AB0057 IN VITRO EFFECT OF CTLA4-IGG ON M1-M2 SHIFT OF MACROPHAGES FROM RHEUMATOID ARTHRITIS PATIENTS

Background:Among the cells involved in the inflammatory process of rheumatoid arthritis (RA) [1], macrophages play a key role through their capacity to polarize into “classically” or “alternatively” activated phenotypes (M1 or M2) and making macrophages important players for the inflammatory cascade or for the anti-inflammatory reaction, respectively [2]. CTLA4-Ig fusion protein (abatacept) has been shown to contribute to macrophage shift from M1 to M2 [3].Objectives:We aimed to investigate the effects of abatacept to induce the polarization from the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype in cultured human macrophages obtained from RA patients’ and healthy subjects’(HS) circulating monocytes.Methods:Cultured monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of three early RA patients and ten HS, after signing informed consent and Ethics Committee approval. Cells were treated with phorbol myristate acetate (PMA) [5ng/ml] for 24 hours (hrs) to induce their differentiation into monocyte-derived macrophages (MDMs). Therefore, cultured HS MDMs were stimulated with lipopolysaccharides [LPS, 1mg/mL] for 4hrs [4] in order to induce their polarization into a pro-inflammatory M1 phenotype and then treated or not with abatacept at the concentrations of 100mg/mL and 500mg/mL for 3, 12, 24 and 48hrs. Cultured RA MDMs, were directly treated with abatacept as previous described. Cultured HS and RA MDMs without any pro-inflammatory stimuli and abatacept treatment were used as respective control.The transition of MDMs from M1 to M2 phenotype was evaluated through gene expression and protein synthesis of M2 macrophage markers, namely scavenger receptors (CD163 and CD204), and mannose receptor-1 (CD206) by quantitative real-time polymerase chain reaction (PCR) and by Western blotting. The statistical analysis evaluation was carried out by GraphPad Prism 8 analysis software using the Wilcoxon non-parametric t-test. Any p-value lower than 0.05 was considered as statistically significant. Results were indicated as median±standard deviation (SD).Results:In cultured RA MDMs (three cases), abatacept upregulated the gene expression of all investigated M2 markers, specifically after 12hrs of treatment with the concentration of 100mg/mL. In these cells, abatacept upregulated only the CD204 protein synthesis with more evidence at 24hrs of treatment and with the 500mg/mL concentration. In cultured HS MDMs (ten cases), abatacept upregulated the gene expression of M2 markers, significantly for that of CD206 [at 3hrs with 100mg/mL concentration, p= 0.0312] and CD163 [at 12hrs with 500mg/mL concentration, p= 0.0312]. Moreover, in these cells, abatacept significantly upregulated the protein synthesis of CD206 [at 48hrs with 500mg/mL concentration, p= 0.0195] and CD204 [at 24hrs with 100mg/mL concentration, p= 0.0156; both at 24 and 48hrs with 500mg/mL concentration, p= 0.0234].Conclusion:Preliminary data seem to indicate that abatacept can promote the in vitro shift from the M1 into the M2 macrophage phenotype, by upregulating specific markers (CD163, CD204, CD206) in cultured M1-MDMs from RA patients and in M1 macrophages induced from HS.References:[1]McInnes IB, et al. N Engl J Med 2011;365:2205–19.[2]Fujii M, et al. Biochem Biophys Res Commun. 2013;438(1):103-9.[3]Cutolo M, et al. Arthritis Res Ther. 2009;11:R176.[4]Pelegrin P., Surprenant, A. EMBO J. 2009 Jul 22; 28(14): 2114–2127.Disclosure of Interests:Samuele Tardito: None declared, Stefano Soldano: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene.

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Differentiation of blood monocytes and features of the cytokine status in patients with lung tuberculosis

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/0031-2991.2020.04.79-87 ◽

2020 ◽

pp. 79-87

Author(s):

Е.Г. Чурина ◽

О.И. Уразова ◽

А.В. Ситникова ◽

В.В. Новицкий ◽

Т.Е. Кононова ◽

...

