GITR/GITRL interaction is pivotal in regulating the activity of Treg and of Th9 effector cells.1 Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by a strong expression of IL-9 and Th9 polarisation in the inflamed gut, synovial tissues and peripheral blood.2 Factors involved in the regulation of Th9 responses in PsA have been not yet investigated.
Objectives
Aim of the study: to investigate the role of GITR/GITRL in PsA.
Methods
GITR, GITRL expression were assessed by rt-PCR and immunohistochemistry in synovial biopsies, gut specimens and bone marrow of PsA patients and healthy controls (HC). The co-localization of CD4 and GITR was analysed by immunofluorescence. The expression of GITR was also assessed by flow cytometry analysis in isolated PBMC of patients and controls (Tregs, CD4 and CD8). Peripheral and intestinal IL-9 producing cell (Th9) percentages and cytotoxic activity of T lymphocytes were also studied by flow cytometry analysis ex vivo and after in vitro stimulation with GITRL. A mouse model of induced arthritis (collagen induced arthritis, CIA) was used to study GITR/GITRL axis and the effect of anti-GITRL blocking agent.
Results
Increased GITR and GITRL expression was observed in the inflamed gut and synovial samples of PsA patients. Analysis of GITR expression among PBMC and LPMC from PsA patients demonstrated its down-regulation among Tregs and upregulation on effector CD4 +and CD8+T cells. In in vitro studies, GITR co-stimulation potently induced Th9 activation and IL-9 production. In particular, GITR ligation subverted the induction of Foxp3(+) Tregs, directing the activated CD4(+) T cells to a Th9 phenotype and enhancing the function of DCs and cytotoxic T lymphocytes. Moreover, in a murine model of CIA massive expression of IL-9 and GITR was observed in the synovial tissues and anti-GITR therapy significantly ameliorated arthritis score.
Conclusions
We demonstrated that GITR/GITRL axis modulates IL-9/Th9 responses in PsA, representing GITR activation a relevant upstream pathway involved in Th9 polarisation. A novel mechanism by which GITR agonist exert an inflammatory response was also demonstrated in PsA indicating GITR blocking agents as possible therapy in PsA.
References
[1] Xiao X, Shi X, Fan Y, Zhang X, Wu M, Lan P, Minze L, Fu YX, Ghobrial RM, Liu W, Li XC. GITR subverts Foxp3(+) Tregs to boost Th9 immunity through regulation of histone acetylation. Nat Commun. 2015Sep 14;6:8266. [2] Ciccia F, Guggino G, Ferrante A, Raimondo S, Bignone R, Rodolico V, Peralta S, Van Tok M, Cannizzaro A, Schinocca C, Ruscitti P, Cipriani P, Giacomelli R, Alessandro R, Dieli F, Rizzo A, Baeten D, Triolo G. Interleukin-9 Overexpression and Th9 Polarization Characterize the Inflamed Gut, the Synovial Tissue, and the Peripheral Blood of Patients With Psoriatic Arthritis. Arthritis Rheumatol2016Aug;68(8):1922–31. [3] Xiao X, et al. GITR subverts Foxp3(+) Tregs to boost Th9 immunity through regulation of histone acetylation. Nat Commun. 2015Sep. [4] Ciccia F, Guggino G, et al. Interleukin-9 Overexpression and Th9 Polarization Characterize the Inflamed Gut, the Synovial Tissue, and the Peripheral Blood of Patients With Psoriatic Arthritis. Arthritis Rheumatol 2016Aug.
Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non-tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome-mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real-time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients' exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre-treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand-receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.
