CCL5 and CCL22. sPLA2 induced the phosphorylation of the MAPK p38 and ERK1/2, and inhibition of these kinases by SB203580 and PD98059, respectively, reduced TNF‐α and CXCL8 release. Suppression of sPLA2 enzymatic activity by a site‐directed inhibitor influenced neither cytokine/chemokine production nor activation of MAPK, whereas alteration of sPLA2 secondary structure suppressed both responses. GIA activated the phosphatidylinositol 3‐kinase (PI3 K)/Akt system and a specific inhibitor of PI3 K (LY294002) reduced sPLA2‐induced release of TNF‐α and CXCL8. GIA promoted phosphorylation and degradation of IκB and inhibition of NF‐κB by MG‐132 and 6‐amino‐4‐phenoxyphenylethylamino‐quinazoline suppressed the production of TNF‐α and CXCL8. These results indicate that sPLA2 induce the production of cytokines and chemokines in human macrophages by a non‐enzymatic mechanism involving the PI3 K/Akt system, the MAPK p38 and ERK1/2 and NF‐κB.

Nonredundant role of CCRL2 in lung dendritic cell trafficking

Chemokine CC motif receptor-like 2 (CCRL2) is a heptahelic transmembrane receptor that shows the highest degree of homology with CCR1, an inflammatory chemokine receptor. CCRL2 mRNA was rapidly (30 minutes) and transiently (2-4 hours) regulated during dendritic cell (DC) maturation. Protein expression paralleled RNA regulation. In vivo, CCRL2 was expressed by activated DC and macrophages, but not by eosinophils and T cells. CCRL2(-/-) mice showed normal recruitment of circulating DC into the lung, but a defective trafficking of antigen-loaded lung DC to mediastinal lymph nodes. This defect was associated to a reduction in lymph node cellularity and reduced priming of T helper cell 2 response. CCRL2(-/-) mice were protected in a model of ovalbumin-induced airway inflammation, with reduced leukocyte recruitment in the BAL (eosinophils and mononuclear cells) and reduced production of the T helper cell 2 cytokines, interleukin-4 and -5, and chemokines CCL11 and CCL17. The central role of CCRL2 deficiency in DC was supported by the fact that adoptive transfer of CCRL2(-/-) antigen-loaded DC in wild-type animals recapitulated the phenotype observed in knockout mice. These data show a nonredundant role of CCRL2 in lung DC trafficking and propose a role for this receptor in the control of excessive airway inflammatory responses.