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Introduction The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. Methods Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). Results Clinically, we found that high numbers of CD163 + M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type ( p <0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. Conclusions This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as \"twin\" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

ObjectivesInterleukin (IL)-38 is a newly characterised cytokine that belongs to the IL-1 family. This cytokine is expressed in the rheumatoid arthritis (RA) synovial tissue and IL-38 deficient mice have exacerbated arthritis. Here, we analysed the effect of IL-38 overexpression in the joints of arthritic mice, in human macrophages and synovial fibroblasts in vitro.MethodsArticular injections of an adeno-associated virus (AAV) 2/8 encoding IL-38 were performed in collagen-induced arthritis (CIA), K/BxN serum transfer-induced arthritis (STIA) and antigen-induced arthritis (AIA) in mice. The effect of IL-38 overexpression was evaluated through clinical scores, immunohistochemistry, microCT, Luminex and RT-qPCR analysis. THP-1 macrophages were transduced with a lentiviral vector to overexpress IL-38.ResultsClinical inflammatory scores were significantly decreased after AAV IL-38 injection in joints of mice with CIA and STIA, but not AIA. This decrease was accompanied by reduced macrophage infiltration and a decreased expression of Th17 cytokines (IL-17, IL-23, IL-22) and TNFα. However, IL-38 overexpression had no effect on cartilage or bone destruction. In vitro, the THP-1 monocytic cell line expressed less IL-6, TNFα and IL-23 after IL-38 overexpression. Conditioned media from these cells, containing released IL-38, also exert an anti-inflammatory effect on human primary macrophages and synovial fibroblasts from patients with RA.ConclusionsThis study shows for the first time that IL-38 overexpression attenuates the severity of experimental arthritis. IL-38 may exert its anti-inflammatory effects by decreasing the production of proinflammatory cytokines by macrophages and synovial fibroblasts. This effect can lead to the development of novel treatment strategies in arthritis.

In bone regeneration induced by the combination of mesenchymal stromal cells (MSCs) and calcium-phosphate (CaP) materials, osteoclasts emerge as a pivotal cell linking inflammation and bone formation. Favorable outcomes are observed despite short-term engraftments of implanted MSCs, highlighting their major paracrine function and the possible implication of cell death in modulating their secretions. In this work, we focused on the communication from MSCs towards osteoclasts-like cells in vitro. MSCs seeded on a CaP biomaterial or undergoing induced apoptosis produced a conditioned media favoring the development of osteoclasts from human CD14+ monocytes. On the contrary, MSCs’ apoptotic secretion inhibited the development of inflammatory multinucleated giant cells formed after IL-4 stimulation. Components of MSCs’ secretome before and after apoptotic stress were compared using mass spectrometry-based quantitative proteomics and a complementary immunoassay for major cytokines. CXCR-1 and CXCR-2 ligands, primarily IL-8/CXCL-8 but also the growth-regulated proteins CXCL-1, -2 or -3, were suggested as the major players of MSCs’ pro-osteoclastic effect. These findings support the hypothesis that osteoclasts are key players in bone regeneration and suggest that apoptosis plays an important role in MSCs’ effectiveness.

Background Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1β, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. Results Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient’s crystals enhanced mRNA expression of pro-IL-1β , as well as IL-18 , NF-kB , and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite. Conclusions As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.

Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients.

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.

Cutibacterium acnes (formerly Propionibacterium acnes ) is recognized as a pathogen in foreign-body infections (arthroplasty or spinal instrumentation). To date, the direct impact of C. acnes on bone cells has never been explored. The clade of 11 C. acnes clinical isolates was determined by MLST. Human osteoblasts and osteoclasts were infected by live C. acnes . The whole genome sequence of six isolates of this collection was analyzed. CC36 C. acnes strains were significantly less internalized by osteoblasts and osteoclasts than CC18 and CC28 C. acnes strains (p ≤ 0.05). The CC18 C. acnes ATCC6919 isolate could survive intracellularly for at least 96 hours. C. acnes significantly decreased the resorption ability of osteoclasts with a major impact by the CC36 strain (p ≤ 0.05). Genome analysis revealed 27 genes possibly linked to these phenotypic behaviors. We showed a direct impact of C. acnes on bone cells, providing new explanations about the development of C. acnes foreign-body infections.

Background Ewing sarcoma (ES), the second main pediatric bone sarcoma, is characterised by a chromosomal translocation leading to the formation of fusion proteins like EWS::FLI1. While several studies have shown that potassium channels drive the development of many tumours, limited data exist on ES. This work therefore aimed to study the transcriptional regulation of KCNA2 and define the involvement of the Kv1.2 channel encoded by KCNA2 in a key function of ES development, cell proliferation. Methods KCNA2 expression in patients and cell lines was measured via bioinformatic analysis (RNA-Seq). The presence of a functional Kv1.2 channel was shown using patch-clamp experiments. Molecular biology approaches were used after EWS::FLI1 silencing to study the transcriptional regulation of KCNA2 . Proliferation and cell count assessment were performed using cell biology approaches. KCNA2 silencing (siRNA) and RNA-Seq were performed to identify the signalling pathways involved in the ability of KCNA2 to drive cell proliferation. The regulation of the Hippo signalling pathway by KCNA2 was studied by measuring Hippo/YAP target genes expression, while YAP protein expression was studied with Western-Blot and immunofluorescence approaches. Results This research identified KCNA2 (encoding for a functional Kv1.2 channel) as highly expressed in ES biopsies and cell lines. The results indicated a correlation between KCNA2 expression and patient survival. The data also demonstrated that KCNA2 /Kv1.2 is a direct target of EWS::FLI1, and identified the molecular mechanisms by which this chimeric protein regulates KCNA2 gene expression at the transcriptional level. Furthermore, the results indicated that KCNA2 expression and Kv1.2 activity regulate ES cell proliferation and that KCNA2 expression drives the Hippo/YAP signalling pathway. Using the specific Kv1.2 channel inhibitor (κ-Conotoxin), the results suggested that two complementary mechanisms are involved in this process, both dependently and independently of Kv1.2 functional channels at the plasma membrane. Conclusion This study is the first to describe the involvement of KCNA2 expression and Kv1.2 channel in cancer development. The study also unveiled the involvement of KCNA2 in the regulation of the Hippo/YAP signalling cascade. Thus, this work suggests that KCNA2 /Kv1.2 could be a potential therapeutic target in ES.

Vascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability. Tissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque. Seventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG. OPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.