Ultrasound, a widely used technique in medical applications for its non-ionizing properties, low cost, and ease of handling, proves its practicality in hyperthermia. High-intensity focused ultrasound (HIFU) is a prime example, as it has been extensively employed to concentrate all its energy at a specific point with millimetric precision. When used in conjunction with other treatment techniques, HIFU can alleviate the pain caused by cancer, thus improving the quality of life for patients with bone metastases. However, monitoring the temperature rising at the target remains a challenge. Here, we propose a new method supported by ultrasonic longitudinal critically refracted (LCR) waves to estimate the temperature at the soft tissue and bone boundary. We tested our model using polymethylmethacrylate (PMMA) to mimic cortical bone tissue. We can monitor the temperature in real-time through ultrasonic waves, providing a practical solution to a longstanding challenge in hyperthermia treatment. The results showed that temperature strongly influences the speed of sound and the time of flight of the propagating wave. The work demonstrated the practical and straightforward use of the ultrasonic method to obtain material constants, which are essential in various fields.
Abstract A fundamental task in cancer research aims at the identification of new pharmacological therapies that can affect tumor growth. Differentiation therapy might exploit this function not only for hematological diseases, such as acute promyelocytic leukemia (APML) but also for epithelial tumors, including lung cancer. Here we show that Retinoic Acid (RA) arrests in vitro and in vivo the growth of Tyrosine Kinase Inhibitors (TKI) resistant Non Small Cell Lung Cancer (NSCLC). In particular, we found that RA induces G0/G1 cell cycle arrest in TKI resistant NSCLC cells and activates terminal differentiation programs by modulating the expression of GATA6, a key transcription factor involved in the physiological differentiation of the distal lung. In addition, our results demonstrate that RA inhibits EGFR and Wnt signaling activation, two pathways involved in NSCLC progression. Furthermore, we uncovered a novel mechanism in NSCLC that shows how RA exerts its function; we found that RA-mediated GATA6 activation is necessary for EGFR and Wnt inhibition, thus leading to 1) increased differentiation and 2) loss of proliferation. All together, these findings prove that differentiation therapy might be feasible in TKI resistant NSCLCs, and shed light on new targets to define new pharmacological therapies.
Abstract Background : The liver is the main metastatic site for patients with colorectal cancer (CRC) and represents the most frequent cause of death for patients affected by this cancer. In recent years, a number of studies have highlighted the crucial role played by small extracellular vesicles (sEVs) released by cancer cells in initiating pre-metastatic niche formation in the liver, specifically affecting the activities of non-parenchymal cells as Kupffer cells and hepatic stellate cells, while the role of the hepatocytes still remains unknown. Even if this cell component is the most conspicuous in the liver and it is responsible for several physiological activities of this complex organ, its role in liver metastasis is only partly described and related to tumour cell colonization, while no data is available about its enrolment during the pre-metastatic niche formation. Methods: sEVs were isolated from SW480 and SW620 CRC cells through differential centrifugation, quantified using the Nanosight technology and characterized by transmission electron microscope and western blot. The presence of TGFβ1 on the surface of CRC_sEVs was assessed by western blot, super-resolution fluorescence microscope, and ELISA. Functional and epithelial to mesenchymal transition (EMT) markers were analyzed through RT PCR, ELISA assay, western blot, and confocal analysis in human healthy hepatocytes (THLE-2 cells) treated with sEVs derived from CRC cells (CRC_sEVs) at different time points. Results: Our study shows for the first time that CRC_sEVs carrying the transforming growth factor‑β1 (TGFβ1) impair the morphological and functional properties of human healthy hepatocytes by triggering their TGFβ1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. Conclusions: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV conditioned hepatocytes could have an active and until now neglected role during the liver metastasis formation.
