Macrophages (MФ) and mast cells are important cell types in the context of tissue remodeling and regeneration. Mast cells participate in the early stages of wound healing and modulate the acute inflammatory responses to biomaterials. Mast cells can secrete a myriad of different cytokines by the process of degranulation; the process of regulated secretion in which preformed contents stored in their granules are rapidly released by exocytosis. Some of these cytokines such as IL-4, IL-13 and TNF-α can modulate the MФ phenotype. Macrophages (MΦ) are innate immune cells, crucial for tissue homeostasis, presentation of foreign and self-antigens following infection/injury, pathogen clearance, inflammation resolution, angiogenesis, and wound healing. MΦ display plasticity and can acquire pro-inflammatory (M1) or angiogenic/wound healing (M2) phenotypes depending upon the environmental stimuli. The phenotypic profile of MФ as M1 or M2 following exposure to the biomaterial can dictate the downstream processes of tissue remodeling and angiogenesis. An analysis of how these two cell types interact with electrospun biomaterials and how different properties of an electrospun biomaterial impacts the MΦ phenotype is the focus of this thesis. Mast cells synthesize several potent angiogenic factors and can also stimulate fibroblasts, endothelial cells and macrophages. An understanding of how they participate in wound healing and angiogenesis is important to further our knowledge about in situ vascular prosthetic regeneration. The adhesion, proliferation and cytokine secretion of bone marrow derived murine mast cells (BMMC) on electrospun polydioxanone (PDO), polycaprolactone (PCL) and silk scaffolds, as well as tissue culture plastic (TCP) has been investigated in the presence or absence of IL-3, SCF, IgE and IgE with a crosslinking antigen, dinitrophenol-conjugated albumin (DNP). It was previously believed that only activated BMMCs exhibit adhesion and cytokine secretion. However, this study shows non-activated BMMC adhesion to electrospun scaffolds. Silk scaffold was not found to be conducive for mast cell adhesion and cytokine secretion. Activation by IgE and DNP significantly enhanced mast cell adhesion, proliferation, migration and secretion of TNF-α, MIP-1α and IL-13. This indicates that mast cells might play a role in MФ polarization (M1/M2), biomaterial integration into the host tissue, regeneration, and possibly angiogenesis. In the next study, bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) electrospun from varying polymer concentrations (60, 100, and 140 mg/ml). Scaffold evaluation showed that large polymer concentrations led to larger fiber diameters, which in turn led to larger pore-sizes and porosity but a smaller surface area to volume ratio. After 24 hrs of culture, the cell lysates were analyzed for Arginase (Arg1) and inducible nitric oxide synthase (iNOS) expression by western blot and cell culture supernatants were analyzed for Nitric oxide (NO2-), Tumor Necrosis Factor – alpha (TNF-α), Interleukin-6 (IL-6), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor – beta1 (TGF-β1) and basic fibroblast growth factor (bFGF) levels. The results indicated a correlation between Arg1 expression and increasing fiber/pore-size, indicating that the larger fiber/pore-sizes polarize towards a M2 phenotype. Also, the expression of iNOS was downregulated on the larger fiber/pore-size. The levels of NO2- were significantly higher on the lower fiber/pore-sizes indicating an M1 phenotype. The levels of VEGF, TGF-β1 and bFGF increased with increasing fiber/pore-sizes. The results showed higher Arg1 expression in M2s on the 60 mg/ml scaffold created by the air-flow impedance method compared to the 60 mg/ml scaffold created on the solid mandrel created by traditional electrospinning. The Arg1 expression was reduced on the compressed 140 mg/ml PDO scaffold compared to the normal 140 mg/ml scaffold. This result indicates that pore-size might be playing a greater role compared to fiber diameter in BMMФ phenotype modulation. In order to assess the angiogenic potential of BMMΦs cultured on PDO scaffolds, a 3D angiogenesis bead assay was performed using conditioned media from the BMMΦ:PDO interaction. The results of the 3D angiogenesis bead assay showed that the conditioned media from BMMΦs of M0 and M2 phenotypes cultured on the 140 mg/ml PDO scaffold induced larger sprouting and higher percentage density of sprouts when compared to the 60 mg/ml PDO scaffold and TCP. To investigate the signaling mechanism involved in this phenotypic switch, BMMΦs were isolated from the bone marrow of MyD88 knockout (KO) mice (Jackson Laboratories) and cultured on PDO (60 and 140 mg/ml) scaffolds (106 /disc) and TCP for 24 hrs and their Arg1 and iNOS expression was analyzed by western blot. The expression of Arg1 and iNOS was severely