CHEMOKINES AND TRAFFICKING (CT)

CC CHEMOKINES SIGNALLING PLAYS A PROTECTIVE ROLE DURING EXPERIMENTAL SEPSIS VIA CCR5

FERNANDA VARGAS E SILVA CASTANHEIRA; FABIANE SÔNEGO; ALEXANDRE KANASHIRO; PAULA GISELE CZAIKOSKI; THIAGO MATTAR CUNHA; JOSÉ CARLOS ALVES-FILHO; FERNANDO QUEIRÓZ CUNHA. UNIVERSITY OF SÃO PAULO, RIBEIRÃO PRETO - SP - BRASIL.

Introduction: Sepsis is a systemic inflammatory response resulted from the inability of the innate immune system to control a microorganism infection. The survival rate during sepsis is dependent on the recruitment of neutrophils to the infection site. Neutrophils from naïve mice express the CXCR family of chemokine receptors, but they do not express the CCR family of receptors. However, it has been showed that chemokine receptors expression profile can be altered on neutrophils under sepsis conditions. Data from our laboratory show that CXCR2 expression is down regulated, impairing the neutrophil migration to infection focus. By contrast, CCR2 appears on the neutrophils surface, mediating the accumulation of these cells in secondary organs. Thus, the aim of this study was to investigate the possible expression of CCR5 receptor on neutrophils and its role on sepsis evolution. Methods and Results:C57BL/6 and CCR5 deficient mice (CCR5

-/-) were used to induce sepsis using cecal ligation and puncture (CLP) model. We showed

that neutrophils from naïve C57BL/6 mice express high levels of CXCR2 and low levels of CCR5 (n=5). However, after CLP, in parallel with CXCR2 internalization, neutrophils express higher levels of CCR5 (n=5). Furthermore, neutrophils expressing CCR5 were negative for annexin 5, indicating that CCR5 expression is not related to neutrophil apoptosis during sepsis (n=3). Interestingly, CCR5

-/- mice subject to CLP show decreased survival rate, reduced neutrophil

migration to the infection site, increased neutrophil infiltration in lung and increased levels of markers of injuries, when compared to C57BL/6 mice (n=5). In addition, the incubation of bone marrow derived-neutrophils with LPS enhances the expression of CCR5 and renders them responsive to CCL4-induced chemotaxis, in a NF-kB dependent mechanism. Moreover, we demonstrated that CCR5 has an important role during neutrophil adhesion to the vascular endothelium before transmigration (n=5). Conclusion: Together, these results indicate that during CLP-induced sepsis, the increase of the expression of CCR5 on neutrophils plays a host protective role, contributing to the emigration of these cells to infection focus, which control the infection locally.

Financial support: CNPq, FAPESP, CAPES, FAEPA

CCR3-DEPENDENT EOSINOPHIL RECRUITMENT IS REGULATED BY SIALYLTRANSFERASE ST3GAL-IV MARKUS SPERANDIO

1; DAVID FROMMHOLD

2; ALEXANDER DOERNER

3; JANINE ABISCH

4; SARAH SCHMIDT

5;

SILKE HUBER6; DAVID VOEHRINGER

7; MARCUS MALL

8; JAMEY D MARTH

9.

1,4,5,6.LUDWIG MAXIMILIANS UNIVERSITY, MUNICH - ALEMANHA; 2,3,8.UNIVERSITÄT HEIDELBERG, HEIDELBERG - ALEMANHA; 7.UNIVERSITAET ERLANGEN, ERLANGEN - ALEMANHA; 9.SANFORD BURNHAM MEDICAL RESEARCH INSTITUTE, SANTA BARBARA - ESTADOS UNIDOS.

Introduction: Sialylated proteins play an important role during leukocyte recruitment into inflamed tissue. This has been demonstrated recently using St3gal4 deficient mice where CXCR-2 mediated firm neutrophil arrest and extravasation were dramatically impaired (Frommhold et al., J.Exp.Med 2008).

Methods and Results: To investigate whether ST3Gal-IV contributes to eosinophil trafficking, we studied eosinophil recruitment into inflamed tissue in the absence of ST3Gal-IV. Eosinophil recruitment in St3gal4-deficient mice was examined using various in vivo models of eosinophilic inflammation: adhesion and extravasation of St3gal4-deficient eosinophils were significantly reduced in CCL11-induced inflammation of the cremaster muscle compared to control mice. In the 24-hour thioglycollate-induced peritonitis model, we found a marked reduction of eosinophil transmigration into the peritoneal cavity in the absence of ST3Gal-IV. In an ovalbumin induced asthma model, we observed a significant reduction in eosinophil migration into the alveolar space in St3gal4

-/- mice. Finally, eosinophil adhesion in

flow chambers coated with P-selectin, VCAM-1, and CCL11 was significantly reduced in the absence of ST3Gal-IV.

Conclusions: These findings show that ST3Gal-IV-dependent sialylation is crucial for eosinophil recruitment during inflammation in vivo. ST3Gal-IV might therefore be an interesting target to limit unwanted eosinophil recruitment in disorders by unwanted eosinophil infiltration.

Supported by Mizutani Grant Ref. Nr. 090063 and FöFoLe #42.

CO-REGULATION BETWEEN LEUKOTRIENE B4 AND CCL2 GENERATION ENHANCES GAMMA DELTA T LYMPHOCYTE MIGRATION DURING INFLAMMATION

CATARINA BASTOS TRIGO DE NEGREIROS

1; MARIA FERNANDA DE SOUZA COSTA

2; RAQUEL DE SOUZA

MARTINS3; THADEU MARAMALDO COSTA

4; LAYSSA ZEITUNE

5; CLAUDIA FARIAS BENJAMIM

6; MARIA DAS

GRAÇAS HENRIQUES7; CLAUDIO DE AZEVEDO CANETTI

8; CARMEN PENIDO

9.

1,2,3,4,5,7.LABORATÓRIO DE FARMACOLOGIA APLICADA - FARMANGUINHOS - FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL; 6.LABORATÓRIO DE INFLAMAÇÃO E CÂNCER - UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL; 8.LABORATÓRIO DE INFLAMAÇÃO, PROGRAMA DE IMUNOLOGIA - UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL; 9.CENTRO DE DESENVOLVIMENTO TECNOLÓGICO EM SAÚDE - FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL.

Introduction: Gamma delta T lymphocytes accumulate in injured tissue and play important roles in immune surveillance. Gamma delta T cell trafficking into sites of inflammation is mainly orchestrated by CCL2 and leukotriene (LT)B4. It has been shown that CCL2 regulates LTB4 synthesis and vice versa. Here, we have investigated the cooperation between CCL2 and LTB4 during LPS-induced inflammation and have examined its impact on gamma delta T cell migration.

Methods and Results: The intra-pleural (i.pl.) injection of LTB4 (0.5 μg/cav) triggered CCL2 production in mouse pleura (SAL 83.6±11.4; LTB4 188.5±45.0 pg/ml, p<0.05, n=8). CCL2 i.pl. stimulus (500 ng/cav) induced LTB4 production (SAL 4.1±0.8; CCL2 17.9±4.1 pg/ml, p<0.05, n=8). During LPS-induced inflammation (250 ng/cav.; i.pl.), BLT1 antagonism by CP105,696 (4 mg/kg; i.p.) diminished CCL2 production (SAL 137.0±10.0; LPS 329.0±105.0; LPS+CP 168.0±54.0 pg/ml, p<0.05, n=8). CCL2 neutralization (anti-CCL2 mAb 10 mg/mouse; i.p.) also impaired LTB4 generation (SAL 6.6±1.2; LPS 24.6±3.1; LPS+anti-CCL2 11.2±4.1 pg/ml, p<0.05, n=8). The i.pl. injection of both mediators induced gamma delta T cell influx (SAL 3.2±0.6; LTB4 7.8±2.1; CCL2 117.0±38.8 x10

3cells/cav, p<0.05, n=8); however LTB4 i.pl. injection into CCR2 KO mice induced a discrete increase in gamma

delta T cell numbers (SAL 3.1±0.6; LTB44.9±0.8 x103cells/cav, p<0.05, n=6). CCL2 i.pl. injection also failed to induce

gamma delta T cell influx into 5-lipoxygenase (5-LO) KO mouse pleura (SAL/WT 23.8±6.9; CCL2/WT 117.0±38.8; SAL/KO 46.5±4.1; CCL2/KO 40.4±8.3 x10

3cells/cav, p<0.05, n≥6). BLT1 and CCR2 expression on gamma delta T

cells was upregulated in vitro under anti-CD3 mAb (5 mg/ml) stimulation (RPMI 10.2±0.3 vs anti-CD3 17.9±0.1% BLT1

+ gamma delta T cells; RPMI 6.2±0.2 vs anti-CD3: 17.5±0.7 % CCR2

+ gamma delta T cells, p<0.05, n=8). LPS

i.pl. stimulation induced increased numbers of BLT1+, CCR2

+ and BLT1

+/CCR2

+ gamma delta T cells in mouse pleura

(SAL 0.5±0.0 vs LPS 2.9±0.4 x103 BLT1

+cells/cav; SAL 0.3±0.0 vs LPS 3.6±0.4 x10

3 CCR2

+cells/cav; SAL 0.8±0.2 vs

LPS 3.1±0.6 x103 BLT1

+/CCR2

+cells/cav, p<0.05, n=8). In vitro stimulation of gamma delta T cells with sub-optimal

concentrations of LTB4 plus CCL2 (0.1 nM - 1 nM) induced intracellular calcium influx.

Conclusion: LTB4 and CCL2 reciprocally upregulate each other's production and synergize to mediate gamma

delta T cell activation and migration.