Keyword(s):

Pulmonary Tuberculosis ◽

Peripheral Blood ◽

Blood Monocytes ◽

Clinical Form ◽

Hla Dr ◽

M1 Phenotype ◽

Anti Inflammatory ◽

Macrophage Precursors ◽

M2 Phenotype

Введение. При клинической манифестации туберкулеза легких альвеолярные макрофаги накапливают микобактерии и перестают выполнять свои эффекторные функции. Это связано с конверсией их провоспалительного фенотипа М1 в противовоспалительный М2, что способствует хронизации инфекции. Научная гипотеза исследования предполагает влияние цитокинового статуса организма на поляризацию моноцитов в крови в процессе их миграции к очагу воспаления, определяя дифференцировку и пути активации макрофагов в тканях. Цель исследования - оценка иммунофенотипа моноцитов крови и исследование in vitro уровня секреции иммунорегуляторных цитокинов мононуклеарными лейкоцитами периферической крови у больных с различными клиническими формами туберкулеза легких. Методика. Обследовано 65 пациентов с впервые выявленным туберкулезом легких. Материалом исследования служили венозная кровь и мононуклеарные лейкоциты периферической крови. Исследование иммунофенотипа моноцитов проводили методом проточной цитометрии (цитофлуориметр Cytoflex, Becman Coulter, США) в цельной крови с использованием моноклональных антител («eВioscience», США). Обработку полученных данных проводили с помощью программы «CytExpert 2.0». Определяли количество клеток экспрессирующих поверхностные маркеры: CD14, CD163, CD204 и HLA-DR. Содержание цитокинов (IL-2, IL-10, TGFβ) в супернатантах клеточных культур оценивали с помощью твердофазного иммуноферментного анализа (ELISA). Результаты. Полученные результаты позволяют предположить, что при общем снижении численности циркулирующих CD14-позитивных моноцитов крови у больных туберкулезом легких, независимо от его клинической формы сохраняется высокая экспрессия маркеров активации клеток как по провоспалительному фенотипу М1 (HLA-DR-позитивные моноциты), так и противовоспалительному фенотипу М2 (CD163-позитивные моноциты). При диссеминированной форме заболевания повышается количество противовоспалительных CD204-позитивных моноцитов, предшественников М2-макрофагов, что свидетельствует о доминировании супрессорного типа иммунного ответа. Анализ цитокинового статуса in vitro показал, что течение болезни сопровождается угнетением эффекторных иммунных реакций и повышением уровня противовоспалительных цитокинов. Выявленные изменения в равной степени могут быть как причиной, так и следствием дефицита секреции IL-2. Показано также, что уровень секреции медиаторов с супрессорными эффектами (IL-10, TGFβ) меняется в зависимости от клинической формы заболевания и чувствительности возбудителя к противотуберкулезным препаратам: гиперсекреция IL-10 отмечается у больных с инфильтративным лекарственно-чувствительным, а TGFβ - при диссеминированном лекарственно-устойчивом туберкулезе легких. Заключение. Особенности дифференциации моноцитов крови у больных туберкулезом легких позволили прийти к заключению, что предшественники макрофагов - моноциты, уже в кровотоке начинают экспрессировать маркеры, характерные для разных по функциям М1- и М2-макрофагов, c поляризацией в направлении М2-иммунофенотипа. Следовательно, при развитии туберкулеза легких реализуются механизмы цитокиновой регуляции, подавляющие активацию врожденного иммунитета, что, возможно, является причиной хронизации воспалительного процесса в легких и формирования иммунодефицита индуцированного Mycobacterium