impaired on the BMMΦs from MyD88-/- mice cultured on the 140 mg/ml scaffold when compared to the 60 mg/ml PDO scaffold and TCP. This result indicates that scaffolds with different fiber/pore-sizes signal differently. A subcutaneous mouse model (described in Chapter 6) was used to evaluate the angiogenic and regenerative potential of PDO scaffolds in vivo. The DIVAA assay showed statistically higher FITC-dextran signal intensity for the 140 mg/ml scaffold compared to the 60 mg/ml scaffold indicating greater angiogenic response in the 140 mg/ml tube. However, problems of high background were observed in this assay with the use of electrospun PDO. The observed high background was probably due to the formation of complexes between dextran and adsorbed plasma proteins on the surface of the PDO. More studies are needed to optimize this assay for use with biomaterials such as PDO. H&E staining of the harvested PDO tubes (60 mg/ml and 140 mg/ml) was also performed. The cross-sections of these tubes showed greater cell recruitment and infiltration into the fibrous structures of the 140 mg/ml tube compared to the 60 mg/ml tube. This result corroborates the in vitro result of BMMФ infiltrating deeper into the structures of the 140 mg/ml scaffold compared to the 60 mg/ml scaffold. The scaffolds were also analyzed by immunostaining for iNOS (indicative of M1 phenotype of MФs). The results showed statistically higher ratios of iNOS positive:negative areas on the 60 mg/ml scaffold compared to the 140 mg/ml scaffold. Overall, these studies indicate that 140 mg/ml scaffold supports greater cell recruitment and cell infiltration in vivo but a smaller ratio of iNOS positive:negative areas compared to the 60 mg/ml scaffold, which supports a predominately M1 MФ phenotype. The studies indicate that varying properties of PDO can alter both the phenotype and function of BMMΦs in vitro and in vivo. We have also shown that the 140 mg/ml scaffold signal BMMΦs through MyD88-dependent mechanisms. A complete understanding of the way materials signal would allow us to control or modulate undesirable immune reactions to biomaterials in vivo. These studies would also help engineer biomaterials that promote angiogenesis and regeneration.

1 Abstract Vitamin D3 is important for keeping the right concetration of Ca2+ in plasma. Therefore it is essential for proper bone growth and development. Nevertheless, vitamin D3 has also a number of immunomodulating effects. Our thesis has been targeted on evaluation and comparison of vitamin D3 influence on expression of chosen surface markers (CD14, CD54, HLA-DR, CD16, CD36 and CD163) with THP-1 cells and monocytes gained from human peripheral blood. Other aims have been analysing the vitamin D3 influence on longevity of THP-1 cells and measuring the soluble CD14 and IL-8 production with THP-1 cells under the vitamin D3 influence. The cells have been stimulated with five different concentrations of vitamin D3 for the time 24, 48 and 72 hours. Higher used concetrations of vitamin D3, i.e. 100 nM and 1000 nM have increased the expression of CD14 with THP-1 cells in the time 48 and 72 hours of the stimulation time. With the monocytes from peripheral human blood the increase of the CD14 expression hasn't been remarkable from the physiological point of view. Together with the vitamin D3 concentration increase the sCD14 production with THP­1 cells was considerably higher. The sCD14 was the highest in the time 72 hours after the stimulation with the highest used vitamin D3 concetration. The IL-8 quantity with...

Monocytes and macrophages are important components of the innate immune response. These mononuclear phagocytes form a heterogeneous cell population, of which phenotype and functions can be modified under the influence of different signals coming from the surrounding microenvironment. The aim of this work was to modulate the phenotype of these cells by a variety of stimulants and to compare the changes induced on the model of THP-1 monocytic cell line and on the human peripheral blood monocytes. Surface marker expression was analyzed by flow cytometry. Further on, IL-8 production was evaluated by Luminex assay and the concentration of soluble calprotectin was assessed by ELISA. The most significant changes in surface marker expression were induced by exposure to IFNγ. This cytokine increased the expression of CD54, CD14 and HLA-DR on the surface of THP-1 cell line. Higher concentrations of IFNγ promoted higher apoptotic rate and augmented calprotectin expression and production in THP-1 cell line. On the surface of monocytes, IFNγ stimulation resulted only in the upregulation of CD54 expression. IL-4 increased the expression of CD36 by THP-1 cell line and inhibited the expression of CD163 by human monocytes. LPS stimulation caused the suppression of HLA-DR activation in monocytes and enhanced IL-8...