ABSTRACT Introduction Multiple myeloma (MM) is a malignant disorder of post-germinal centre B cells, characterised by the clonal proliferation of malignant plasma cells within the bone marrow (BM). The hypoxic condition that develops in the BM niche during progression of MM, has been shown to play a major role in i) the dissemination of MM, ii) the proliferation of MM cells and iii) the induction of drug resistance finally determining a poor prognosis for MM patients. The molecular mechanisms driving hypoxic responses is the activation and nuclear translocation of the Hypoxia-inducible factor 1-alpha, (HIF1α) that, in turns, induce the expression of genes controlling angiogenesis, hypermetabolism, stemnes maintenance, resistance to chemotherapy, and tumour metastasis. The lncRNA H19, an imprinted non-coding RNA which expression was found up-regulated in many tumours including MM, is among the targets of HIF1α. We previously attributed to the lncH19 a role into control of hypoxic response; in particular we demonstrated, in both glioblastoma and colon cancer, that the lncH19, is necessary and required to sustain HIF1α activity. Here we propose to investigate the role of lncH19 in hypoxia mediated MM progression. Material and methods Transcriptional analysis (RT-PCR) of MM cell lines (RPMI and MM1S) exposed to normoxia or hypoxia (1% O2) was done in order to evaluate lncH19 levels under hypoxic stimulation. To investigate the role of lncH19 in hypoxia mediated MM progression, transcriptional, protein and functional assays have been performed on MM cell lines, silenced or not for lncH19, under normoxia or 24 hour hypoxic stimulation in low oxygen chamber Results and discussions Our data indicate that MM cell lines respond to hypoxic stimulation by HIF1α nuclear translocation and activation of hypoxic responsive genes including the lncH19. Our data revealed that lncH19 silencing inhibits HIF1a nuclear translocation with a subsequent reduction in the expression of hypoxia induced genes, associated to MM progression, such as snail and VEGF. Moreover, adhesion assay of MM cells on Mesenchymal Stromal Cells revealed that lncH19 silencing abrogates the increased adhesion induced by hypoxic condition. Conclusion LncH19 is required for the induction of hypoxic responses in MM cells thus representing a new therapeutic target for MM. Further studies are required to better define the molecular mechanism through which H19 may control HIF1α activity.
Abstract CML is an uncontrolled proliferation of bone marrow myeloid cells driven by the constitutively active fusion product tyrosine kinase BCR/ABL. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is newly recognized as a factor in CML progression. Exosomes, released by a broad spectrum of cells, are microvesicles that play an important role in cell-to-cell communication both in physiological and pathological conditions. The role of exosomes released by CML cells in angiogenesis is emerging; however, little is known about the mechanisms involved in this process. We first isolated and characterized exosomes released by K562 CML cells and we demonstrated their ability to stimulate human vascular endothelial cells (HUVECs) tube differentiation on Matrigel. K562 exosomes induced an increase of the cumulative tube length in a dose-dependent manner, with a maximum effect at 10ug/ml (p=0.003). Next, we evaluated the effect on exosome behavior of imatinib and dasatinib, two tyrosine kinase inhibitors in use in CML treatment. K562 CML cell treatment with either imatinib or dasatinib reduced exosome release by 58% and 56%, respectively (p<0.01). Equivalent exosome concentrations isolated from imatinib or dasatinib treated K562 cells maintained their ability to stimulate HUVEC tube formation, compared to those isolated from untreated cells. Dasatinib treatment of HUVECs strongly reduced exosome-induced vascular differentiation (p=0.0002). On the contrary, little effect was observed following treatment with imatinib. K562 exosomes stimulated vascularization when added to Matrigel plug in vivo; angiogenesis was markedly inhibited by oral administration of dasatinib (p<0.01), but not imatinib. Consistent with the differential effects of dasatinib and imatinib, exosome-induced HUVEC Src and FAK phosphorylation was only inhibited by dasatinib. Confocal images showed that both FAK and Src phosphorylation were increased at points of membrane-matrix contact. Immunoblot analysis confirmed that K562 exosomes induced a dasatinib-sensitive phosphorylation of Src and FAK and their downstream effectors, Erk and Akt. Again, imatinib was minimally active against exosome stimulation of HUVEC cell signaling. Thus, K562 CML exosomes stimulate angiogenesis in vitro and in vivo in a dasatinib-sensitive fashion. This credentials exosomes and angiogenesis as molecular targets in CML via activation of Src both in leukemia and its microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4372. doi:1538-7445.AM2012-4372
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