tuberculosis. In clinical manifestation of pulmonary tuberculosis, alveolar macrophages perform as a reservoir where they accumulate mycobacteria and lose their effector functions due to the pathological conversion of macrophage pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which provides transition to chronic infection. The study hypothesis suggested that the cytokine status, as evaluated by leukocyte secretion of cytokines in vitro, influences the monocyte polarization in the blood during their migration to the inflammatory focus, thereby determining differentiation and pathways of macrophage activation in tissues. The aim of this work was to assess the immunophenotype of blood monocytes and the in vitro secretion of immunoregulatory cytokines by mononuclear peripheral blood leukocytes from patients with different clinical forms of pulmonary tuberculosis taking into account the pathogen sensitivity to major anti-tuberculosis drugs. Methods. 65 patients with newly diagnosed pulmonary tuberculosis were evaluated. The study material was venous blood and peripheral blood mononuclear leukocytes. Monocyte immunophenotype was determined in whole blood by flow cytometry on a Cytoflex flow cytometer (Becman Coulter, USA) with monoclonal antibodies (eBioscience, USA). Results were processed with a CytExpert 2.0 software. The number of cells expressing surface markers, CD14, CD163, CD204 and HLA-DR, was determined. Content of cytokines (IL-2, IL-10, TGFβ) in supernatants of cell cultures was measured by enzyme-linked immunosorbent assay (ELISA). Results of the study were processed with a SPSS v.11.0 standard software package. Results. The study results suggested that with an overall decrease in the number of circulating CD14-positive blood monocytes in patients with pulmonary tuberculosis regardless of its clinical form, high expression of cell activation markers remained both for the pro-inflammatory M1 phenotype (HLA-DR-positive monocytes) and the anti-inflammatory M2 phenotype (CD163-positive monocytes). In disseminated tuberculosis, the number of anti-inflammatory CD204-positive monocytes, M2 macrophage precursors, increases indicating predomination of the immunosuppressive response. In vitro analysis of the cytokine status showed that tuberculosis progression is accompanied by inhibition of effector immune responses and increases in anti-inflammatory cytokine concentrations in vitro. These changes may be equally either a cause or a consequence of deficient IL-2 secretion. We also found that the secretion of mediators with suppressor effects (IL-10, TGFβ) varied depending on both the clinical form of tuberculosis and the pathogen sensitivity to anti-TB drugs; IL-10 hypersecretion was observed in patients with drug-sensitive, infiltrative tuberculosis whereas TGFβ hypersecretion was observed in disseminated, drug-resistant tuberculosis. Conclusion. Features of blood monocyte differentiation in patients with pulmonary tuberculosis allowed us to conclude that monocytes, the macrophage precursors, start expressing markers for different functions of M1 and M2 macrophages with polarization toward the M2 immunophenotype already in the bloodstream. Therefore, in the development of pulmonary tuberculosis, cytokine regulation mechanisms become involved in suppressing the activation of innate immunity, which possibly causes chronic inflammation in the lungs and formation of Mtb-induced immunodeficiency.