Macrophages are a natural target cell for HIV-1 infection, and they contribute to the development of disease as they are important for transmission, dissemination and persistence of the virus in an infected patient. Macrophages are less well-studied than T cells and cell lines in relation to HIV-1 infection, yet macrophages are highly specialised and key aspects of the HIV-1 life cycle in these cells are already known to differ compared to other cell types. HIV-1 entry into macrophages has been suggested to occur by macropinocytosis, however the entry route in these cells has not been fully characterised. In this thesis I have tested a panel of pharmacological inhibitors of cellular proteins and uptake pathways, in order to delineate the requirements for HIV-1 entry into macrophages and to determine the nature of the entry route. My findings suggest that the following host factors are important for entry; membrane cholesterol, actin rearrangements, dynamin, sodium-hydrogen exchange, Pak1, and Rac. Other factors including clathrin, PI-3 kinase, Rho kinase and some isoforms of PKC were found to be dispensable for infection or to inhibit infection. Macrophages are a heterogeneous group of cells, and tissue macrophages from different parts of the body differ in their morphology, phenotype and function. I have used the growth factors M-CSF and GM-CSF to direct monocytes to differentiate into distinct types of macrophage. This allowed me to determine that different macrophages differ in their susceptibility to infection and in their ability to support replication. This is likely to be due to variation in HIV-1 receptor expression and the levels of key HIV-1 transcription factors, respectively. Overall this thesis contributes to existing knowledge regarding HIV-1 infection of macrophages. These findings may assist with the design of entry inhibitors, and with therapies designed to eradicate HIV-1 from infected individuals.

As infecções do trato respiratório inferior lideram entre as principais causas de morbidade e mortalidade no mundo. Um dos grandes problemas associados ao tratamento das infecções do sistema respiratório, como as pneumonias, advém da crescente resistência aos mais modernos antibióticos adquirida pelos microrganismos. A terapia fotodinâmica, uma técnica baseada na interação da luz com uma substância fotoativa para causar dano oxidativo a células, tem se destacado como uma interessante alternativa para diversas doenças como diferentes tipos de câncer e infecções. Neste trabalho foi realizada, com experimentos in vitro, uma prova de princípio da possibilidade de inativar, com um protocolo eficiente e seguro, uma das bactérias mais comumente encontradas em quadros de pneumonia, a Streptococcus pneumoniae, com terapia fotodinâmica infravermelha mediada pela indocianina verde. Duas fontes de luz, uma a base de lasers emitindo 780 nm e outra construída com LEDs emitindo 850 nm, foram comparadas para avaliar sua eficiência. Experimentos com a bactéria foram realizados para determinação dos melhores parâmetros de inativação microbiana. Em seguida, ensaios de citotoxicidade foram feitos com macrófagos RAW 264.7 com o intuito de averiguar se as condições microbicidas não apresentavam atividade tóxica para células fagocitárias do sistema imune. Foi possível delinear os parâmetros de concentração de indocianina, tempo de incubação e dose de luz que apresentassem atividade microbicida e que não fossem tóxicas para as células. A interação da terapia fotodinâmica com a ação fagocitária dos macrófagos sobre as bactérias foi avaliada pelo estabelecimento de co-cultura dessas espécies. Concluiu-se que, utilizando-se LEDs de 850 nm fornecendo uma dose de luz de 10 J/cm2 as amostras contendo indocianina verde 5μM, é possível inativar S. pneumoniae de modo eficiente e auxiliar a ação fagocitária de macrófagos. / The lower respiratory tract infections lead among the main causes of morbidity and mortality worldwide. A major problem associated with respiratory tract infections, e.g. pneumonia, stems from from the increasingly resistance to most modern antibiotics developed by microorganisms. Photodynamic therapy, a technique based on the interaction of light and a photoactive substance to cause oxidative damage to cells, has emerged as an attractive alternative for several diseases such as different kinds of cancer and infections. In this work, with in vitro experiments, we accomplished a proof of concept for the possibility of inactivating, with an efficient and secure protocol, one of the most commonly found bacteria in pneumonia cases, Streptococcus pneumoniae, with infrared photodynamic therapy mediated by indocyanine green. Two light sources, one based on 780 nm lasers and the other built with 850 nm LEDs, were compared to evaluate their efficiency. Experiments with bacteria determined the best parameters microbial inactivation. Then, cytotoxicity assays with RAW 264.7 macrophages analyzed if the microbicidal parameters had toxic effects on immune cells. It was possible to delineate the indocyanine concentration parameters, incubation time and dose of light to obtain microbicidal results that weren´t toxic to the cells. Interaction of photodynamic therapy with the phagocytic action of macrophages on the bacteria was assessed by establishing a co-culture with these species. We concluded that, using 850 nm LEDs providing a light dose of 10 J/cm2 to samples containing 5μM indocyanine green, it is possible to inactivate S. pneumoniae and efficiently assist the phagocytic action of macrophages.

