Inflammasomes NLRP3 and AIM2 in peri-implantitis: A cross-sectional study

Background: Inflammasome components NLRP3 and AIM2 contribute to inflammation development by the activation of caspase-1 and IL-1β. They have not been yet evaluated in samples from patients with active peri-implantitis. Thus, the aim of the present study is to analyze the expression of inflammasomes NLRP3 and AIM2 and subsequent caspase 1 and IL-1β assessing the microenvironment of leukocyte subsets in samples from patients with active peri-implantitis. Methods: Biopsies were collected from 33 implants in 21 patients being treated for peri-implantitis. Biopsies from gingival tissues from 15 patients with healthy periodontium were also collected for control. These tissues were evaluated through conventional histological stainings. Then, immunohistochemical detection was performed to analyze NLRP3, AIM2, caspase-1, and IL-1β and markers of different leukocyte subsets. PCR for inflammasomes and related genes was also done. Results: This manuscript reveals a high immunohistochemical and mRNA expression of NLRP3 and AIM2 inflammasomes, caspase-1, and IL-1β in biopsies collected from human peri-implantitis. The expression of the tested markers was significantly correlated with the increase in inflammatory infiltrate, probing depth, presence of biofilm, and bleeding on probing. In these peri-implantitis lesions, the area of biopsy tissue occupied by inflammatory infiltrate was intense while the area occupied by collagen was significantly lower. In comparison with periodontal healthy tissues, the inflammatory infiltrate was statistically significantly higher in the peri-implantitis biopsies and was mainly composed of plasma cells, followed by T and B lymphocytes. Conclusion: In human peri-implantitis, chronic inflammation can be explained in part by the action of IL-1β/ caspase 1 induced through NLRP3 and AIM2 inflammasome activation.Junta de Andalucía, Grant/Award Number: CTS-138CTS-1028; Universidad de Granada, Grant/Award Number: B-CTS- 504- UGR18Universidad de Granada/CBU

Maxillary sinus floor augmentation comparing bovine versus porcine bone xenografts mixed with autogenous bone graft. A split-mouth randomized controlled trial

This investigation was conducted under an Investigator--Initiated Study partially funded by Dentsply Sirona Implants (Molndal, Sweden) through a research transfer agreement with the Technology Transfer Office of the University of Granada (number I--BI--17--026) and the Research Cathedra "Dentsply Sirona-UGR" agreed between Dentsply Sirona Iberia S.A.U. and the University of Granada. The authors are also supported by funding from Research Groups #CTS-138 and #CTS--1028 (Junta de Andalucia, Spain) The authors are grateful to Justin G. Davis for assistance with the English translation and to Emilio Couso-Queiruga for assistance with the volumetric radiographic analyses.Aim: To compare the effectiveness of two xenografts for maxillary sinus floor augmentation in terms of clinical, radiographical, histologic, and molecular outcomes. Materials and methods: A split-mouth randomized clinical trial was conducted at the University of Granada. Ten consecutive patients in need of bilateral two-staged maxillary sinus floor augmentation were included. Each patient received both biomaterials (porcine bone mineral and anorganic bovine bone), which were randomly assigned for bilateral sinus augmentation. The maxillary autogenous bone scraped from the sinus access window was mixed with each xenograft at a 20:80 ratio. After a healing period of 6 months, bone biopsies were collected with a trephine during the implant placement in the regenerated area. Histologic, histomorphometrical, immunohistochemical, and molecular outcomes were analyzed. Clinical and radiographical data throughout the treatment phases were also evaluated. Results: The resulting anatomic features were similar between both groups. After six months of graft consolidation, the graft resorption rates were similar between both biomaterials. The histologic, histomorphometrical, and immunohistochemical results showed no statistical differences between groups. Conclusion: Anorganic bovine bone and porcine bone mineral combined with maxillary autogenous cortical bone show similar biologic and radiologic features in terms of biomaterial resorption, osteoconduction, and osteogenesis when used for maxillary sinus floor augmentation.Dentsply Sirona Implants (Molndal, Sweden)Technology Transfer Office of the University of Granada I-BI-17-026Dentsply Sirona Iberia S.A.U.University of GranadaJunta de AndaluciaEuropean Commission CTS-138 CTS-102

GARP promotes the proliferation and therapeutic resistance of bone sarcoma cancer cells through the activation of TGF-β

Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-β-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.Instituto de Salud Carlos IIIEuropean Union (EU) PI15/00794 PI18/00826 CPII15/00032 PI15/02015Junta de Andalucía C-0013-2018Spanish Government PEJ-2014-A-46314Agencia Estatal de Investigación (AEI) [MICINN/Fondo Europeo de Desarrollo Regional (FEDER)] SAF-2016-75286-RISCIII/FEDER [Miguel Servet Program] CPII16/00049ISCIII/FEDER [Sara Borrell Program] CD16/00103Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III PT17/0015/0023Fundación Bancaria Cajastur PT17/0015/0023ISCIII/FEDER [Consorcio CIBERONC] CB16/12/0039

Importancia de las moléculas que interactúan con TGF-B1 sobre la biología y capacidad terapéutica de las células mesenquimales estromales (MSCs): papel de CD105 y GARP

Las células mesenquimales estromales (MSCs) son células multipotentes no hematopoyéticas presentes en prácticamente todos los tejidos y que presentan un alto potencial de inmunomodulación y de secreción de factores tróficos. Debido a estas propiedades, las MSCs han mostrado su potencial terapéutico en diferentes ensayos clínicos. Sin embargo, el beneficio clínico no ha sido homogéneo, obteniéndose resultados diferentes tanto en función de la patología como del grupo de investigación. Esto es en parte debido a la ausencia de una uniformidad en la caracterización de las MSCs así como a la necesidad de su expansión que está asociada con numerosos problemas como son la pérdida de su capacidad de migración y diferenciación así como el incremento en mutaciones en el DNA. El factor de crecimiento transformante beta (TGF-β) es una molécula pleiotrópica involucrada en múltiples procesos biológicos como proliferación, diferenciación, migración y respuesta inmunitaria. Se sabe que las MSCs son capaces de producir y de responder a TGF-β lo que tiene un gran impacto en su biología y potencial terapéutico. Dada la importancia de esta molécula sobre la biología de las MSCs, en la presente tesis nos hemos centrado en el estudio de dos moléculas presentes en las MSCs y que interactúan con TGF-β: CD105 (endoglina) y GARP (del inglés Glycoprotein A repetitions predominant). El objetivo de la presente tesis es entender las implicaciones de estas dos moléculas en la biología y capacidad terapéutica de las MSCs. En primer lugar nos centramos en estudiar las subpoblaciones de MSCs CD105+ y CD105- y encontramos que la subpoblación CD105- presenta una mayor capacidad de diferenciación adipo y osteogénica, así como una mejor capacidad de inhibir la proliferación de las células T in vitro. Estos resultados podrían ser de gran utilidad a la hora de diseñar terapias con MSCs para el tratamiento de enfermedades inflamatorias y autoinmunes así como para regeneración ósea. En segundo lugar, estudiamos las implicaciones de GARP sobre la biología de las MSCs. Demostramos por primera vez que GARP se encuentra en la superficie de las MSCs en cultivo uniendo TGF-β a la superficie celular y regulando su secreción y activación. Por lo tanto GARP podría ser utilizado como un nuevo marcador de superficie para la identificación de las MSCs. El silenciamiento de GARP mediante shRNA demostró el papel fundamental que juega esta proteína no solo en la capacidad de inmunomodulación del las MSCs (como cabría esperar por su papel como proteína de unión de TGF-β) sino también en su capacidad para proliferar. Efectivamente, la eliminaciónde GARP de las MSCs hacía que estas células perdieran parte de su capacidad de inhibir la proliferación de las células T y que dejaran de proliferar casi por completo. Dado el efecto tan drástico que tenía la eliminación de GARP sobre la proliferación de las MSCs, procedimos a investigar los mecanismos involucrados. El silenciamiento de GARP en las MSCs dió como resultado un aumento de MSCs bloqueadas en la fase G2/M del ciclo celular y mayores niveles de roturas de DNA de doble cadena (DSBs). También observamos niveles elevados de especies reactivas de oxígeno mitocondrial (mtROS) en las células GARP- y que al añadir inhibidores de estas mtROS éramos capaces de revertir el bloqueo en proliferación. Por último, también vimos que la inhibición de la señalización por TGF-β en las células GARP- producía una disminución de los niveles de mtROS y de DSBs y restauraba la habilidad de proliferación de estas células. Todos estos datos indicaban que GARP es imprescindible para la viabilidad de las MSCs evitando que los niveles de mtROS se incrementen en exceso como consecuencia de la señalización por TGF-β1. Por último demostramos que la sobreexpresión de GARP proporciona una ventaja selectiva de crecimiento al evitar la muerte por daño en el ADN. Por lo tanto la sobreexpresión de GARP en MSCs podría ser una estrategia para la generación de MSCs con mejores características terapéuticas, mejorando su capacidad proliferativa e incrementando su resistencia a apoptosis.Tesis Univ. Granada

CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities

<div><p>Administration of in vitro expanded mesenchymal stromal cells (MSCs) represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs) with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105) expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105⁺ and CD105^- MSCs. CD105⁺ and CD105^- mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105⁺ mMSCs was affected by passage number and cell confluency while CD105^- mMSCs remained negative. The CD105⁺ and CD105^- mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29⁺CD44⁺Sca1 ⁺ MHC-I⁺ and CD45^-CD11b^-CD31^-) but varied in their differentiation and immunoregulatory properties. Interestingly, CD105^- mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4⁺ T cells more efficiently compared to CD105⁺ mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.</p> </div

Comparison of the immunomodulatory capacity of CD105^- and CD105⁺ mASCs.

<p>(A) The levels of PGE₂ and TGF-β1 were measured in supernatants from ASC^tot, CD105^- and CD105⁺ mASCs using specific ELISAs (see materials and methods). (B) ASC^tot, CD105^- and CD105⁺ mASCs were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR. (C) Increasing numbers of mitomycin C-treated ASC^tot, CD105^- and CD105⁺ mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 µg/ml) for 3 days. The cells were harvested and acquired on a FACS Canto II flow cytometer and the proliferation of CD4⁺ splenocytes was quantified using the FlowJo software. (D) BM-MΦs were cultured with or without mASC for 48 hours and then restimulated with LPS (1 µg/ml) for 24 hours. The levels of IL-10 (left graph) and IL-12 (right graph) in the supernatants of the co-cultures were measured using specific ELISAs. Data is shown as mean (SEM) of at least 3 independent experiments. *=p<0.05 vs. CD105⁺ mASCs.</p

CD105 defines two distinct MSC subpopulations in mouse.

<p>(A) Balb/c ASCs were double-stained for CD105 in combination with CD29, CD44, CD45, CD49f, sca-1 and MHC class I and analyzed by flow cytometry. One representative experiment out of 3 independent experiments is shown. (B) MACS-purified CD105^- and CD105⁺ mASCs were cultured separately for 9 passages and CD105 expression was analyzed at each passage by flow cytometry. (C) CD105^- and CD105⁺ mASCs were starved for 24 hours and then stimulated with 10 ng/ml recombinant human TGF-β1 for 0, 2, 6 and 24 hours. At each time point total RNA was purified and reverse transcribed and the expression of CD105L was measured using qPCR. (D) ASC^tot, CD105^- and CD105⁺ cells were plated at 2500 cells/cm in T25 cell culture flasks and cultured at 5% O₂ until reaching 70-80% confluency. Cells were harvested, counted and reseeded at the same density for 10 passages. (E) ASC^tot, CD105^- and CD105⁺ cells were cultured in 10 cm petri dishes (2000 cells/plate) for 14 days for CFU-F assay as described in materials and methods. Cells were subsequently fixed with PFA and stained with cresyl violet to visualize CFU-F. (F) Total RNA was extracted from ASC^tot, and MACS-separated CD105^- and CD105⁺ mASCs and reverse transcribed into cDNA. qPCR analysis was performed on genes involved in ASC multipotency (nanog), adipogenesis (LPL, PPAR-γ), osteogenesis (osteocalcin, ALP) and chondrogenesis (Sox-9). Results are shown as mean (SEM) of three independent experiments.</p

CD105 expression is downregulated on confluent mASCs.

<p>Total mASCs were plated at a low density (5000 cells/cm²) and grown to confluence during 7 days. Cells were harvested on day 1, 3, 4 and 7 and (A) CD105 expression was analyzed by flow cytometry and confluence was assessed using the ImageJ software and (B) total mRNA was purified, reverse transcribed and used for analyzing the expression levels of CD105 and MMP-14. (C) Total ASCs were seeded at 5000 cells/cm² in 6 well plates and 4 days later, GM6001 (25 µM) or vehicle (DMSO) were added to the cells. Cells were harvested after 72 hours and CD105 expression was analyzed by flow cytometry. Data is shown as mean (SEM) of at least 3 independent experiments.</p

CD105^- ASCs are more prone to differentiate into adipocytes and osteocytes.

<p>Adipogenesis (A) and osteogenesis (B) were induced in ASC^tot, CD105^- and CD105⁺ mASC populations. Differentiation data is shown as one representative experiment out of 3-4 independent experiments. The extent of adipocyte differentiation was visualized using OilRED O and the amounts of OilRED O extracted from the different ASC populations were measured using a spectrophotometer (450 nm). Bars are shown as mean (SD) from experimental triplicates. *=p<0.05 vs. CD105^- mASCs. The deposition of calcium by differentiating osteocytes was visualized using alizarin red and quantified using the ImageJ software. Bars represent mean (SD) from experimental triplicates. **=p<0.01 vs. CD105^- mASCs. (C) CD105^- and CD105⁺ mASC were stained for TGF-β receptor II (TGF-βRII) and analyzed by flow cytometry. (D) Total RNA was purified from ASC^tot, CD105^- and CD105⁺ mASCs, reverse transcribed and the expression levels of β-glycan, ALK1, ALK2 and ALK5 assayed using qPCR. (E) ASC^tot, CD105^- and CD105⁺ mASCs were starved for 24 hours and then stimulated with 10 ng/ml recombinant human TGF-β1 for 0, 2, 6 and 24 hours. At each time point total RNA was purified and reverse transcribed and the expression of IL-11 was measured using qPCR. Results are shown as mean (SEM) or two independent experiments. Expression values are plotted relative to ASC^tot 0h.</p

Murine MSCs are heterogeneous for CD105L expression.

<p>(A) Adipose tissue (subcutaneous and epididymal) from Balb/c mice were digested with collagenase type I for 30 minutes and the resulting cell suspension was stained for several cell surface markers before (day 0), 1, 2 and 6 days after plating on tissue-culture treated plastic and analyzed by flow cytometry. (B) ASC cultures were harvested with EDTA (2mM in PBS), TrypLE, or EDTA followed by treatment with collagenase type I or TrypLE followed by treatment with collagenase type 1 and analyzed by flow cytometry. The expression level (MFI) of each marker was normalized to the corresponding staining on EDTA-harvested cells. (C) Passage 4 mASCs were stained for a panel of surface antigens and analyzed by flow cytometry. Percentages of positive cells are represented as means (SEM) of 4 independent experiments. (D) ASCs from Balb/c and C57Bl/6 mice and BM-MSC from Balb/c mice were stained for CD105 and analyzed by flow cytometry. (E) Total RNA was purified from three independent mASC-cell preparations (passage 4-5), reverse transcribed and the expression of CD105L, CD105S and β-actin were analyzed by qPCR. (F) Total mASCs were cultured at either 5% O₂ or 21% O₂ for five passages and CD105 expression was analyzed for each passage by flow cytometry. (G) ASCs were cultured with or without TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 hours after which the cells were harvested and analyzed by flow cytometry for a panel of MSC markers. Results in A, B and F are shown as mean (SEM) of 3 independent experiments; in C and D a representative experiment is shown out of at least 4 independent experiments; data in G is represented as mean (SEM) of 2 independent experiments.</p