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CTLA4-Ig treatment induces M1–M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

Arthritis Research & Therapy ◽

10.1186/s13075-021-02691-9 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Maurizio Cutolo ◽

Stefano Soldano ◽

Emanuele Gotelli ◽

Paola Montagna ◽

Rosanna Campitiello ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Protein Synthesis ◽

Healthy Subjects ◽

Mononuclear Cells ◽

Costimulatory Molecule ◽

High Concentration ◽

Peripheral Blood Mononuclear ◽

Alternatively Activated

Abstract Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

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THU0199 ABX464, A NOVEL DRUG IN THE FIELD OF INFLAMMATION, INCREASES MIR-124 AND MODULATES MACROPHAGES AND T-CELL FUNCTIONS.

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4959 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 321-322

Author(s):

C. Begon-Pescia ◽

J. Mielle ◽

N. Campose ◽

K. Chebli ◽

L. Manchon ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Ulcerative Colitis ◽

T Cells ◽

Strong Increase ◽

Inadequate Response ◽

Soluble Receptor ◽

Research Support ◽

Anti Inflammatory ◽

Rectal Biopsies

Background:ABX464 is a small oral molecule with a novel mode of action. It binds the Cap Binding Complex, involved in the biogenesis of RNAs and predominantly upregulates the expression of a microRNA miR-124 in PBMCs and T cells (1). miR-124 has been widely described for its anti-inflammatory properties, with many confirmed targets i.e. monocyte chemoattractant protein 1 (MCP-1, CXCL-1, SERPIN-E1, STAT-3, IL-6 receptor. It post-transcriptionally regulates the expression of MCP-1 in rheumatoid arthritis (RA) synoviocytes and decreases their proliferation (2). While miR-124 is decreased in synoviocytes of RA patients, its injection in joint improved arthritis in rats (3). miR-124 expression in macrophages leads to the induction and maintenance of anti-inflammatory M2 phenotype (4). Its effect in T cells remains controversial.Objectives:(i) To assess the effect of ABX464 on miR-124 expressionin vitroin macrophages andin vivoin patients; (ii) to assess the effect of ABX464 on arthritis in mice and (iii) to decipher the effect of ABX464 on human macrophages and T cells.Methods:miR-124 was measured in human monocyte-derived macrophages (huMDM) treated with ABX464 for 4 days and in patients with ulcerative colitis included in a phase IIa RCT in blood and rectal biopsies at day 56 by TaqMan qPCR. Collagen-induced arthritis (CIA) was induced using usual protocol and ABX464 was given by gavage 2 weeks at 40 mg/kg after the 2ndinjection of collagen and Freund adjuvant. HuMDM were exposed to 5 µM of ABX464 or DMSO (control) for 4 days, during a M1-polarization. Cytokines and chemokines were assessed in supernatants using both Proteome Profiler Array and Luminex. PBMCs were exposed to ABX464 (5 µM) for 6 days. Th1 (IFN-g+), Th17 (CCR6+IL-17+), Th2 (CRTH2+ IL-4+) and Tregs (CD25+CD125-/loFoxP3+) were assessed by flow cytometry. IL-6 receptor was assessed in CD4+ supernatant using ELISA.Results:ABX464 increased miR-124 in vitro by 3.41 folds in huMDM (p=0.001) compared to DMSO. The phase IIa RCT conducted in 32 patients with moderate to severe active ulcerative colitis showed a good safety profile and significant clinical efficacy. A strong increase of miR-124 was observed both in blood and rectal biopsies of patients treated with ABX464 (637 and 7.69 folds respectively, compared to placebo, p<0.05). The use of ABX464 drastically decreased the incidence of arthritis from 52% (15/ 29 mice) to 10% (3/30 mice) in a CIA model. Macrophages treated with ABX464 produced significantly less MCP-1 (median decrease -67%, p=0.004), CXCL-1 (-18%, p=0.004) and SERPIN-E1 (-53%, p=0.004), as confirmed by the two technics (n=9). ABX464 significantly decreased Th17 (-56%, p=0.02), while increasing Th2 (+21%, p=0.01). IL-6 soluble receptor was significantly decreased in supernatant of PBMCs treated with ABX464 (-43%, p=0.04).Conclusion:We demonstrated that ABX464 increases miR-124 bothin vitroand in ulcerative colitis patients.In vitro, ABX464 decreased the expression of miR-124 target genes, that is MCP-1, CXCL-1, SERPIN-E1 in macrophages and decreases the number of Th17 as well as IL-6 soluble receptor in CD4+ T cells. A phase IIa RCT is currently ongoing in patients with rheumatoid arthritis and inadequate response to methotrexate and/or TNF-inhibitors (n=60). Results are expected during 2020 summer.References:[1]Vautrin A et al. Sci Rep. 2019;9:792[2]Nakamachi Y et al. Arthritis Rheum 2009; 60:1294-304[3]Nakamachi Y et al. Ann Rheum Dis 2016; 75:601-8[4]Veremeyko T et al. PLoS ONE 2013; 8:e81774Disclosure of Interests:Christina BEGON-PESCIA: None declared, Julie Mielle: None declared, Noélie Campose Employee of: ABIVAX, Karim Chebli Consultant of: ABIVAX, Laurent Manchon: None declared, Julien Santo Employee of: ABIVAX, Cécile Apolit Employee of: ABIVAX, Kévin Martin Grant/research support from: ABIVAX, Laure Lapasset Employee of: ABIVAX, Audrey Vautrin Employee of: ABIVAX, Didier Scherrer Employee of: ABIVAX, Aude Garcel Employee of: ABIVAX, Jamal Tazi Shareholder of: ABIVAX, Grant/research support from: ABIVAX, Consultant of: ABIVAX, Employee of: ABIVAX, Paid instructor for: ABIVAX, Speakers bureau: ABIVAX, Claire DAIEN Grant/research support from: from Pfizer, Abbvie, Roche-Chugaï, Novartis, Abivax, Sandoz, Consultant of: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis, Speakers bureau: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis

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Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190619111118 ◽

2019 ◽

Vol 20 (11) ◽

pp. 920-933 ◽

Cited By ~ 3

Author(s):

Lucía Gato-Calvo ◽

Tamara Hermida-Gómez ◽

Cristina R. Romero ◽

Elena F. Burguera ◽

Francisco J. Blanco

Keyword(s):

Gene Expression ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Cartilage Explants ◽

Ex Vivo Model ◽

Chondrocyte Viability ◽

Platelet Concentration ◽

The Absolute ◽

Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

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871 In vitro immunoregulatory effect from cervical cancer derived mesenchymal stromal cells over molecules expression in monocyte derived macrophage polarization

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0871 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A923-A923

Author(s):

Víctor Cortés-Morales ◽

Juan Montesinos ◽

Luis Chávez-Sánchez ◽

Sandra Espíndola-Garibay ◽

Alberto Monroy-García ◽

...

Keyword(s):

Cervical Cancer ◽

Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Macrophage Polarization ◽

Induction Medium ◽

M2 Macrophage ◽

List Type ◽

M2 Phenotype

BackgroundMacrophages are immunological cells that sense microenvironmental signals that may result in the polarized expression of either proinflammatory (M1) or anti-inflammatory (M2) phenotype.1 Macrophages M2 are present in tumoral microenvironment and their presence in patients with cervical cancer (CeCa) is related with less survival.2Mesenchymal Stromal Cells (MSCs) are also present in tumor microenvironment of cervical cancer (CeCa-MSC), which have shown immunoregulatory effects over CD8 T cells, decreasing their cytotoxic effect against tumoral cells.3 Interestingly, MSCs from bone marrow (BM-MSC) decrease M1 and increase M2 macrophage polarization in an in vitro coculture system.4 Macrophages and MSCs are present in microenvironment of cervical cancer, however it is unknown if MSCs play a role in macrophage polarization. In the present study, we have evaluated the immunoregulatory capacity of CeCa-MSCs to induce macrophage polarization.MethodsCD14 monocytes were isolated from peripheral blood and cultivated in the absence or presence of MSCs from BM, normal cervix (NCx) and CeCa. Two culture conditions were included, in the presence of induction medium to favors M1 (GM-CSF, LPS and IFNg) or M2 (M-CSF, IL-4 and IL-13) macrophage polarization. M1 (HLA-DR, CD80, CD86 and IFNg) or M2 (CD14, CD163, CD206, IDO and IL-10) macrophage molecular markers were evaluated by flow cytometry. Finally, we evaluated concentration of IL-10 and TNFa in conditioned medium form all coculture conditions.ResultsWe observed that CeCa-MSCs and BM-MSCs in presence of M1 induction medium, decreased M1 macrophage markers (HLA-II, CD80, CD86 and IFNg), and increase the expression of CD14 (M2 macrophage marker). Interestingly, in presence of M2 induction medium, BM-MSCs and CaCe-MSCs but not CxN-MSC increased CD163, CD206, IDO and IL-10 (M2 macrophage markers). We observed a decreased concentration of TNFa in the supernatant medium from all cocultures with MSCs, but only in presence of CeCa-MSCs, increased IL-10 concentration was detected in such cocultures.ConclusionsIn contrast to NCx-MSCs, CeCa-MSCs similarly to BM-MSCs have in vitro capacity to decrease M1 and increase M2 macrophage phenotype.AcknowledgementsAcknowledgments The authors are indebted to gratefully acknowledge to CONACYT (Grant No. 272793) and IMSS (Grant no. 1731) for support to Juan J. Montesinos research.ReferencesMartinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 2014;6-13.Petrillo M, Zannoni GF, Martinelli E, et al. Polarization of tumor-associated macrophages toward M2 phenotype correlates with poor response to chemoradiation and reduced survival in patients with locally advanced cervical cancer. PLoS One 2015;10: e0136654.Montesinos JJ, Mora-García Mde L, et al. In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity. Stem Cells Dev 2013;22:2508-2519.Vasandan AB, Jahnavi S, Shashank C. Human mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE 2-dependent mechanism. Sci Rep 2016;6:38308.

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POS0330 NINTEDANIB (TYROSINE KINASE INHIBITOR) DOWNREGULATES THE TRANSITION OF CULTURED SYSTEMIC SCLEROSIS FIBROCYTES INTO MYOFIBROBLASTS AND THEIR PRO-FIBROTIC ACTIVITY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2411 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 392.2-392

Author(s):

S. Soldano ◽

P. Montagna ◽

E. Gotelli ◽

S. Tardito ◽

S. Paolino ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Kinase Inhibitor ◽

Adherent Cells ◽

Research Support ◽

Fibrotic Process ◽

Circulating Fibrocytes

Background:Fibroblast-to-myofibroblast transition is one of the fundamental steps involved in the fibrotic process that characterise systemic sclerosis (SSc) [1]. Myofibroblasts are α-smooth muscle actin (αSMA) positive cells that contribute to fibrosis through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, primarily fibronectin (FN) and type I collagen (COL1) [2].Among the cells involved in the fibrotic process of SSc, circulating fibrocytes seem to have an emerging role as an important source of fibroblasts and myofibroblasts [3].Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis (4). Nintedanib was recently demonstrated to have a beneficial effect in patients with interstitial lung disease (ILD) associated with SSc (5).Objectives:To investigate nintedanib effect in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.Methods:Circulating fibrocytes were obtained from 14 SSc patients (mean age 64±14 years), who fulfilled the 2013 ACR/EULAR criteria for SSc and that underwent complete disease staging in a day-hospital setting at the Rheumatology Division of Genoa University. Five age-matched healthy subjects (HSs) were also analysed. All SSc patients and HSs signed the informed consent and the local EC approved the study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and plated on FN-coated dishes. After overnight culture, non-adherent cells were removed, and adherent cells were maintained in growth medium for 8 days (T8) to obtain fibrocytes [6]. T8-cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1μM and 1μM for 3 and 24 hours. Fibroblast specific protein-1 (S100A4) and αSMA, as markers of fibroblast/myofibroblast phenotype, together with COL1 and FN, were investigated by qRT-PCR and Western blotting. Non-parametric Mann-Whitney and Wilcoxon tests were used for the statistical analysis.Results:Significantly elevated gene and protein expressions of αSMA, S100A4, COL1 and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p<0.001; others p<0.0001; protein: all p<0.05). In accordance with the antibody positivity for Scl70 and the presence or absence of ILD at CT scan, SSc patients were grouped as either Scl70 positive patients with ILD (Scl70+ILD+) or Scl70 negative patients without ILD (Scl70-ILD-). Significant αSMA, S100A4, COL1 and FN gene expressions were found in fibrocytes from Scl70+ILD+ compared to HS fibrocytes (αSMA p<0.001; others p<0.0001). Moreover, fibrocytes from Scl70+ILD+patients showed a more significant gene expression of fibroblasts/myofibroblasts markers compared to Scl70-ILD-patients (p<0.01 for S100A4), whereas no differences were observed for ECM gene expression.Nintedanib reduced the gene and protein expression of αSMA, COL1 and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated αSMA, S100A4, COL1 and FN gene expression (all p<0.05) in Scl70+ILD+fibrocytes, whereas only that of S100A4 and FN was significantly downregulated (p<0.05) in Scl70-ILD- fibrocytes compared to untreated cells.Conclusion:Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+SSc patients.References:[1]Cutolo M et al. Exp Rev Clin Immunol. 2019;15:753-64.[2]Van Caam A et al. Front. Immunol. 2018;9:2452.doi:10.3389/fimmu.2018.02452.[3]Distler JH et al. Arthritis Rheumatol. 2017;69:257-67.[4]Distler O et al. New Eng J Med. 2019; 380:2518-28.[5]Maher TB et al. Arthritis Rheumatol.2020.doi:10.1002/art.41576.[6]Cutolo M et al. Arthritis Res Ther. 2018;20:157.doi:10.1186/s13075-018-1652-6.Acknowledgements:We thank Stefano-Lutz Willing for the scientific support through the study.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Emanuele Gotelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli: None declared, Carlotta Schenone: None declared, Greta Pacini: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene

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