O exercício físico regular contribui para prevenção e redução da aterosclerose, em grande parte, por melhorar o perfil lipídico e o transporte reverso de colesterol (TRC). O TRC é um sistema antiaterogênico que promove a remoção do excesso de colesterol de macrófagos pelas apo A-I e HDL e seu transporte ao fígado, com eliminação de colesterol na bile e fezes. Em camundongos selvagens e transgênicos para proteína de transferência de colesterol esterificado (CETP-tg), o treinamento físico aumentou a transferência de colesterol radioativo de macrófagos para o plasma, fígado e fezes e o conteúdo dos receptores B-E e SR-BI no fígado. Em leucócitos, hepatócitos e enterócitos, o exercício físico aumentou a expressão do receptor de HDL, ABCA-1. Entretanto, não é claro se o exercício físico modula o fluxo de lípides em macrófagos, o que seria determinante para a primeira etapa do TRC. Assim sendo, avaliou-se, em camundongos selvagens e CETP-tg, o efeito do treinamento físico aeróbio sobre: 1) a expressão de genes envolvidos no fluxo de lípides, modulação da resposta inflamatória, vasodilatadora e antioxidante: Pparg (PPAR?), Nr1h3 (LXRalfa), Nr1h2 (LXRbeta), Abca1 (ABCA-1), Abcg1 (ABCG-1), Scarb1 (SR-BI), Cd36 (CD-36), Olr1 (LOX-1), Ccl2 (MCP-1), Tnf (TNFalfa), Il6 (IL-6), Il10 (IL10), Nos3 (eNOS) e Cat (Catalase) na parede arterial e em macrófagos peritoneais; 2) o efluxo de 14C-colesterol de macrófagos peritoneais para HDL2 e apo A-I e 3) a captação de 3H-colesteril oleoil éter-LDL (3H-COE-LDL) acetilada por macrófagos peritoneais. Camundongos machos com 12 semanas de idade, recebendo dieta padrão e água ad libitum, foram aleatoriamente divididos em grupo sedentário e treinado. O treinamento físico foi realizado em esteira, 15m/min, 30 min/dia, 5 vezes/semana, durante 6 semanas. Arco aórtico e macrófagos da cavidade peritoneal foram isolados dos animais sedentários e treinados, imediatamente (0 h) e após 48 h da última sessão de exercício. A expressão de genes foi avaliada por RT-PCR e o efluxo de colesterol, por meio da sobrecarga de macrófagos peritoneais com LDL acetilada e 14C-colesterol, seguindo-se incubação com apo A-I ou HDL2. A captação de LDL foi determinada pela incubação de macrófagos com 3H-COE-LDL acetilada. Não foram observadas alterações sistemáticas na expressão de genes envolvidos no fluxo de lípides em macrófagos e na aorta comparando-se animais sedentários e treinados, selvagens ou CETP-tg. De modo semelhante, não houve diferença no efluxo de colesterol celular e na captação de LDL. Em conclusão, não foram evidenciadas alterações em macrófagos peritoneais e na parede arterial frente ao treinamento físico aeróbio que possam contribuir para o TRC em modelo experimental de camundongos não dislipidêmicos, sem intervenção farmacológica ou alimentar. Sendo assim, o efeito do treinamento físico na melhora do transporte reverso de colesterol, observado em estudos anteriores, deve ser consequente à sua ação sistêmica sobre mediadores deste transporte e expressão de receptores no fígado e intestino / Regular physical exercise prevents and reduces atherosclerosis mainly by improving lipid profile and reverse cholesterol transport (RCT). RCT is an antiatherogenic system that promotes excess cholesterol removal from macrophages by apo A-I and HDL and its transport to the liver. Then, cholesterol can be secreted into bile and excreted in feces. In wild type and cholesteryl ester transfer protein transgenic (CETP-tg) mice exercise training increased the transfer of 14C-cholesterol from macrophages to plasma, liver and feces and elevated SR-BI and B-E receptor content in the liver. In leucocytes, hepatocytes and enterocytes, physical exercise increased mRNA of HDL receptor, ABCA-1. Nonetheless, it is not clear if exercise can modulate lipid flux in macrophages that can be important for the first phase of the RCT. It was analyzed in wild type and CETP-tg mice the effect of aerobic exercise training in: 