Gwenola Bougras

Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients

Objectives Interleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA). Methods Immunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1β. Wild-type, jnk1−/−–jnk2−/− and nemo−/− murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect. Results IL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1β stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway. Conclusion This work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1β, may contribute to inflammation and bone erosions in RA.

Apoptotic body–loaded dendritic cells efficiently cross-prime cytotoxic T lymphocytes specific for NA17-A antigen but not for Melan-A/MART-1 antigen

DCs hold promise for cancer immunotherapy due to their functional ambivalence: iDCs internalize antigens, then mDCs trigger naive T‐cell activation. However, no consensus has been reached concerning the optimal mode of antigen acquisition for efficient cross‐priming of TAA‐specific CTLs, and this remains a field of investigation. Here, we used highly purified apobodies derived from an HLA‐A*0201‐negative melanoma line as a source of tumor antigens for HLA‐A*0201 DCs. We compared in vitro mDCs loaded with apobodies to DCs loaded with antigenic peptides, NA17‐A1–9 and Melan‐A/MART‐126–35 A27L analogue, for their capacity to stimulate melanoma antigen‐specific T cells from autologous PBLs. Apobody phagocytosis did not induce spontaneous DC maturation, but phagocytic DCs were still responsive to maturation signals, resulting in a functional ability to activate antigen‐specific lymphocytes. NA17‐A‐specific T lymphocytes were activated by both types of stimulation, whereas only peptide‐pulsed DCs stimulated the growth of Melan‐A/MART‐1‐specific lymphocytes. We also observed a lack of staining of melanoma‐derived apobodies with a Melan‐A‐specific MAb, suggesting protein alteration during apoptosis induction. After HLA‐A*0201/NA17‐A multimer sorting, antigen‐specific lymphocytes induced by mature DCs loaded with either peptide or apobodies displayed similar functional capacity against peptide‐pulsed T2 cells and melanoma cells. Therefore, apobody‐loaded DCs can achieve T‐cell priming similar to that induced by peptide‐pulsed DCs, provided that the apoptotic process allows the preservation of antigen expression.

INCREASED EXPRESSION OF INDUCIBLE HSP70 IN APOPTOTIC CELLS IS CORRELATED WITH THEIR EFFICACY FOR ANTITUMOR VACCINE THERAPY

Apoptosis is a physiologic process in normal development, tissue remodeling and cell turnover. This cell death is noninflammatory and nonimmunogenic, but when associated with a danger signal, it can activate the immune system. However, the capacity of apoptotic cells to activate the immune system is not clearly established, although dead tumor cells have been largely exploited as a source of TAA in cellular therapy against cancer. From these cellular preparations, contradictory results have been reported on the effect of apoptotic cells as an effective source of TAA and their immunologic properties. These conflicting data strongly suggest that the optimal preparation of apoptotic cells derived from tumor cells remains to be determined. In this work, we studied and compared the efficacy of antitumor immune responses derived from repeated injections using different preparations of apoptotic cells. We investigated the importance of HSP70 and TGF‐β expression in apoptotic cells used in the treatment of an established and nonimmunogenic rat carcinoma. UVB‐mediated apoptosis did not affect TGF‐β expression in tumor cells, whereas HS treatment sharply downregulated it. Thus, downregulation of TGF‐β permits normal DC activation and maturation and the induction of tumor immunity. We conclude that HS followed by UVB irradiation is a superior source of tumor antigen for the treatment of established tumors. Future work will determine whether HS independently upregulates HSP70, thereby suppressing expression of active TGF‐β, or whether the 2 are linked via a still undefined mechanism.