Financial Support: FAPERJ, CNPq, CAPES, FIOCRUZ

CRITICAL ROLE FOR CXCR7 IN CONTROLLING INFLAMMATION AND ANGIOGENESIS DURING SKIN WOUND

HEALING IN MICE

CAMILA PEREIRA ALMEIDA (IC)1; LEANDRO CEOTTO FREITAS-LIMA

2; LUIZA DIAS DA CUNHA LIMA (PG)

3;

MARIA CECILIA CAMPOS CANESSO (IC)4; ANGELICA THOMAZ VIEIRA

5; ROBSON AUGUSTO SOUZA DOS

SANTOS6; MAURO MARTINS TEIXEIRA

7; CHARLES MACKAY

8; LUCIOLA DA SILVA BARCELOS

9.

1,2,3,4,5,6,7,9.UFMG, BELO HORIZONTE - MG - BRASIL; 8.MONASH UNIVERSITY, CLAYTON - AUSTRÁLIA.

Introduction: The CXCL12/CXCR4 axis plays an important role in wound healing, especially by recruiting inflammatory cells and stimulating angiogenesis. CXCR7 was recently identified as a second high-affinity receptor for CXCL12, but its role in wound healing is unknown. In this study, we investigated the role of CXCR7 in cutaneous wound healing.

Methods and Results: 8-10 weeks old C57Bl/6 mice received either IgG2 isotype control antibody or anti-CXCR7 antibody (1.7 mg/mL, i.p.). Treatments started 24 h before the creation of excisional wounds on the dorsum of animals and were repeated once a week. Wound area was measured with a digital caliper and the results were expressed as percentage closure to original size. Wound blood flow was evaluated by laser Doppler perfusion imaging. Animals were euthanized at varied time-points and wounds collected for analysis (n=5-7 animals/group). Myeloperoxidase and N-acetyl-b-D-glucosaminidase activities were used to assess accumulation of neutrophils and macrophages, respectively. Cytokines were quantified by ELISA. Anti-CXCR7-treated animals showed a slower wound closure rate compared to the control group (day 7: Control (C) 70±7% vs anti-CXCR7 antibody (A) 43±8%*; day 14: C 98±2 vs A 90±3%*, p<0.05). Interestingly, we observed a marked increase in the content of neutrophils (day 3: C 0.3±0.03 vs A 0.7±0.1*; day 7: C 0.1±0.03 vs A 0.6 ± 0.2***, p<0.05 and p<0.001, respectively) and macrophages (day 7: C 4.1±0.3 vs A 5.6±0.3*; day 14: C 4.3±0.5 vs A 6±0.3*, p<0.05) in the wounds of treated mice. Moreover, we found increased wound levels of cytokines such as CXCL12 (p<0.01, at days 7 and 14 post-wounding), TNF-α (p<0.01, at days 7 and 14), TGF-β1 (p<0.01, at days 7 and 14) and IL-10 (p<0.01, at days 3, 7 and 14) in treated animals. Regarding vascularization, we observed a reduced blood supply in wounded area (day 7: C 228±14 vs A 168±11 perfusion units, p<0.01) as well as reduced vascular area (day 7: C 3.4±0.6 vs A 1.9±0.2 mm

2, p<0.05) in wounds from anti-CXCR7-

treated mice. Finally, scar tissue area at day 14 post-injury was increased in anti-CXCR7-treated mice skin compared to control (p<0.05).

Conclusion: Our data suggests the blockade of CXCR7 delays the closure of skin wounds. This is due to the accumulation of cells and chronic inflammation mediators in wounds. Thus, CXCR7 receptor seems to have a critical role in wound healing.

Financial suport: FAPEMIG, CNPq, CAPES, NHMRC-Australia, INCT-Nanobiofar.

DEFICIENCY OF THE ATYPICAL CHEMOKINE RECEPTOR D6 PROTECTS MICE FROM LETHALITY INDUCED

BY INFLUENZA A VIRUS (IAV) INFECTION THROUGH INCREASED LYMPHOCYTE INFLUX AND ATTENUATED VIRAL BURDEN

ARIANE KARLA CRUZ GOMES

1; CRISTIANA COUTO GARCIA

2; GABRIEL AUGUSTO OLIVEIRA

3; BRAULIO

FREIRE LIMA4; ANDIARA CARDOSO PEIXOTO

5; ALEXANDRE MAGALHAES MACHADO

6; DANILO BRETAS

OLIVEIRA7; GABRIEL FREITAS ALMEIDA

8; ALBERTO MANTOVANI

9; MASSIMO LOCATI

10; MAURO MARTINS

TEIXEIRA11

; REMO CASTRO RUSSO12

. 1,2,3,4,5,7,8,11,12.FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 6.RENE RACHOU RESEARCH CENTER - OSWALDO CRUZ FOUNDATION, BELO HORIZONTE - MG - BRASIL; 9,10.HUMANITAS CLINICAL AND RESEARCH CENTER, MILAN - ITÁLIA.

Introduction: Influenza A virus (IAV) infection has major relevance in worldwide epidemic episodes being responsible for several cases of respiratory diseases and the recent severe pandemic H1N1. Once infected, the immune response of the subject is essential for overcome pathogen proliferation, but in many cases an unbalanced immune response leads to lung damage, which may result in lethality due to fibrosis. The D6 receptor has an atypical scavenger function, being able to selectively bind and degrade various inflammatory CC chemokines. Recent literature has shown that this receptor also has controversial action in organ-specific diseases. This study aimed to evaluate the role of the receptor D6 during IAV infection.Methods and Results:D6 deficient (D6 -/-)(n=7) and Wild Type (WT)(n=7) mice were instilled intranasally with 10

4 PFU of WSN/33 virus or instilled with PBS only (Mock)(n=7) and weight loss

and lethality were checked daily for 21 days. The infection of WT mice with WSN/33 virus led to 30% of death and weight loss, which was not completely recovered at the end of the experiment. However the D6 -/- mice were protected, showing 100% survival and weight recovered ( p=0.0559). We found reduced viral burden in D6-/- mice but not in WT mice (WT:7.28±1.74;D6: 2.3±0.77). D6-/- mice presented increased total cell number in BAL compared to WT mice (WT:7.75±0.63; D6:10.75±0.65), caused by substantially increased lymphocyte into the airways (WT:0.55±0.07; D6: 1.93±0.83). We also have found higher levels of IFN-γ (WT:328.3±105.5;D6:788.7±102.5), CCL5 (WT:1415±116.9;D6:2249±230.5),CCL11(WT:175.1±36.13; D6:489.2±48.9) and lower IL6 levels (WT:98,1±18.1; D6:47.2 ±14.3) in D6 -/- infected mice, as well as higher expression of IFN-beta (WT:02±0.008; D6:1.22 ±0.03) and OAS (WT:0.07±0.01; D6:1.53±0.08) genes.Conclusions:We concluded that the absence of anti-inflammatory D6 receptor led to increased levels of CCL5 and CCL11, and consequent increased influx of lymphocytes, which may have caused the protection against the lethality and weight loss induced by IAV by controlling viral proliferation. As well as increased IFN- γ may have led to enhanced expression of IFN-b and OAS, "antiviral genes” that also have contributed to this reduced viral replication in D6 -/- mice.This study can contribute to the knowledge of the pulmonary inflammatory response caused by influenza A and developing of new pharmacological targets for the treatment of this virus in humans.

Financial support:CAPES, CNPq and FAPEMIG

DENGUE VIRUS REQUIRES THE CC-CHEMOKINE RECEPTOR CCR5 FOR REPLICATION AND DEVELOPMENT

OF INFECTION RAFAEL ELIAS MARQUES

1; RODRIGO GUABIRABA

2; JULIANA LEMOS DEL SARTO

3; ANA LUIZA QUEIROZ

4;

REBECA ROCHA FRÓES5; DANIEL CISALPINO

6; CAROLINA PACCA

7; PEDRO ELIAS MARQUES

8; GUSTAVO

BATISTA DE MENEZES9; DANIELLE DA GLORIA SOUZA

10; MAURO MARTINS TEIXEIRA

11.

1,3,4,5,11.IMMUNOPHARMACOLOGY, ICB, UFMG, BELO HORIZONTE - MG - BRASIL; 2.INFLAMMATION INFECTION AND IMMUNITY, UNIVERSITY OF GLASGOW, GLASGOW - REINO UNIDO; 6,10.HOST-MICRORGANISM INTERACTION, ICB, UFMG, BELO HORIZONTE - MG - BRASIL; 7.LAB. PESQUISA EM VIROLOGIA, FAMERP, SÃO JOSÉ DO RIO PRETO - SP - BRASIL; 8,9.IMMUNOBIOPHOTONICS, ICB, UFMG, BELO HORIZONTE - MG - BRASIL.

Introduction: Dengue is the most important human arbovirosis. Symptomatic infection by Dengue virus (DENV) is characterized by a systemic inflammatory response and hematological alterations that may evolve with shock and death in severe cases. There are no vaccines or specific treatment available for dengue, whose development requires further studies on the disease pathogenesis. Interestingly, the CC-chemokine receptor CCR5 has been implicated in many viral diseases, which led us to investigate the role of CCR5 in DENV infection. Methods and Results: In vitro infection of a human monocytic cell line (THP-1) with DENV-2 showed that the virus and CCR5 co-localize at the macrophage membrane during infection. This interaction is necessary to maintain viral replication, as treatment before infection with Met-RANTES, a CCR5 antagonist, prevented CCR5/DENV-2 interaction and reduced DENV RNA abundance inside macrophages. Importantly, the effects of CCR5 blockade were not related to an impaired viral entry. Further, in vivo experiments using a mouse-adapted DENV-2 strain, showed that CCR5 deficiency or blockade before infection prevented DENV-induced disease and mortality. Treatment with CCR5 antagonists after infection was not effective in reducing disease or mice mortality. Notably, CCR5 abrogation before infection decreased DENV load in mice tissues, contrary to the DENV load observed in wild type (WT) mice, which continuously increased during infection. This data suggests that DENV replication in tissues of Met-RANTES-treated or CCR5

-/- mice were impaired.

In accordance, in vitro assays with primary murine macrophages confirmed that DENV replication was impaired in CCR5

-/- and in WT cells treated before infection with Met-RANTES, when compared to WT controls. Interestingly,

pertussis toxin, which blocks CCR5 signaling, reduced DENV-2 load when compared to non-treated WT cells. Conclusion: CCR5 is a host factor required for DENV replication early in the infection. This requirement is crucial for dengue pathogenesis, as the lack of CCR5 in vivo prevented DENV replication and the development of disease. Further studies will focus on the signaling events triggered by the virus/CCR5 interaction, and how they affect the macrophage and DENV. Our findings add to the known association between CCR5 and viral infections, and might contribute to the development of treatment for dengue in the future.

Financial support: CNPq, FAPEMIG, INCT em Dengue

DOWNREGULATION OF CXCR4 BY RNA INTERFERENCE INHIBITS THE GROWTH AND PULMONARY METASTASIS OF B16 MELANOMA.

NAYARA DELGADO ANDRÉ BORTOLETO

1; VIVIANE ALINE OLIVEIRA SILVA

2; FERNANDO LUIZ DE LUCCA

3.

1.UNIVERSIDADE FEDERAL DE SÃO JOÃO DEL REI, DIVINÓPOLIS - MG - BRASIL; 2,3.FACULDADE DE MEDICINA DE RIBEIRÃO PRETO, RIBEIRÃO PRETO - SP - BRASIL.

Introduction: The incidence of malignant melanoma has increased during the last decades with a high mortality. Although surgery can be curative in localized disease, a large number of patients develop distant metastases

(1). In

recent years, it was found that the CXC chemokine receptor-4 (CXCR4) is expressed in many different types of human cancers and is involved in chemotaxis, invasion, angiogenesis, proliferation, tumor aggressiveness and resistance to chemotherapy

(2-5). These findings suggest that CXCR4 is a potentially attractive therapeutic target. In

this study we examined the effect of CXCR4 downregulation by RNA interference (RNAi) on the growth and pulmonary metastasis of B16 melanoma in C57BL/6 mice. Methods and Results: We have used a short hairpin RNA (shRNA) expressing plasmid (psiSTRIKE U6 hairpin cloning systems, Promega) controlled by Pol III U6 promoter. The B16-F10 melanoma cells were transfected (5, 24 and 48 hours) with 30ug CXCR4 specific shRNA and lipofectamine 2000 (Invitrogen). A scrambled shRNA was used as control. After transfection, the levels of CXCR4 mRNA and protein were evaluated by RT-PCR and Western blot, respectively. We observed that the maximum reduction of CXCR4 mRNA (96%) and CXCR4 protein (92%) levels occurs 48h after transfection of B16-F10 melanoma cells. To evaluate the role of CXCR4 on metastasis, B16-F10 melanoma cells transfected with non-related shRNA or CXCR4-specific shRNA were injected into the tail vein (1x10

5cells/animal) of C57BL/6 mice (n=10). Our results showed that

the pulmonary metastatic nodules were significantly reduced (97%) only in animals treated with CXCR4 shRNA transfected cells. We also found that the tumor growth is inhibited (66%) in mice (n=10) that received a subcutaneous injection of CXCR4 shRNA transfected cells when compared to animals inoculated with cells transfected with scrambled shRNA (4x10

5 cells/animal). Conclusion: Our findings suggest that CXCR4 is involved in the tumorigenesis

and metastasis of B16 melanoma. Thus, this study may contribute to develop new strategies for the treatment of melanoma.

References

1.Clin Cancer Res. 11:1835-1841,2005.

2. Blood 107:1761–1767,2006.

3. Immunol Rev. 177:175-184, 2000.

4. Clin Cancer Res.17:2074–2080, 2011.

5.Diagn Pathol. 24;8(1):104, 2013.

Financial Support: CAPES and FAPEMIG

EXPRESSION OF HLA-E AND CCR5 IS ASSOCIATED WITH WORSE OUTCOME IN KIDNEY TRANSPLANTATION

ANTONNYO PALMIELLY LIMA

1; ÁDALLA HAIANNE ANDRADE

2; WENNA GLEYCE NASCIMENTO

3; ROBERTA

APARECIDA DUARTE4; LUIZ PAULO FAGGIONI

5; CHRISTIANE PIENNA SOARES

6; ISABELA JUBÉ

WASTOWSKI7; LUCIANA TANAJURA SABER

8; ROBERTO SILVA COSTA

9; JOÃO SANTANA SILVA

10; EDUARDO

ANTÔNIO DONADI11

; JANAÍNA OLIVEIRA CRISPIM12

. 1,2,3,12.UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, NATAL - RN - BRASIL; 4,6.UNIVERSIDADE ESTADUAL PAULISTA JÚLIO DE MESQUITA FILHO, ARARAQUARA - SP - BRASIL; 5.AGÊNCIA NACIONAL DE SAÚDE SUPLEMENTAR, RIBERÃO PRETO - SP - BRASIL; 7.UNIVERSIDADE ESTADUAL DE GOIÁS, MORRINHOS - GO - BRASIL; 8,9,10,11.UNIVERSIDADE DE SÃO PAULO - FMRP, RIBERÃO PRETO - SP - BRASIL.

Introduction: HLA-E is a non-classical MHC (class Ib) molecule that binds to a limited number of peptides, and modulates the activity of NK-cell-mediated lysis by means of either inhibition or activation. In the context of transplantation, the presence of anti-HLA-E antibodies in kidney transplanted patients has been associated with a worse prognosis, although the kidney in situ expression of HLA-E has not been evaluated. Furthermore, studies suggest that the expression of chemokines and their interaction with chemokine receptors expressed on T cells plays a key role in the development of renal allograft rejection. This study aimed to evaluate whether HLA-E and chemokines expression may play a role in kidney allograft outcome and whether it there is relationship with clinical and laboratory variables.

Methods and Results: A sectional study was performed evaluating the immunohistochemical expression of HLA-E and chemokines (CXCR3, IP-10, MCP-1, CCR5) in 94 and 51 kidney allograft specimens, respectively. The specimens were stratified according to the presence or absence of rejection. Overall, HLA-E expression was detected in 23 (24.5%) cases, 12 (52.2%) of which exhibited acute or chronic rejection, and the remaining 11 (47.8%) exhibited no signs of rejection (p=0.039). Regarding chemokines expression, CCR5 was detected in 23 cases of which 10 (43,4%) had rejection (p>0.05), CXCR3 was detected in 26 cases of which 9 (35%) exhibited rejection (p>0.05), IP-10 was detected in 21 cases of which 8 (38%) had rejection (p>0.05) and MCP-1 was detected in 12 cases of which 5 (41,2%) exhibited rejection (p>0.05). Among specimens that presented HLA-E expression, 80% also had CCR5 expression (p=0.031).

Conclusion: Our data suggest that HLA-E expression in kidney allograft is correlated with CCR5 expression and are

associated with worse allograft outcome.

Financial support: We acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

by supported this study.

FREE HEME ACTIVATES MICE MESOTHELIAL CELLS: IMPLICATIONS FOR THE INFLAMMATORY RESPONSE

ASSOCIATED TO PLEURAL EFFLUX ANDRE LUIS PEIXOTO CANDEA

1; RAPHAEL LOUREIRO SIMÕES

2; THEREZA CRISTINA BARJA-FIDALGO

3;

MARIA DAS GRAÇAS MULLER DE OLIVEIRA HENRIQUES4.

1,4.FARMANGUINHOS / FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 2,3.UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.

Introduction: Heme, a ubiquitous iron-containing compound, is present in large amounts in many cells and is inherently dangerous, particularly

when it escapes from intracellular sites. We have demonstrated that free heme is a

pro-inflammatory molecule. The release of heme from damaged cells and tissues is supposed to be higher in

diseases such hemolytic anemia or in trauma, hemorrhage and pleural effusion. Pleural effusion is a common clinical

manifestation that is associated with the presence of a variety of cytokines/chemokines in pleural fluid. Mesothelial cells (MC) are metabolically active cells that line the pleura in a continuous monolayer and are able to release several chemokines after inflammatory stimuli. The aim of this work was to investigate evaluating the cytokine/chemokine production and ROS production by heme-stimulated MC, as well the molecular mechanisms modulated by this activation.

Methods and Results: The mesothelial cells were incubated with heme (10-100 µM) for different periods of

time. Initially, we observed that, differently of air pouch, a cavity without MC monolayer, the injection of heme in

pleural cavity, induced a neutrophil accumulation. As demonstrated by confocal microscopy analysis, heme induces a

progressive increase in HIF-1 and HO-1 expression as well NF-Kb nuclear translocation. We observed a significant

increase of KC and TNF-alpha releasing in mesothelial cells induced by heme at 6 and 12h, without changes in IL-

1beta, CCL2 and IL-6 levels, as demonstrated by ELISA. Additionally, the treatment with PDTC (1µM), a nuclear

factor (NF)-kB inhibitor was able to reduce these mediators releasing. Also, heme increases p47 translocation to

membrane and triggered ROS production in a NADPHox-dependent manner.

Conclusion: Our results indicate that free heme evokes mesothelial cells activation, which in turn release mediators

that may contribute for the onset of pleural inflammatory processes, particularly those associated with pleural efflux. A

better knowledge of this process may corroborate to the development of new strategies in order to ameliorate the

inflammatory response associated to pleural efflux.

HUMAN INTESTINAL EPITHELIAL CELLS SECRET INCREASED LEVELS OF IL-8 AFTER INTERACTION WITH

SULPHATE-REDUCING BACTERIA (SRB) ALESSANDRA ALVES ABALO

1; HAYANDRA FERREIRA NANINI

2; GIANI FRANCO SANTORO

3; ANGELA

SANTOS4; NILDA BRITTES

5; VINICIUS COTTA-DE-ALMEIDA

6; ROBSON COUTINHO-SILVA

7; CLAUDIA MARA

LARA MELO COUTINHO8.

1,2,3,4,5,6.IOC/FIOCRUZ/LABORATÓRIO DE INOVAÇÕES EM TERAPIAS, ENSINO E BIOPRODUTOS (LITEB), RIO DE JANEIRO - RJ - BRASIL; 7.UFRJ/IBCCF/LABORATÓRIO DE IMUNOFISIOLOGIA, RIO DE JANEIRO - RJ - BRASIL; 8.UFF/INSTITUTO DE BIOLOGIA, NITERÓI - RJ - BRASIL.

Introduction: Sulphate-reducing bacteria (SRB) are anaerobic organisms found colonizing the human gut, besides many others environments, and have been associated with the development of chronic inflammatory bowel diseases (IBD). Intestinal epithelial cells form the first line of defense against pathogenic luminal microbes and are recognized as active participants in mucosal immune and inflammatory reactions. In the present work, we aimed to study the outcome of in vitro interaction between human intestinal epithelial cells (HCT8 cell line) and Desulfovibrio indonesiensis, a pure strain of SRB, in order to check the secretion of cytokines.

Methods and Results: Cell-free supernatants obtained from HCT8 cell culture alone (control) or co-incubated with SRB for 24h were collected to investigate the presence of human cytokines by flow cytometry using the CBA kit, including tumor necrosis factor (TNF), interleukin-1 β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-12 (IL-12). Among the six tested cytokines, only IL-8 levels were detected on control cell-free supernatant (1305.05 ± 10.03 pg/ml; n=3), and, interestingly, they were increased on cell-free supernatant obtained from HCT8 culture co-incubated with SRB for 24h (1854.06 ± 30.15 pg/ml; n=3).

Conclusion: Our data show that intestinal epithelial cells challenged by SRB release increased levels of IL-8. The recruitment and activation of leukocytes in inflamed tissues is a complex process driven by cytokines and chemokines that induce cell adhesion and locomotion. Several studies have provided evidence for the role of IL-8 in mediating infiltration of neutrophils. Previous data of our group showed that soluble chemotactic factors released by HCT8 cells after in vitro interaction with SRB are able to attract human monocytes and neutrophils. So, we can speculate that the increased levels of IL-8 we detected in the cell-free supernatant obtained from HCT8 intestinal epithelial cells after interaction with SRB might be one of the chemotactic factors involved in attracting neutrophils. Finally, taking all together, our data suggest that SRB might be involved both in the initiation and in the perpetuation of IBD, playing a crucial role in the pathogenesis of such inflammatory diseases.

Financial support: PETROBRAS, CNPq/Proep, FAPERJ.

IL-4/CCL22/CCR4 AXIS MEDIATES REGULATORY T CELL MIGRATION INTO PERIODONTAL TISSUES AND THE ARREST OF EXPERIMENTAL PERIODONTITIS PROGRESSION

ANA CLAUDIA ARAUJO PIRES

1; ANDREIA E VIEIRA

2; CLAUDIA C BIGUETTI

3; ANA PAULA FAVARO

TROMBONE4; ANDREW J GLOWACKI

5; SAYURI YOSHIZAWA

6; CHARLES S SFEIR

7; STEVEN R LITTLE

8;

GUSTAVOPOMPERMAIER GARLET9.

1,2,3,9.FOB/USP, BAURU - SP - BRASIL; 4.USC-BAURU, BAURU - SP - BRASIL; 5,6,7,8.UNIVERSITY OF PITTSBURGH, PITTSBURGH - ESTADOS UNIDOS.

Introduction: Periodontal diseases (PD) are characterized by the progressive destruction of gingival soft tissue and alveolar bone, which is mediated by a complex network of pro-inflammatory, Th1 and Th17 cytokines produced in response to a chronic bacterial insult. While regulatory T cells (Tregs) are described to play a protective role in PD pathogenesis, the exact immunological mechanisms involved in such protection remain unknown, and in this study we investigated the mechanisms involved in its migration to periodontal tissues.

Methods and Results: C57Bl/6 (WT), IL4KO and CCR4KO mice infected with A. actinomycetemcomitans, treated or not with anti-GITR to inhibit Tregs function, were analyzed regarding inflammatory cell and Tregs (Foxp3+) influx, alveolar bone loss and chemokine/cytokine expression/production (analyzed by RealTimePCR and ELISA) throughout experimental periodontitis. Our results demonstrated that Foxp3+ cells extracted from periodontal tissues express high levels of CCR4, CCR8, IL-10, TGF-b and CTLA-4, and low levels of CCR5 and CCR2. Accordingly, CCR4KO mice presented a reduced influx of Foxp3+ cells in periodontal tissues, associated with increased inflammatory reaction and bone loss. Interestingly, IL4KO mice present a similar experimental PD phenotype; where impaired Tregs migration was associated to a lower expression/production of CCL22, a ligand of CCR4. The administration of formulations designed to controlled release of CCL22 in IL4KO resulted in the recovery of the increased disease severity phenotype, but was ineffective attenuating disease severity in CCR4KO mice. CCL22-releasing particles were also found to arrest disease progression in WT mice (being its effect counteracted by anti-GITR treatment), as well in a canine pre-clinical model of PD.

Conclusion: Our results demonstrate that IL-4/CCL22/CCR4 axis mediates regulatory T cell migration into periodontal tissues and the arrest of experimental periodontitis progression, and that CCL22-targeted therapy can comprise a valuable clinical strategy to treat periodontitis.

Supported by NIH R01 DE021058-01A.

IMPORTANT ROLE FOR CCR2 IN INFLAMMATORY ANGIOGENESIS IN MICE

POLLYANA RIBEIRO CASTRO; SUZANE MOTA MARQUES; CELSO TARSO RODRIGUES VIANA; LUCÍOLA DA

SILVA BARCELOS; SILVIA PASSOS ANDRADE.

FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: The chemokine system is involved in the control, migration, activation, proliferation and apoptosis of

leukocytes and endothelial cells and one of its members, the chemokine ligand 2 (CCR2) is expressed in a number of

inflammatory processes. However, the role of this chemokine in inflammatory angiogenesis has not been totally

characterized. Here, we evaluated sponge-induced angiogenesis, inflammation and fibrogenesis in CCR2 receptor

deficient mice.

Methods and Results: We have performed the kinetics of angiogenesis, inflammation and fibrogenesis in polyether-

polyurethane sponge discs implanted subcutaneously in female C57/BL6 mice with and without CCR2 (CCR2-WT and

CCR2-KO) (n=8). These parameters were evaluated in implants removed at days 1, 4, 7, and 14 post implantation

(n=5-7 animals/group). Angiogenesis was reduced in implants of KO animals at day 14 as determined by hemoglobin

(µg/mg) (KO=2.17+0.52 vs WT=3.95+0.38) and blood flow (perfused unit) (KO=123.1+6.44 vs WT=138.6+17.9).

Neutrophils accumulation was markedly increased in CC2R-KO implants at day 14, as determined by

myeloperoxidase activity (OD/mg) (KO=24.6+0.24 vs WT= 5.21+1.12). On other hand macrophages accumulation

was decreased in CCR2-KO implants at days 4 and 7, as determined by N-acetyl-b-D-glucosaminidase activity

(OD/mg) (KO=2.70+0.30 vs WT=4.06+0.43; day 4). Furthermore, the levels of nitrite (µg/mg) were also reduced in

CCR2-KO implants (KO=0.91+0.01 vs WT=3.27+0.50; day 7). CCL2/MCP-1 levels (ρg/mg), however, were higher in

KO implants (0.06+0.003 vs 0.05+0.002, day 14). Similarly, collagen deposition (µg/mg), a fibrogenic parameter, was

higher in KO implants compared with control animals (KO= 0.87+0.07 vs WT=0.64+0.04; day 14).

Conclusion: We have shown that inflammation, angiogenesis and fibrogenesis are clearly different between CCR2-

WT and CCR2-KO mice, indicating that CCR2 is a critical endogenous regulator of the fibroproliferative tissue induced

by the sponge implant.

Financial support: Coordination of Improvement of Higher Education Personnel (Capes); National Council for

Scientific and Technological development (CNPq).

INDUCIBLE EXPRESSION OF BRAFV600E IN CX3CR1+ CELLS TRIGGERS MYELOID EXPANSION AND

DEVELOPMENT OF A LCH-LIKE DISEASE

MONIQUE FERRAZ DE SA BELTRAO; MICHELLE E PACER; RAFAEL S CZEPIELEWSKI; JINGJING JIAO; LILI

CHEN; ALAN SOTO; GLAUCIA C FURTADO; SERGIO A LIRA.

IMMUNOLOGY INSTITUTE, ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, NEW YORK - ESTADOS UNIDOS.

Introduction. Langerhans cell histiocytosis (LCH) is a rare disease characterized by an accumulation of histiocytes

and Langerhans cells. LCH can involve single or multiple organs, and has a worse prognosis when it affects risk-

organs such as, liver, spleen and lung. The origin of LCH is unclear. Studies have shown that LCH is promoted by an

uncontrolled clonal proliferation of dendritic cells with LC characteristics, suggesting that these cells share a common

myeloid progenitor. An activating mutation of the BRAF gene (V600E) has been detected in many LCH biopsy

samples, but it is unclear if it is sufficient to induce disease.

Methods and Results. To test the hypothesis that BRAFV600E is sufficient to cause LCH, we generated FRBRAF

mice. These mice express BRAFV600E in the mononuclear phagocyte system (CX3CR1+ cells) upon administration

of tamoxifen (TAM). After 3 months of TAM treatment, mice appeared hunched, had an enlarged abdomen and were

neurologically affected (hind limb weakness and spasticity). Upon necropsy, the mice had marked

hepatosplenomegaly. Flow cytometric analysis of the blood showed an expansion of the myeloid compartment.

Histological analyses revealed the presence of marked inflammatory infiltrates in the liver, lung, and spleen and

occasional mild inflammatory infiltrates in the spinal cord and the brain. In some instances, granuloma-like

aggregates, rich in giant cells, myeloid cells, eosinophils and lymphocytes were detected in liver and the lungs.

Remarkably, CD11b+MHCII+ Langerin+ cells, typically found within LCH lesions in humans, were also present in

these infiltrates. Preliminary analysis of the liver transcriptome of the FRBRAF mice showed an increase in the

expression of several cytokines, chemokines, and chemokine receptors known to be expressed within LCH lesions

(TNFa, IL-1a, M-CSF, IL-7, CCL17 and CCR6).

Conclusion. We show that expression of BRAFV600E in CX3CR1+ cells in mice promotes expansion of the myeloid

compartment and development of a disease that resembles human LCH. These results directly implicate

BRAFV600E in the pathogenesis of LCH.

Support: FACEPE

LEUKOCYTES TRAFFICKING AT BLOOD BRAIN BARRIER: A NEW CONCEPT FOR ENDOGENOUS "PROTECTIN" MOLECULE

ELISA MAGGIOLI; EGLE GIULIANA SOLITO. WILLIAM HARVEY RESEARCH INSTITUTE, SMD, QUEEN MARY UNIVERSITY, LONDON - REINO UNIDO.

Introduction: The blood–brain barrier (BBB) is the brain-specific capillary barrier that is critical for preventing toxic substances from entering the central nervous system (CNS). In contrast to vessels of peripheral organs, the BBB limits the exchange of inflammatory cells and mediators under physiological and pathological conditions. Clarifying these limitations would provide new insights into neuroprotective strategies in neuroinflammatory diseases. We have previous shown that the resolving endogenous molecule Annexin A1 (ANXA1) is expressed in the endothelial cells of the brain microvasculature, where it plays a major role in promoting the integrity of the blood-brain barrier (BBB). In particular, ANXA1

-/- mice exhibit significantly increased BBB permeability due to disrupted inter-endothelial cell tight

junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. Furthermore, ANXA1 is selectively lost in the BBB endothelium in multiple sclerosis (MS), but not in other neuropathologies such as Parkinson’s.

Methods and Results: Here we studied the involvement of ANXA1 in leukocyte-BBB interactions and migration, utilising the immortalised human brain microvascular cell line (hCEMC/D3) stably infected with shRNA for ANXA1, in a transwell system under shear stress conditions to ensure the correct cellular polarity and formation of tight junctions. Significantly greater numbers of peripheral blood mononuclear cells (PBMCs) (25±3.1 vs 13±1.6; n=3) adhered to endothelial monolayers lacking ANXA1 under inflammatory conditions. Furthermore endothelial ANXA1 loss correlated with higher expression of ICAM1 measured by flow cytometry (MIF 903±17 vs 600±15; n=3) and constitutively higher CD4 migration in non-inflammatory condition.

Conclusions: We present here evidence for a dual role of ANXA1 in leukocyte migration into the central nervous system: 1) as a safeguard at the level of the endothelium in non-inflammatory conditions and 2) as a blocker of leukocytes extravasation in the presence of circulating cytokines and inflammatory conditions.

Supported by the Wellcome Trust and ARUK

LFA-1 IS CRITICAL FOR T-CELL MEDIATED PROTECTIVE IMMUNITY ELICITED BY HETEROLOGOUS PRIME-

BOOST VACCINATION STRATEGY AGAINST A SYSTEMIC PARASITIC INFECTION

FERNANDO VIRGILIO1; MARIANA R DOMINGUEZ

2; ALEXANDRE V MACHADO

3; RICARDO T GAZZINELLI

4;

OSCAR BRUNA–ROMERO5; MAURICIO M RODRIGUES

6; JOSÉ RONNIE CARVALHO VASCONCELOS

7.

1,2,6,7.UNIFESP, SÃO PAULO - SP - BRASIL; 3,4.FIOCRUZ, MINAS GERAIS - MG - BRASIL; 5.UFSC,

FLORIANÓPOLIS - SC - BRASIL.

INTRODUCTION: Heterologous prime-boost vaccination strategy using recombinant plasmid DNA followed by

replication-defective human recombinant adenovirus 5 (AdHu5) is a powerful strategy to elicit specific CD4+ and

CD8+ T cells which play a key role in protective immunity against intracellular pathogens as for example

Trypanosoma cruzi, the causative agent of the neglected Chagas' disease. Based on evidences that recirculation after

challenge is critical for T-cell mediated protective immunity, herein we tested whether certain integrins, cell adhesion

molecules (CAM) and chemokines were key mediators of this process playing a critical role during immunity against

infection.

RESULTS: To study the role of integrins, A/Sn mice were immunized with heterologous prime-boost vaccine (rec.

plasmid DNA/AdHu5), challenged with parasites (Y strain) and treated with blocking antibodies to LFA-1 and/or VLA-

4. Immunized mice treated with control Rat IgG or anti-VLA-4 antibodies controlled the parasitemia and survived the

lethal challenge. In contrast, immunized mice treated with anti-LFA-1 or anti-LFA-1 and anti-VLA-4 displayed high

parasitemia and all of them died after challenge. LFA-1 blockage neither diminished the frequency nor compromised

the capacity of specific CD8+ T cells to respond in vitro (IFN-g and TNF). We performed similar experiments by

vaccinating icam-1-/-

or cccr5-/-

mice. These mice controlled the infection at similar extension as wild type

animals. CONCLUSION: We concluded that LFA-1 integrin, but not VLA-4, ICAM-1 or CCR5 play a critical role in the

protective immune response generated by the heterologous prime-boost vaccination possibly by blocking T cell

recirculation after challenge.

Financial support: INCTV (CNPq) and FAPESP.

MAMMALINA STERILE 20-LIKE KINASE 1 – A NEW PLAYER IN NEUTROPHIL RECRUITMENT IN VIVO

ANGELA KURZ1; MONIKA PRUENSTER

2; SUSANNE BIERSCHENK

3; JOHANNES WIESSNER

4; DEA-SIK LIM

5;

MARKUS MOSER6; BARBARA WALZOG

7; MARKUS SPERANDIO

8.

1,2,3,4,7,8.LUDWIG MAXIMILIANS UNIVERYITY, MUNICH - ALEMANHA; 5.NATIONAL CREATIVE INITIATIVES

CENTER FOR CELL DIVISION AND DIFFERENTIATION, DAEJEON - CORÉIA; 6.MAX PLANCK INSTITUTE OF

BIOCHEMESTRY, MARTINSRIED - ALEMANHA.

Introduction: Mammalian sterile 20-like kinase 1 (MST1) is a ubiquitously expressed serine/threonine kinase involved

in LFA-1-induced T-cell adhesion. In this study, we investigated the role of MST1 in leukocyte recruitment in different

mouse models under inflammatory conditions in vivo.

Methods and Results: We studied leukocyte rolling, adhesion and extravasation in TNF-alpha-stimulated cremaster

muscle venules of Mst1-deficient and wild type mice using intravital microscopy. We observed a significant increase in

leukocyte adhesion 2h after TNF-alpha stimulation, while we could not find any difference in the rolling behaviour of

leukocytes. Extravasation of neutrophils into inflamed tissue was 4-fold elevated in Mst1-deficient mice compared to

wild type mice 2h after intrascrotal injection of TNF-alpha. FACS analysis of relevant surface molecules, involved in

the rolling and adhesion process, showed no differences between Mst1-deficient and wild type neutrophils. Similarly,

Mst1-/-

and wild type neutrophils were equally able to bind E-selectin, P-selectin, and ICAM-1 under different

conditions. This indicates a similar surface expression and function of integrins and selectins in the absence of MST1.

In addition, experiments with bone marrow chimeras using the TNF-alpha-stimulated cremaster model, demonstrated

a contribution of both, endothelial as well as leukocyte compartment, in leukocyte extravasation.

Conclusion: We identified MST1 as an important negative regulator during the inflammatory response demonstrating

leukocyte adhesion and extravasation in vivo. Additional studies are currently in process to further unravel the

molecular mechanisms by which MST1 influences leukocyte recruitment in vivo. (Supported by DFG Collaborative

Research Center SFB914)

NEUTROPHIL NECROTAXIS TO INJURED LIVER INCREASES INFLAMMATION AND TISSUE DAMAGE

PEDRO ELIAS MARQUES1; RAFAELA VAZ PEREIRA

2; BRUNA ARAUJO DAVID

3; ANDRE GUSTAVO OLIVEIRA

4;

LINDISLEY FERREIRA GOMIDES5; RAFAEL ELIAS MARQUES

6; VICENTE DE PAULO MARTINS

7; MAURO

MARTINS TEIXEIRA8; GUSTAVO BATISTA MENEZES

9.

1,2,3,4,5,6,8,9.UFMG, BELO HORIZONTE - MG - BRASIL; 7.UNB, BRASILIA - DF - BRASIL.

Introduction: Acetaminophen (APAP) is a safe analgesic and antipyretic drug. However, at high doses, APAP causes

acute liver failure (ALF), a disorder associated with massive hepatocyte necrosis. In this context, spilled intracellular

contents, cytokines and chemokines would induce neutrophil recruitment and activation, which could lead to additional

liver injury. Therefore, our objective was to investigate the molecules and mechanisms involved in neutrophil

recruitment during APAP-induced liver necrosis.

Methods and Results: ALF was induced by APAP overdose (600mg/kg) in C57BL/6 and lysm-eGFP (GFP+

neutrophils) (n≥5 for all experiments). Liver injury was quantified by serum aminotransferase levels (U/L). Hepatic

cytokines and chemokines were measured by ELISA (pg/mL). The mechanisms of neutrophil recruitment were

assessed by liver intravital microscopy; Neutrophils were GFP+ and hepatic necrosis was shown by propidium iodide

(PI; 0.1mM) (% area). The parameters evaluated include: neutrophil count, trajectory lenght (µm), mean velocity

(µm/s), displacement (µm) and trajectory time span (s). Data is shown as (mean±SEM). During APAP overdose, mice

developed liver injury (6961±1223). The levels of chemokines CXCL1 and CXCL2 were increased by 3-fold in liver

and serum after APAP challenge. Likewise, IL-1-beta and TNF-alfa were increased by 5-fold in both tissues. Liver

imaging revealed PI-positive necrosis 12h after APAP, reaching its maximum at 24h (17.4±2.6). We observed

significant neutrophil recruitment 6h after APAP (50.4±4.2), peaking also at 24h (86.0±8.1). Neutrophils adhered within

liver vasculature after 6h and gradually migrated into necrosis at later time-points (12 and 24h; 52.6±8.3). All

neutrophil tracking parameters were significantly increased 24h after APAP, however, neutrophil movement was

almost completely restricted to necrotic areas. Elimination of neutrophils by anti-GR1 depletion significantly prevented

liver injury (70% reduction). Blockage of CD18 did not reduce neutrophil counts, but neutrophil recruitment to the liver

was dependent on the chemokine receptor CXCR2 and the formyl-peptide receptor FPR1 (both 50% reduction).

Conclusion: APAP-induced liver injury triggers neutrophil recruitment to necrotic areas, which is dependent on

CXCR2 and FPR1, and contributes to liver damage.

Ethical Approval: CETEA UFMG (051/11)

Financial Support: CAPES, CNPq, FAPEMIG

OLIGOFRUCTOSE SUPPLEMENTATION (10%) DURING PREGNANCY AND LACTATION CAUSED A

PROINFLAMMATORY EFFECT ON RETROPERITONEAL ADIPOSE TISSUE AND SOLEUS MUSCLE ON

LACTATING RATS.

JULIANA LOPEZ DE OLIVEIRA1; LAIS VALES MENNITTI

2; ANA CLAUDIA LOSINSKAS HACHUL

3; LILA MISSAE

OYAMA4; ELIANE BERALDI RIBEIRO

5; LUCIANA PELLEGRINI PISANI

6; CLAUDIA MARIA OLLER DO

NASCIMENTO7.

1,3,4,5,7.UNIVERSIDADE FEDERAL DE SÃO PAULO - DEPARTAMENTO FISIOLOGIA, DISCIPLINA DE

FISIOLOGIA DA NUTRIÇÃO, SÃO PAULO - SP - BRASIL; 2,6.UNIVERSIDADE FEDERAL DE SÃO PAULO -

DEPARTAMENTO DE BIOCIÊNCIAS, INSTITUTO DE SAÚDE E SOCIEDADE, SANTOS - SP - BRASIL.

Introduction: During pregnancy and lactation, the maternal intake of prebiotics and dietary fibers is considered

important and beneficial for the mother and the offspring. Several studies in humans and animals have confirmed that

dietary fibers such as oligofructose (OF), fructooligosaccharides (FOS) and inulin influence glucose metabolism,

specifically by reducing glucose serum concentrations (Br J Nutr 1996, 76:881–890). Gestation is a critical period for

development of insulin resistance and gestational diabetes. It has been reported that high maternal serum TNF-α

levels were associated with insulin resistance (J Endocrinol Invest. 2005;28(9):779–86). Objectives: The aim of this

study was to evaluate the effect of the supplementation of the diet of the dams with 10% oligofructose during

pregnancy and lactation on the tissues cytokines content of lactating rats. Methods and Results: On the first day of

pregnancy, rats were divided into two groups: Control group - received a control diet and C-OFS group received a

control diet supplemented with 10% oligofructose. On 21 days of lactation the dams were euthanized. The serum

glucose, insulin, adiponectin, lipopolissacarideos concentrations were analyzed. The IL-6, IL-10 and TNF-α contents

of the retroperitoneal white adipose tissue, liver, soleus and extensor digital longus muscles were analyzed by ELISA.

The results are presented as the means ± standard error of the mean. Statistical significance was assessed using

Student “t” test and considered significant at p < 0.05. Results: All serum parameters analyzed were similar between

groups. In the retroperitoneal adipose tissue, the IL-6 content was increased in C-OFS group relative to the Control,

and the TNF-α content and TNF-α/IL-10 ration were higher in soleus muscle in C-OFS group than in the C group.

Similar cytokines content was observed in the liver and extensor digital longus muscles between groups. Conclusions:

Supplementation of the diet of the dams with 10% of oligofructose during pregnancy and lactation, contributed to

development of a pro-inflammatory status. Further studies are necessary to analyze the effect of dose response of the

oligofructose supplementation during the gestation and lactation period. Financial support: FAPESP, CAPES and

CNPq.

PLASMIN INDUCES IN VIVO MONOCYTE RECRUITMENT THROUGH PROTEASE-ACTIVATED RECEPTOR-1,

MEK/ERK AND CCR2 MEDIATED SIGNALING

BRUNO COSTA; ALINE CARMO; JULIANA VAGO; LEONARDO OLIVEIRA; LUCIANA TAVARES; CAMILA

NOGUEIRA; BRUNO BRASIL; LUCIMARIA DUSSE; LUCÍOLA BARCELOS; CLÁUDIO BONJARDIM; MAURO

TEIXEIRA; LIRLÂNDIA SOUSA.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: The Plasminogen/Plasmin (Plg/Pla) system is associated with a variety of biological activities beyond

the classical dissolution of fibrin clots, including cell migration and inflammation. Although the capacity of Plg to induce

cell migration is well defined, the in vivo mechanism underlying the process is elusive. In this study we investigate the

effect of Pla on cell migration in vitro and in vivo and the role of MAPK ERK1/2, PAR-1 and CCL2/CCR2 axis on the

process.

Methods and Results: We performed an in vitro migration assay in cultures of fibroblasts (MEFs) and macrophages

(RAW). The cells were treated with Pla (2µg/mL) at different times or pretreated with a MEK1/2 inhibitor (U0126), a

serine protease inhibitor (Leupeptin) or the lysine analog tranexamic acid (TXA) 1h prior to and throughout Pla

treatment. The cells were processed for migration count or WB analysis for P-ERK1/2. BALB/C mice were challenged

by intrapleural injection of Pla (2µg). The cells present in the cavity were harvested at different times or 48h after pre-

treatment with specific inhibitors (U0126 60µg/cav/i.pl.; Leup. 100µg/mouse/i.p.; SCH79797 5mg/kg/i.p). The inhibitors

were administrated 1h before Pla. Cells were processed for leukocyte count or WB analysis for P-ERK1/2 and P-IkB-

a. Pleural levels of IL-1β, IL-6, TNF-α and CCL-2 were analyzed by ELISA. CCR2-/-

and WT mice were injected with

Pla and the cells present in the pleural cavity harvested after 48h and processed for count. Pla induced in

vitro migration of MEF and RAW depending on MEK/ERK pathway and by requiring its proteolytic activity and lysine

binding sites on cell surfaces. Pla injection into the pleural cavity induced a time-dependent influx of mononuclear cells

associated with enhanced P-ERK1/2 and P-IkB-α and increased levels of CCL2 and IL-6. In vivo inhibition of protease

activity by Leupeptin or the PAR-1 antagonist SCH79797 prior to Pla injection abolished Pla-induced mononuclear

recruitment and ERK1/2 and IkB-α phosphorylation. Interestingly, inhibition of MEK/ERK pathway impaired Pla-

induced CCL2 upregulation and mononuclear cell influx. According to the requirement of CCL2 for the Pla-induced cell

trafficking, CCR2-/-

mice were not responsive to Pla-induced mononuclear recruitment.

Conclusion: Pla induces mononuclear cell recruitment in vivo depending on PAR-1 activation via MEK/ERK/NF-kB

pathway, what leads to CCL2 release and CCR2 activation.

Financial support: FAPEMIG, CNPq, PQPq-UFMG

PROTECTIVE ROLE OF ST2 IN BONE REMODELING INDUCED BY MECHANICAL LOADING

IZABELLA LUCAS DE ABREU LIMA1; DAVIDSON FROIS MADUREIRA

2; ADRIANA PEDROSA MOURA

3; CELSO

MARTINS QUEIROZ-JUNIOR4; SILVANA RODRIGUES DE ALBUQUERQUE TADDEI

5; SORAIA MACARI

6; JOSÉ

CARLOS FARIAS ALVES FILHO7; SANDRA YASUYO FUKADA ALVES

8; MAURO MARTINS TEIXEIRA

9; TARCÍLIA

APARECIDA SILVA10

.

1,2,3,4,6,9,10.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL;

5.UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR - BA - BRASIL; 7,8.UNIVERSIDADE DE SAO PAULO,

RIBEIRAO PRETO - SP - BRASIL.

Introduction: Some cytokines and chemokines regulate bone remodeling; one of them is interleukin-33 and its

receptor, ST2. This study aimed to investigate the protective role of IL-33 in bone remodeling induced by mechanical

loading.

Methods and Results: The orthodontic tooth movement (OTM) was induced in wild-type (WT) (Balb/c) mice and ST2

knockout (ST2−/−

) mice, then compared. Our results showed that bone remodeling was significantly increased in

ST2−/−

mice, as well the number of TRAP positive cells. While, mRNA levels of receptor activator of nuclear factor

kappa-B ligant (RANKL) were decreased at periodontal tissues in ST2−/−

compared to WT mice, the levels of

osteoprotegerin (OPG) were increased in ST2−/−

after 72 hours of mechanical loading. Tumor necrosis factor alpha

(TNF-α) and interleukin-10 (IL-10) levels were significantly lower in ST2-/-

mice when compared to WT. There was no

significant difference in the levels of receptor activator of nuclear factor kappa-B (RANK) and runt-related transcription

factor 2 (RUNX2). In addition, mRNA levels of osteoclasts markers, such as Cathepsin K (CtsK), metalloproteinase-2

(MMP2), MMP-9 and MMP-13 were increased in ST2-/-

mice when compared to WT mice.

Conclusion: These findings show that IL-33 reduces bone resorption, representing a potential therapeutic option to

prevent disease associated with excessive osteoclastogenesis and consequent bone loss.

We are grateful to the Fundação de Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG, Brazil), the

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for financial support.

REDUCED LEVELS OF ADIPONECTIN IN FRUCTOSE-FED MICE ARE ASSOCIATED WITH INCREASED LIVER

NEUTROPHIL ADHESION

DÉBORA FERNANDES RODRIGUES; MILENE CRISTINA DO CARMO HENRIQUES; MARINA CHAVES DE

OLIVEIRA; JAQUELINE PEREIRA LANA; LAÍS BHERING MARTINS; PEDRO ELIAS MARQUES; GUSTAVO

BATISTA MENEZES; MAURO MARTINS TEIXEIRA; ADALIENE VERSIANI MATOS FERREIRA.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction

Chronic consumption of fructose is associated with a state of low-grade chronic inflammation and metabolic

disorders. Adiponectin, the most abundant adipokine produced by the adipose tissue, has been demonstrated to exert

profound anti-inflammatory and antiatherosclerotic effects, in part via diminishing endothelial activation and leukocyte

endothelial interaction. Considering the proinflammatory response arising from the high-fructose diet (HFD)

consumption, we aimed to investigate the impact of HFD intake on adiponectin levels and neutrophil recruitment in the

liver of mice in the postprandial state.

Methods and Results

The experimental protocol was approved by the Ethics Committee in Animal Experimentation at the Universidade

Federal de Minas Gerais (protocol N°: 63/2010). Male BALB/c mice (n=8) at 12-14w were divided into 3 groups: fasted

(F), control (C, mice fed an AIN-93 diet containing 20% sucrose) and HFD (mice fed a modified AIN-93 diet containing

20% fructose). Then, 2 hours postprandial, mice were killed and samples of blood and liver were collected. Mice fed

the HFD showed a systemic inflammatory response characterized by increased numbers of leukocytes (F: 37.44 ±

1.80 x 105/ mL; C: 41.25 ± 7.71 x 10

5/ mL; HFD: 107.92 ± 9.49 x 10

5/ mL) and high serum concentration of pentraxin 3

(F: 3.39 ± 0.10 ng/ mL; C: 3.88 ± 0.40 ng/ mL; HFD: 4.87 ± 1.26 ng/ mL). The adiponectin concentration dropped after

fructose intake (F: 921.76 ± 113.35 µg/mL; C: 1074.33 ± 71.20 µg/mL; HFD: 680.94 ± 61.61 µg/mL), while the MPO

activity were higher in the liver of mice fed HFD (0.43 ± 0.08, relative unit) compared with F (0.02 ± 0.004, relative

unit) and C (0.03 ± 0.01, relative unit). To investigate the impact of low serum adiponectin concentration in neutrophil

recruitment, LysM-eGFP mice (green fluorescent neutrophils, n=4) were submitted to intravital microscopy for

hepatocyte visualization. Mice with low serum adiponectin concentration had more than 2-fold increase on neutrophil

adhesion in liver sinusoids (17.07 ± 1.96 adhered neutrophils/ field), compared with F (6.00 ± 0.65 adhered

neutrophils/ field) and C (7.80 ± 1.06 adhered neutrophils/ field).

Conclusion

Our data demonstrate that HFD led to a high inflammatory response in the postprandial state, with a drop in serum

adiponectin concentration. These events may have contributed to the increased neutrophil adhesion in liver sinusoids.

Financial support: CNPq, FAPEMIG, CAPES.

ROLE OF NEURONAL P2X3 RECEPTOR ON RELEASE OF INFLAMMATORY CYTOKINES

MARIA CLÁUDIA OLIVEIRA-FUSARO1; CLÁUDIO FUSARO

2; CARLOS AMÍLCAR PARADA

3.

1.SCHOOL OF APPLIED SCIENCES, STATE UNIVERSITY OF CAMPINAS, LIMEIRA - SP - BRASIL; 2.SAO

FRANCISCO UNIVERSITY, BRAGANÇA PAULISTA - SP - BRASIL; 3.INSTITUTE OF BIOLOGY, STATE

UNIVERSITY OF CAMPINAS, CAMPINAS - SP - BRASIL.

Introduction: It has been described that the P2X3 receptor is expressed preferentially on nociceptive C-fibers and we

have demonstrated that P2X3 receptors contribute to mechanical hyperalgesia induced by carrageenan mediated by

the previous release of inflammatory cytokines (Pain.141:127-134, 2009). Therefore, the aim of this study was to

analyze the role of neuronal P2X3 receptor on the release of TNF-α, IL-1β and CINC-1 in the subcutaneous tissue of

a rat´s hind paw. Methods and results: First, we intrathecally (i.t.) administered oligonucleotides antisense against

P2X3 receptors or capsaicin in order to induce depletion of C-fibers and evaluated, using the ELISA test, the local

concentration of inflammatory cytokines induced by carrageenan on the subcutaneous tissue. Second, we tested

whether the activation of P2X3 receptors induced the release of the inflammatory cytokines on the subcutaneous

tissue of rats previously treated with capsaicin. Third, we tested whether the selective P2X3 receptor antagonist,

A317491, reduced the release of inflammatory cytokines induced by carrageenan on the subcutaneous tissue of rats

previously treated with capsaicin. Male Wistar rats were used and all procedures were approved by the Ethics

Committee in Animal Research at UNICAMP (# 1558-1). Antisense against P2X3 receptors (80µg/10µl, 4 days)

reduced the P2X3 receptor expression on the saphenous nerve (p<0.05, t-test) and did not affect (p>0.05, t-test) the

increase on the concentration of TNF-α, IL-1β and CINC-1 induced by carrageenan (100µg, 24.5±2.3; 38.5±2.9;

26.3±3.3pg/mg, n=16) when compared to mismatch. Pretreatment with capsaicin (6µg/10µl, 48h) depleted the

capsaicin sensitive fibers and did not affect (p>0.05, t-test) the increase on the concentration of TNF-α, IL-1β and

CINC-1 induced by carrageenan (100µg, 24.9±2.1; 36.9±1.8; 24.0±1.5pg/mg, n=20) or by non-selective P2X3

receptor agonist αβmeATP (50µg, 11.2±1.2; 19.9±2.1; 2.2±0.4pg/mg, n=10). Finally, A317491 (60µg, 14.1±1.8;

16.5±1.2pg/mg, n=12) reduced (p<0.05, t-test) the increase on the concentration of TNF-α and CINC-1, but not of IL-

1β, induced by carrageenan in rats previously treated with capsaicin. Conclusion: These data have demonstrated

that neuronal P2X3 receptors did not contribute to the release of inflammatory cytokines induced by carrageenan.

Therefore, it is plausible to suggest that P2X3 receptors expressed in non-neuronal cells contribute to inflammation.

Support: FAPESP-2009/53589-3.

THE SUPPLEMENTATION OF OLIGOFRUCTOSE DURING PREGNANCY AND LACTATION INCREASES SERUM LIPOPOLYSACCHARIDES AND DECREASES SERUM CONCENTRATION OF ADIPONECTIN, ON 21 DAYS OLD

OFFSPRING.

LAIS VALES MENNITTI; JULIANA LOPEZ DE OLIVEIRA; ANA CLAUDIA LOSINSKAS HACHUL; ALINE ALVES DE SANTANA; ALINE BOVETO SANTAMARINA; LILA MISSAE OYAMA; CLAUDIA MARIA DA PENHA OLLER DO NASCIMENTO; ELIANE BERALDI RIBEIRO; LUCIANA PELLEGRINI PISANI. UNIFESP, SANTOS - SP - BRASIL.

INTRODUCTION: Maternal nutrition during pregnancy and lactation plays a decisive role in the development of the fetus by epigenetic modifications resulting in a programming that induces changes in phenotype until adulthood. Prebiotics are fermented by the colonic bacteria and can alter the intestinal environment, reducing serum lipopolysaccharides (LPS) concentrations, benefiting the mother and the newborn. The objective this study was to evaluate the effect of oligofructose 10% supplementation during pregnancy and lactation, on the inflammatory status of 21 days old offspring. METHODS AND RESULTS: On the first day of pregnancy, rats were distributed into two groups: Control (C) or control supplemented with 10% of oligofructose (CF). The diets were maintained during pregnancy and lactation. At 21

th, pups were decapitated. Trunk blood was collected, centrifuged and serum was

separated. Adiponectin and LPS serum concentrations were quantified by ELISA. Results are presented as means±standard error of the mean. Statistical analysis was performed with t test Student. p<0.05 was considered significant. The oligofructose supplementation (10%) during pregnancy and lactation, increased serum concentration of LPS (n= 12-13), accompanied by a decreased in serum adiponectin (n= 16) of the 21 days old pups compared to control group. CONCLUSION: Oligofructose 10% during the pregnancy and lactation, probably impairs the intestinal permeability and alters the microbial composition, promoting an increase in serum LPS which can induces inflammatory responses through TLR4 activation, on 21 days old offspring.

Financial support: FAPESP, CAPES E CNPq.

APO - THE USE OF LABEL FREE ELECTRICAL IMPEDANCE SENSING TECHNOLOGIES FOR REAL TIME

ANALYSIS OF PRIMARY MURINE MACROPHAGE CHEMOTAXIS AND SIGNALLING

ASIF JILANI IQBAL1; DANIEL REGAN-KOMITO

2; IVY CHRISTOU

3; GEMMA WHITE

4; EILEEN MCNEILL

5; LEWIS

TAYLOR6; THEODORE KAPELLOS

7; KEITH CHANNON

8; DAVID GREAVES

9.

1,2,3,4,6,7,9.SIR WILLIAM DUNN SCHOOL OF PATHOLOGY, UNIVERSITY OF OXFORD, OXFORD - REINO

UNIDO; 5,8.CARDIOVASCULAR MEDICINE, UNIVERSITY OF OXFORD, OXFORD - REINO UNIDO.

Introduction

Leukocyte migration plays a fundamental role in immune homeostasis and inflammation. Chemokines direct

macrophage migration by binding to Gi-coupled receptors and inducing cytoskeletal rearrangement. Chemotaxis

assays have proven to be an invaluable system for studying macrophage responses to a range of inflammatory

mediators including CC chemokines. Conventional chemotaxis assays such as the modified Boyden chamber are

limited in terms of the data captured given that the assays are analysed at a single time-point. We have optimized and

validated a label-free, real-time cell migration/signalling assay based on electrical cell impedance (xCELLigence

RTCA-DP, developed by ACEA) to measure chemotaxis and adhesion of different primary murine macrophage

populations in response to a wide range of chemoattractant signalling molecules.

Methods and Results

Murine macrophages were placed in the upper chamber of a CIM-16 plate separated from the chemoattractant in the

lower chamber by a porous filter. Cell migration through the pores was measured by following changes in electrical

impedance (i.e. cell index; CI) on the gold electrode on the underside of the filter. The 96-E plate was used to monitor

adhesion and signalling responses. Bio-gel elicited macrophages displayed a rapid dose dependent response to

CCL3 with a max CI of 0.32 recorded at 5nM (p<0.001) with background CI of 0.05 (n=5 independent experiments).

Bone marrow derived macrophages displayed a similar dose dependent migration profile, with a max CI of 0.64 in

response to 5nM CCL3 (p<0.01), however a background impedance of 0.3 was observed indicating a high level of

non-directed migration. Pre-incubation of Bio-gel elicited macrophages with pertussis toxin (PTX) (200ng/ml) or

200nM cytochalasin D was shown to significantly ablate chemotaxis and adhesion in response to 10nM CCL2 or

CCL5 highlighting the role of G-protein dependent chemokine signaling (n=3 independent experiments; p<0.001).

Conclusion

Real time chemotaxis demonstrates important differences in the migratory behavior of different murine macrophage

populations and an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement. We are now using

this technology to study how macrophage chemotaxis and signaling is altered by a range of inflammatory mediators.

Funding: This research was supported by the British Heart Foundation (RG/10/15/28578)

TOPICAL MACADAMIA OIL AND PALMITOLEIC ACID ACCELERATE WOUND HEALING PHASES

ELEINE WEIMANN1; GILSON MASAHIRO MURATA

2; JOSÉ RICARDO BORTOLON

3; MAYSA BRAGA BARROS

SILVA4; RUI CURI

5; ELAINE HATANAKA

6.

1,3,4,6.INSTITUTE OF PHYSICAL ACTIVITY AND SPORT SCIENCE, CRUZEIRO DO SUL UNIVERSITY, SÃO

PAULO - SP - BRASIL; 2,5.INSTITUTE OF BIOMEDICAL SCIENCES, UNIVERSITY OF SÃO PAULO, SÃO PAULO -

SP - BRASIL.

Introduction: Wound healing defines a series of events involved in the restoration of integrity to injured tissue. This

process is divided into four phases: coagulation, inflammation, formation of granulation tissue with deposition of

extracellular matrix and remodeling. The healing process is finely regulated by a series of specific signals that are

triggered by different cell types that can be modulated by linoleic and oleic acids. Macadamia oil contains

approximately 60% oleic, 19% palmitoleic, 2% linoleic and 1% linolenic acids. Palmitoleic acid is found in the skin,

especially in young skin, and decreases with aging. Macadamia oil is used as a cosmetic, but little is known about the

mechanisms of action of this substance and its effects on wound healing. Macadamia oil presents high oxidative

stability, making it particularly suitable for heavy creams and other pharmaceutical formulations. Derivatives of

macadamia oil in cosmetics include the light emollient ethyl macadamiate and water soluble PEG-16 macadamia

glycerides. The aim of this study is to determine the effects of macadamia oil and palmitoleic acid on the healing

process based on in vivo studies.

Methods and Results: A wound was inflicted upon the dorsal surface of adult rats and macadamia oil, palmitoleic

acids and PBS were then administered topically. Both palmitoleic acid and macadamia oil increased the wound

healing ratio, accelerating all the wound healing phases. In the inflammatory phase, the number of neutrophils in the

inflammatory foci was found to decrease by 99% through the release of proinflammatory cytokines: IL-1b (by 84%),

CINC (by 91%), MIP (by 90%), IL-6 (by 98%) and L-selectin (by 26%) in response to treatment with palmitoleic acid

(±SEM of 8 animals per group).

Conclusion: These data suggest that topical macadamia oil and palmitoleic acids accelerate the phases of wound

healing by acting as immunomodulatory agents.

Financial support: FAPESP (11/15360-7) and CNPq (306041/2011-1 and 475841/2011-5).

TREADMILL EXERCISE INDUCES NEUTROPHIL RECRUITMENT INTO MUSCLE TISSUE IN A REACTIVE

OXYGEN SPECIES-DEPENDENT MANNER. AN INTRAVITAL MICROSCOPY STUDY.

ALBENÁ NUNES SILVA1; PRISCILA TELLES TOLEDO BERNARDES

2; BARBARA MAXIMINO REZENDE

3; ELISA

COUTO GOMES4; PEDRO ELIAS MARQUES

5; PEDRO HENRIQUE RODRIGUES

6; GUSTAVO BATISTA

MENEZES7; MAURO MARTINS TEIXEIRA

8; VANESSA PINHO SILVA

9.

1,2,3,5,6,7,8,9.ICB - UFMG, BELO HORIZONTE - MG - BRASIL; 4.EDINBURGH NAPIER UNIVERSITY,

EDINBURGH - REINO UNIDO.

ABSTRACT

Introduction: Intense exercise is physiological stress capable of inducing the interaction of neutrophils with muscle

endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are

not known. Here we investigate whether production of reactive oxygen species is relevant for neutrophil interaction

with endothelial cells and recruitment into muscle in mice subjected to treadmill exercise to fatigue. Methods and

Results: Mice exercised until fatigued running for 56.3 ± 6.8 minutes on an electric treadmill. Skeletal muscle was

evaluated by intravital microscopy at different time points after exercise and then removed to assess local oxidative

stress and histopathological analysis. Results show an increase in the plasma lactate (4,0 ± 1,0 to 5.9 ± 1,3 mmol/dL)

immediately after exercise. The serum levels of CK also increased 12 h (691,6 ± 114,6 U/L) after exercise. The

numbers of total cells (28 ± to 71 x 105/ml), monocytes (0.8 ± 0,7 to 27,2 ± 8,1 10

5/ml), neutrophils (8,2 ± 2,8 to 22,4 ±

3,4 105/ml) and lymphocytes (18,7 ± 7,8 to 29,8 ± 21,8 10

5/ml) was elevated in the blood at 12h after exercise.

Intravital microscopy analysis of the post-capillary venule of quadriceps muscle demonstrates that the number of

rolling (10,2 ± 2,6 to 129,5 ± 20,2 cells/min) and adherent (0,5 ± 0,5 to 4,2 ± 1,0 cells/min) leukocytes increased 12

hours after exercise. Using LysM-eGFP mice and confocal intravital microscopy technology, we could show that the

number of transmigrating neutrophils (1,2 ± 1,2 to 2,7 ± 1,3 cell/20min) increased 12h post-exercise. Mutant gp91phox-/-

mice and mice treated with apocynin impaired neutrophil recruitment 12 h after exercise. SOD treatment promoted

further adhesion (0 to 8,2 ± 1,9 cell/min) of leukocytes. In a presence of SOD the number of neutrophil in a

transmigration process also increased (1,2 ± 1,2 to 5,2 0,9 cells/20min) at the same time-point. Conclusion: These

findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary

venules and NADPH oxidase-induced ROS play an important role in this process.

Financial support: CAPES, FAPEMIG E CNPq.

TROPHOBLAST INVASION IS TRIGGERED BY SERUM AMYLOID A VIA TOLL-LIKE RECEPTOR-4

SILVANA SANDRI1; ALEXANDRE URBAN BORBELY

2; ISABELLA FERNANDES

3; EDSON MENDES DE

OLIVEIRA4; FRANCIELE HINTERHOLZ KNEBEL

5; FABIOLA BRANCO FILIPPIN-MONTEIRO

6; ESTELA

BEVILACQUA7; ANA CAMPA

8.

1,4,5,8.DEPTO DE ANÁLISES CLÍNICAS E TOXICOLÓGICAS, FAC DE CIÊNCIAS FARMACÊUTICAS,

UNIVERSIDADE DE SÃO, SÃO PAULO - SP - BRASIL; 2,7.DEPTO DE BIOL. CELULAR E DO

DESENVOLVIMENTO, INST. DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP -

BRASIL; 3.DEPTO DE CIRURGIA, FAC. DE VETERINÁRIA E ZOOTECNIA, UNIVERSIDADE DE SÃO PAULO, SÃO

PAULO - SP - BRASIL; 6.DEPARTAMENTO DE ANÁLISES CLÍNICAS, FAC. DE FARMÁCIA, UNIVERSIDADE

FEDERAL DE SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL.

Introduction: Well-known functions of the protein serum amyloid A (SAA) are associated with acute phase response

and immunoregulation. Recently, it has been described its involvement in cell proliferation, differentiation and

migratory behavior of different cell types. The wide range of cells that express SAA and in which it triggers some

effects include leukocytes, adipocytes, gliomas and synoviocytes. Trophoblast cells also express SAA although its

biological effects are still unknown. Herein, it is evaluated whether exogenous SAA has effect in trophoblast invasion

using the trophoblast-like BeWo cell line and primary term basal plate extravillous trophoblast cells (EVT). Material

and Results: BeWo cells and EVT isolated of basal plate from term placenta were maintained in DMEM/F12

supplemented with 10% of FBS. The invasion was assessed by Matrigel-coated transwell. mRNA and activity of

metaloproteinases were evaluated by qPCR and Biotrak Activity assay system (Ge-healthcare), respectively. SAA

increased the BeWo cell invasion and metalloproteinases expression and activity. BeWo cell expressed Toll-like

receptor 4 (TLR-4), a known receptor for SAA and its participation in the SAA-induced invasion was identified using a

TLR-4 neutralizing antibody. As observed for BeWo, the invasion of isolated term EVT was improved by SAA and

restrained by anti-TLR4. Also, SAA expression was observed in BeWo cells, and syncytiotrophoblast, decidual cells

and EVT in term placenta.Conclusion: SAA was identified as a functional molecule in placental microenvironment,

controlling MMPs and trophoblast invasion, key process in placentation and placenta homeostasis.

Financial support: FAPESP