1) the expression of genes involved in lipid flux, inflammation, oxidation and vasodilation: Pparg (PPAR?), Nr1h3 (LXRalfa), Nr1h2 (LXRbeta), Abca1 (ABCA-1), Abcg1 (ABCG-1), Scarb1 (SR-BI), Cd36 (CD-36), Olr1 (LOX-1), Ccl2 (MCP-1), Tnf (TNFalfa), Il6 (IL-6), Il10 (IL10), Nos3 (eNOS) and Cat (Catalase) in arterial wall and peritoneal macrophages; 2) the apo A-I and HDL2-mediated cholesterol efflux from macrophages and 3) the uptake of 3H-cholesteryl oleoyl ether- acetylated LDL (3H-COE-LDL) by macrophages. Twelve week old male mice fed a chow diet and water ad libitum were randomly assigned to sedentary and trained groups. Exercise training was performed in a treadmill (15m/min, 30 min/day, 5 times/week, during 6 weeks). Aortic arch and peritoneal macrophages were isolated from sedentary and trained animals immediately (time 0) and 48 h after the last exercise session. Gene expression was analyzed by RT-PCR and cholesterol efflux mediated by apo A-I or HDL2 after macrophage overloading with acetylated LDL and 14C-cholesterol. LDL uptake by macrophages was determined by incubation with 3H-COE-acetylated LDL. There were no systematic changes in the expression of macrophages and aortic genes comparing sedentary and trained wild type or CETP-tg mice. Similarly, there were no changes in cholesterol efflux and LDL uptake by macrophages. In conclusion, it was not found alteration in gene expression and cholesterol flux in macrophages and arterial wall that can contribute to the RCT in experimental model of non-dyslipidemic mice without pharmacological or dietary interventions. Therefore, the benefits of aerobic training in improving RCT, observed in previews studies, should be consequent to its systemic action on mediators of this transport and on the expression of hepatic and intestinal receptors

Crotalfina (CRF) é um peptídeo produzido do veneno da serpente Crotalus durissus terrificus com efeito antinociceptivo tipo opióide detectado apenas na presença de inflamação. Estudos sobre mecanismos de analgesia relatam a ação de opióides em células imunes e macrófagos são apontados como alvos para os efeitos destes. Este estudo caracterizou a ação da CRF sobre funções de macrófagos e avaliou a importância de seus receptores opióides nestas ações. Os resultados indicam que CRF inibe fagocitose, liberação de H2O2 e produção de NO• e TNF-a, modula IL-6 e estimula IL-1b por macrófagos residentes e inflamatórios. Ensaios de expressão e ativação mostram que macrófagos residentes expressam receptores opióides k seguido de m e d e macrófagos inflamatórios expressam receptores m seguido de k e d. A CRF interferiu, de maneira distinta, com a expressão destes receptores, dependendo do estado de ativação destas células. Antagonistas destes receptores bloquearam a ação da CRF, mostrando que este peptídeo possui ação imunomodulatória mediada por receptores opióides. / Crotalphine (CRF) is a peptide isolated from the venom of Crotalus durissus terrificus with type opioid antinociceptive effect only detected in the presence of inflammation. Studies on mechanisms of analgesia reported the action of opioids in immune cells and macrophages are considered as targets for these effects. This study characterized the action of CRF on functions of macrophages and assessed the importance of opioid receptors in these actions. The results indicate that CRF inhibits phagocytosis, release of H2O2 and production of NO• and TNF-a, modulates IL-6 and stimulates IL-1b production by resident and inflammatory macrophages. Expression and activation assays show that resident macrophages express k receptors followed by m and d. Inflammatory macrophages express m receptors followed by k and d. CRF interfered differently with the expression of these receptors, depending on the activation state of these cells. Antagonists of these receptors blocked the action of CRF, showing that this peptide has immunomodulatory action mediated by opioid receptors.