Overexpression of Smad7 Blocks Primary Tumor Growth and Lung Metastasis Development in Osteosarcoma

Purpose: Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastasis is present at diagnosis. Because transforming growth factor-β (TGFβ) has been shown to promote metastasis in many solid tumors, we investigated the effect of the natural TGFβ/Smad signaling inhibitor Smad7 and the TβRI inhibitor SD-208 on osteosarcoma behavior. Experimental Design: By using a mouse model of osteosarcoma induced by paratibial injection of cells, we assessed the impact of Smad7 overexpression or SD-208 on tumor growth, tumor microenvironment, bone remodeling, and metastasis development. Results: First, we demonstrated that TGFβ levels are higher in serum samples from patients with osteosarcoma compared with healthy volunteers and that TGFβ/Smad3 signaling pathway is activated in clinical samples. Second, we showed that Smad7 slows the growth of the primary tumor and increases mice survival. We furthermore demonstrated that Smad7 expression does not affect in vitro osteosarcoma cell proliferation but affects the microarchitectural parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of osteolytic factors such as RANKL, suggesting that Smad7 overexpression affects the “vicious cycle” established between tumor cells and bone cells by its ability to decrease osteoclast activity. Finally, we showed that Smad7 overexpression in osteosarcoma cells and the treatment of mice with SD208 inhibit the development of lung metastasis. Conclusion: Taken together, these results demonstrate that the inhibition of the TGFβ/Smad signaling pathway may be a promising therapeutic strategy against tumor progression of osteosarcoma, specifically against the development of lung metastasis. Clin Cancer Res; 20(19); 5097–112. ©2014 AACR.

Distinct roles of Bcl-2 and Bcl-Xl in the apoptosis of human bone marrow mesenchymal stem cells during differentiation.

Background Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells. Principal Findings Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs. Significance In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions.

Efficient monocyte-derived dendritic cell generation in patients with acute myeloid leukemia after chemotherapy treatment: Application to active immunotherapy

OBJECTIVE While complete remission in acute myeloid leukemia (AML) can be achieved after chemotherapy (CT), relapses occur for the majority of patients, underlying the need to eliminate residual disease. Based on dendritic cell (DC) vaccination, the triggering of an immune response against residual leukemia cells after CT could maintain patients in remission. The aim of our study was to assess, for vaccine preparation, generation of monocyte-derived DCs in AML patients after CT. MATERIALS AND METHODS We evaluated efficiency of the production, yields, maturation, and functional properties of DCs from 22 AML patients at different CT stages compared to those from 15 healthy donors. RESULTS We demonstrated that monocyte-derived DC production is successful later than 3 weeks after the last CT cycle, whatever the CT was. Immature DCs demonstrated functional phagocytic activity. Mature DCs displayed migratory, T-cell stimulatory and Th1-activation capacities. Our results also suggest a favorable period from 20 to 60 days after CT for potent monocyte-derived DC production and immune activation. CONCLUSION In defining patient-sampling conditions, this preclinical study has direct implications for AML DC-based immunotherapy.

Loss of connexin43 expression in Ewing's sarcoma cells favors the development of the primary tumor and the associated bone osteolysis☆

Ewings sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS-FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC). Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS-FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption. Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.

Proteoglycans and osteolysis.

Osteolysis is a complex mechanism resulting from an exacerbated activity of osteoclasts associated or not with a dysregulation of osteoblast metabolism leading to bone loss. This bone defect is not compensated by bone apposition or by apposition of bone matrix with poor mechanical quality. Osteolytic process is regulated by mechanical constraints, by polypeptides including cytokines and hormones, and by extracellular matrix components such as proteoglycans (PGs) and glycosaminoglycans (GAGs). Several studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial: some studies showed a repressive effect of GAGs on osteoclastic differentiation, whereas others described a stimulatory effect. The controversy also affects osteoblasts which appear sometimes inhibited by polysaccharides and sometimes stimulated by these compounds. Furthermore, long-term treatment with heparin leads to the development of osteoporosis fueling the controversy. After a brief description of the principal osteoclastogenesis assays, the present chapter summarizes the main data published on the effect of PGs/GAGs on bone cells and their functional incidence on osteolysis.

Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system.

BackgroundThe relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood.MethodsThe rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo.ResultsIn vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells.ConclusionsWe show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune response