Imatinib-loaded gold nanoparticles inhibit proliferation of fibroblasts and macrophages from systemic sclerosis patients and ameliorate experimental bleomycin-induced lung fibrosis.

Interstitial lung involvement in Systemic Sclerosis (SSc-ILD) is a complication with high morbidity and mortality. Specifically, engineered gold nanoparticles (GNPs) are proposed as targeted delivery system increasing efficacy of drugs with antifibrotic effect, such as tyrosine kinases. We aimed to test in vitro and in vivo the activity of targeted Imatinib (Im)-loaded GNP on SSc-ILD patients derived cells and in experimental model of lung fibrosis. GNPs functionalized with anti-CD44 and loaded with Im (GNP-HCIm) were synthesized. Lung fibroblasts (LFs) and alveolar macrophages from bronchoalveolar lavage fluids of SSc-ILD patients were cultured in presence of nanoparticles. GNP-HCIm significantly inhibited proliferation and viability inducing apoptosis of LFs and effectively reduced IL-8 release, viability and M2 polarization in alveolar macrophages. Anti-fibrotic effect of tracheal instilled GNP-HCIm was evaluated on bleomycin lung fibrosis mouse model comparing effect with common route of Im administration. GNP-HCIm were able to reduce significantly lung fibrotic changes and collagen deposition. Finally, electron microscopy revealed the presence of GNPs inside alveolar macrophages. These data support the use of GNPs locally administered in the development of new therapeutic approaches to SSc-ILD.

Basophil Recruitment into Tumor-Draining Lymph Nodes Correlates with Th2 Inflammation and Reduced Survival in Pancreatic Cancer Patients.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell-derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792-803. ©2016 AACR.

Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer.

Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a predominant Th2 (GATA-3+) over Th1 (T-bet+) lymphoid infiltrate. We found that the ratio of GATA-3+/T-bet+ tumor-infiltrating lymphoid cells is an independent predictive marker of patient survival. Patients surgically treated for stage IB/III disease with a ratio inferior to the median value had a statistically significant prolonged overall survival, implying an active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP), which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor necrosis factor α and interleukin 1ß. TSLP-containing supernatants from activated CAFs induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro. In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-expressing DCs were present in the tumor stroma and in tumor-draining but not in nondraining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type inflammation which associates with reduced patient survival. Thus, blocking TSLP production by CAFs might help to improve prognosis in pancreatic cancer.

Ferroportin gene silencing induces iron retention and enhances ferritin synthesis in human macrophages.

Missense mutations in the ferroportin gene (SLC11A3) result in haemochromatosis type 4 [HFE4, Online Mendelian Inheritance in Man (OMIM) reference 606069] or ferroportin disease, an autosomal dominant disorder characterized by predominantly reticuloendothelial iron accumulation. To verify whether HFE4 is caused by defective iron recycling because of loss of functionality of ferroportin, we down-regulated SLC11A gene expression in human macrophages by using small interfering RNAs (siRNAs). Transfection experiments with ferroportin siRNAs resulted in a marked reduction (about two-thirds on average) in ferroportin mRNA levels as detected by quantitative real time polymerase chain reaction. When macrophages were grown in medium supplemented with iron, cells transfected with siRNAs displayed three- to eightfold increases in staining intensities following Perls reaction. These macrophages also showed significant increases in H-ferritin content. The observation that ferroportin mRNA down-regulation to levels compatible with haplo-insufficiency causes increased iron retention and H-ferritin synthesis in cultured macrophages has important implications. First, this indicates that ferroportin levels must be finely regulated in order to maintain cellular iron homeostasis, and that both copies of SLC11A3 must function efficiently to prevent iron accumulation. Second, this observation supports the hypothesis that reticuloendothelial iron overload in patients with ferroportin disease is caused by loss-of-function mutations in the SLC11A3 gene that mainly impair macrophage iron recycling.

A method of analyzing procoagulant activity in monocytes at single cell level.

BACKGROUND AND OBJECTIVES: Procoagulant activity (PCA) of monocytes is known to play a pivotal role in a variety of physiologic and pathophysiologic processes, such as disseminated intravascular coagulation, atherosclerosis, arterial and venous thromboembolism, cancer-related hypercoagulability and immunopathologies. Until now, PCA has been studied by clotting assays of a whole cell population or at single cell level by analyzing tissue factor antigen, the protein that initiates PCA but does not always correlate with it. Here, we describe a new simple flow cytometric method that allows the PCA of monocytes to be studied at a single cell level by quantifying the fibrin formed around the cells in suspension. DESIGN AND METHODS: Purified fibrinogen was tagged with FITC and added to a recalcified developer plasma containing suitable amounts of heparin in order to inhibit the expansion of clotting, thus limiting the formation of fibrin to the surface of cells with PCA. With appropiate amounts of heparin, in 10 min, large sea urchin-like cells with fibrin needles around some monocytes were formed and, after fixation, cytofluorimetrically analyzed. RESULTS: Blood mononuclear cells isolated and immediately analyzed showed less than 0.1% sea urchin cells. Adherence alone, lipopolysaccharides or ionomycin stimulated expression of PCA in a dose- and time-dependent relationship: after 30 min, 1-3% of the MNC showed PCA, and after 20 h this reached 5-10%. Density separation of monocytes showed that different stimulators act on different maturation stages. Subjects with diabetes express more monocytes with PCA than normal subjects after 30 min stimulation. INTERPRETATION AND CONCLUSIONS: This method allows PCA analysis of monocytes at single cell level and requires only a low number of cells. The signal produced by the fluorescent fibrin is strong and easily analyzed by flow cytometry. The method is suitable for analyzing blood from patients with different pathologies and many conditions under different stimuli.

Modulation of chemokine receptor expression and chemotactic responsiveness during differentiation of human naive T cells into Th1 or Th2 cells.

Chemokines and their receptors direct movements and encounters of lymphocytes and professional APC into specific microenvironments of lymphoid tissues. Chemokine receptors such as CCR7, CXCR5 and CCR4 that are differentially expressed and modulated in distinct subsets of T cells contribute to establish functionally and spatially segregated microenvironments within secondary lymphoid tissues where T cell activation and differentiation occur. Here, we have explored the modulation of CCR7, CCR4, CCR8 and CXCR5 expression and chemotactic responsiveness to their ligands during commitment of human naive T cells along the Th1 or Th2 differentiation pathway in vitro. Our results document that activation of human naive T cells and differentiation in Th1 or Th2 cells result in progressive down-modulation of CCR7 expression and CCL19 responsiveness. By contrast, expression of CCR4 and responsiveness to CCL22 is rapidly induced at the early stages of both Th1/Th2 cell development. However, while CCR4 expression is further up-regulated upon differentiation into Th2 cells, it is lost on fully differentiated Th1 cells. CCR8 is detected at later time points than CCR4 and exclusively on differentiated Th2 cells as revealed by analysis of mRNA expression and responsiveness to CCL1. Expression of CXCR5 is transiently induced at the early stages of Th cell differentiation, but with distinct kinetics in developing Th1 and Th2 cells. Analysis of human tonsillar CD4(+) T cells reveals a consistent pattern of chemotactic responsiveness and chemokine receptor expression in distinct transitional stages of human T cell activation and differentiation in vivo.