O gene Slc11a1 regula a resistência contra S. entérica, L. donovani e M. tuberculosis. Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R e S do gene Slc11a1 (AIRmax,RR, AIRmaxSS, AIRminRR e AIRminSS) foram utilizadas para avaliar o efeito destes alelos na ativação de macrófagos peritoneais (M<font face=\"Symbol\">F), induzida pelo tioglicolato (TIO) ou infecção por M. bovis BCG.O TIO induz baixa ativação celular, a estimulação com LPS aumentou a ativação nos macrófagos dos animais AIRmaxRR e AIRmaxSS. Assim, na inflamação com TIO, a ativação do M<font face=\"Symbol\">F foi dependente do fundo genético selecionado nos animais AIRmax, independente do alelo do gene Slc11a1. Na infecção com BCG, a sublinhagem AIRmaxRR foi a única capaz de controlar a proliferação da bactéria, secretando altos níveis de IL-1<font face=\"Symbol\">b, IL-12, TNF<font face=\"Symbol\">a e IL-6 que ativam o macrófago. Por outro lado, os AIRminSS susceptíveis à infecção produziram maiores concentrações de NO, H2O2 e IL-10, mostrando o fundo genético para alta ou baixa resposta inflamatória, e os alelos do gene Slc11a1 interferem na resistência à infecção. / The Slc11a1 gene regulates resistance against S. enterica, L. donovani and M. tuberculosis. AIRmax and AIRmin mouse sublines, homozygous for Slc11a1 R and S alleles (AIRmaxRR, AIRmaxSS, AIRminRR and AIRminSS) were used in this work to evaluate the effect of this gene in peritoneal macrophage (M<font face=\"Symbol\">F) activation induced by thioglycollate (TIO) or M. bovis BCG infection. TIO induced weak cellular activation, LPS stimulation increased AIRmaxRR and AIRmaxSS macrophages activation. These results indicate that inflammation and M<font face=\"Symbol\">F activation induced by TIO were dependent on the genetic background for acute inflammatory response, while Slc11a1 alleles have little effect on this phenotype. In BCG infection only AIRmaxRR mice were capable of controlling bacterial proliferation, which was accompanied with high levels of IL-1<font face=\"Symbol\">b, IL-12, TNF and IL-6 produced by activated macrophages. On the other hand, susceptible AIRminSS mice produced higher amounts of NO, H2O2 and IL-10 suggesting that both inflammatory background and Slc11a1 alleles interfere on resistance to BCG infection.

The alveolar bone repair process may be influenced by several local and systemic factors that include mediators and immune system cells. Among these cells, macrophages are essential to trigger the repair process, and may acquire an inflammatory (M1) or anti-inflammatory and pro-reparative profile (M2). In this context, we evaluated the effects of FTY720 on macrophage polarization towards the M2 profile and its effects on the alveolar bone repair process. In this study, we used 8 weeks old male C57BL / 6 mice (N = 5 / time / group). The animals were divided in FTY720 group receiving the drug orally at a dose of 3mg / kg / 24h during the whole experimental period, and the control group receiving only the equivalent vehicle. All animals were submitted to extraction of the right upper incisor and were evaluated at 0, 1, 3, 7 and 14 days after extraction, followed by computed tomography (CT), histomorphometry, birefringence, immunohistochemical and molecular analyzes (PCRArray). Our results demonstrated that in the 14-day period, the FTY720 group presented higher bone tissue density, higher bone tissue volume (BV), greater tissue volume fraction (BV / TV), greater number and thickness of trabeculae (Tb.1 and Tb.Th, respectively) (p<0.05). In the 14-day period, the FTY720 group had a higher number of osteoblasts and osteoclasts than the control group (p<0.05). Accordingly, the expression of various bone markers such as BMP2, BMP7, ALPL, SOST and RANK had their mRNA expressions increased in the FTY720 group. This increase may be related to the potentiation in the formation of the bone tissue compared to the control group. The levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206 + cells in the 14 days period, suggesting a participation of M2 macrophages in the potentiation of the alveolar bone repair process. The FTY720 group also showed increased expression levels of CCR2, CCR5, CXCR1, CXCL1, CXCL3, CCL20 and CCL25 mRNA, chemokines and chemokine receptors involved in the recruitment of inflammatory cells and undifferentiated mesenchymal cells (MSCs) most notably was the up CXCL12 up regulation (p<0.05). CXCL12 is responsible in the recruitment of MSCs to the repair site. The increase in CXCL12 expression was accompanied by an increase in CD34 expression over a period of 14 days (p<0.05), indicating a higher presence of MSCs in the repair site. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair in C57BL / 6 mice, possibly because it increased the expression of markers related to bone tissue development (ALPL, SOST, RANK), tissue repair (CXCL12, CD34) and inflammatory cells (CCR2, CCR5) and apparently in the induction of macrophages to an M2 profile (ARG2, FIZZ). / O processo de reparo ósseo alveolar pode ser influenciado por vários fatores locais e sistêmicos que incluem mediadores e células do sistema imunológico. Dentre essas células, os macrófagos são essenciais para desencadear o processo de reparo, podendo adquirir um perfil inflamatório (M1) ou anti-inflamatório e pró-reparativo (M2). Nesse contexto, avaliamos os efeitos do FTY720 na polarização de macrófagos para o perfil M2 e seus efeitos no processo de reparo ósseo alveolar. Nesse estudo foram utilizados camundongos C57BL/6 (N=5/tempo/grupo), machos, com 8 semanas de idade. Os animais foram divididos em grupo que recebeu o fármaco FTY720 via oral, na dosagem de 3mg/Kg/24h, durante todo o período experimental, e grupo controle que recebeu apenas o veículo em regime equivalente. Todos animais foram submetidos à extração do incisivo superior direito e avaliados nos períodos de 0, 1, 3, 7 e 14 dias pós extração, seguido por análises de tomografia computadorizada (CT), histomorfométrica, birrefringência, imuno-histoquímica e molecular (PCRArray). Nossos resultados demonstraram que no período de 14 dias, o grupo FTY720 apresentou maior densidade de tecido ósseo, maior volume de tecido ósseo (B.V), maior volume de fração de tecido ósseo pelo tecido total (BV/TV), maior número e espessura de trabéculas (Tb.1 e Tb.Th, respectivamente) (p<0.05). Ainda no período de 14 dias, o grupo FTY720 apresentou maior número de osteoblastos e osteoclastos em relação ao grupo controle (p<0.05). Em concordância, a expressão de vários marcadores de tecido ósseo como, BMP2, BMP7, ALPL, SOST e RANK, tiveram suas expressões de mRNA aumentadas no grupo FTY720. Esse aumento pode estar relacionado com a potencialização na formação do tecido ósseo comparado ao grupo controle. Os níveis de mRNA de FIZZ, ARG2 e IL-10, sofreram aumento no grupo FTY720 em conjunto com a presença de células CD206+ no período de 14 dias, podendo sugerir uma participação dos macrófagos M2 na potencialização do processo de reparo ósseo alveolar. O grupo FTY720 também mostrou aumento nos níveis de expressão de mRNA de CCR2, CCR5, CXCR1, CXCL3, CCL20 e CCL25, quimicionas e receptores de quimiocinas envolvidos no recrutamento de células inflamatórias e células mesenquimais indiferenciadas (MSCs) com destaque para o aumento da expressão de CXCL12 (p<0.05), quimiocina responsável no recrutamento de MSCs para o local de reparo. O aumento na expressão de CXCL12 foi acompanhado pelo aumento na expressão de CD34 no período de 14 dias (p<0.05) podendo indicar maior presença de MSCs no sitio de reparo. Assim, os nossos resultados demonstram que o FTY720 favoreceu o processo de reparo ósseo alveolar em camundongos C57BL/6, possivelmente por ter aumentado a expressão de marcadores relacionados com o desenvolvimento do tecido ósseo(ALPL, SOST, RANK), no reparo tecidual (TGF-1, IL-10), no recrutamento de células indiferenciadas (CXCL12, CD34) e células inflamatórias (CCR2, CCR5) e aparentemente na indução de macrófagos para um perfil M2 (ARG2, FIZZ).

L'insuffisance cardiaque consécutive à un infarctus du myocarde est une pathologie complexe qui apparaît suite au remodelage du ventricule gauche. Le renouvellement et la dégradation de la matrice extracellulaire cardiaque et l'inflammation sont impliqués dans la mise en place du remodelage post infarctus du myocarde. La métalloprotéinase matricielle 9 (MMP9) et le récepteur de l'immunité innée Toll-like 4 (TLR4) sont des médiateurs clés du remodelage ventriculaire. L'adénosine est un nucléoside cardioprotecteur et anti-inflammatoire dont les effets sur le remodelage sont encore mal caractérisés. Nous avons émis l'hypothèse que l'adénosine pourrait moduler les voies de la MMP9 et du TLR4 dans les macrophages, et ainsi réguler le remodelage ventriculaire. Nos résultats montrent que l'activation du récepteur A3 à l'adénosine augmente la production de MMP9 par les macrophages primaires humains. D'autre part, nous avons observé que l'adénosine réduit l'inflammation par une diminution de l'expression de surface du TLR4. Cet effet se traduit par une inhibition de la synthèse de cytokines pro-inflammatoires et est induit par l'activation du récepteur A2A. Ces résultats ont permis de caractériser certains mécanismes par lesquels l'adénosine agit sur le remodelage ventriculaire. Ils suggèrent de tester, dans un modèle animal, l'administration d'analogues de l'adénosine pour prévenir ou limiter le remodelage. La capacité à prédire la survenue du remodelage ventriculaire après un infarctus du myocarde est importante d'un point de vue clinique et bénéficierait de la découverte de nouveaux biomarqueurs. L'analyse par protéomique du plasma de 30 patients d'une cohorte test ayant développé un infarctus du myocarde a permis d'identifier l'haptoglobine comme biomarqueur pronostique potentiel. L'haptoglobine humaine possède 3 isoformes : [alpha]1-[alpha]1, [alpha]2-[alpha]1 et [alpha]2-[alpha]2. Nos résultats suggèrent que la présence du phénotype [alpha]2 et d'un taux plasmatique d'haptoglobine faible sont associés à l'apparition d'une insuffisance cardiaque. Cette étude preuve-du-concept suggère que l'haptoglobine pourrait être ajoutée à la liste existante des biomarqueurs de l'insuffisance cardiaque / Heart failure following myocardial infarction is a complex pathology that occurs as a result of left ventricular remodeling. Left ventricular remodeling is mediated in part by extracellular cardiac matrix turnover and inflammation. Matrix metalloproteinase 9 (MMP9) and innate immune receptor Toll-like 4 (TLR4) are key mediators of left ventricular remodeling. Cardioprotective and anti-inflammatory nucleoside adenosine acts on left ventricular remodeling through still poorly characterized mechanisms. We hypothesized that adenosine regulates left ventricular remodeling through modulation of MMP9 and TLR4 pathways in macrophages. Using human primary macrophages, we showed that adenosine increases MMP9 production through the A3 receptor. In a second set of experiments, we observed that adenosine dampens inflammation through decreased cell-surface expression of TLR4. This effect, which inhibits pro-inflammatory cytokines production, is induced by adenosine A2A receptor. All together, these results contribute to a better understanding of the mechanisms underlying left ventricular remodeling and suggest a potential therapeutic use of adenosine. Our data suggest testing a therapeutic strategy with different adenosine analogs to prevent or limit left ventricular remodeling. Prediction of left ventricular remodeling after myocardial infarction is clinically relevant and would benefit from the discovery of new biomarkers. Proteomic analysis of plasma samples from a test cohort of 30 myocardial infarction patients identified haptoglobin as a potential prognostic biomarker. Human haptoglobin has 3 distinct isoforms: [alpha]1-[alpha]1, [alpha]2-[alpha]1 and [alpha]2-[alpha]2. Our results suggest that the presence of [alpha]2 isoform, together with low plasma levels of total haptoglobin, is associated with the development of heart failure. This proof-of-concept study suggested that haptoglobin could be added to the panel of existing biomarkers of heart failure