Neutrophils as key effector cells of the innate immune system migrate from the circulation into sites of inflammation and are essential for the containment, killing and clearance of invading pathogens through a variety of highly regulated cell functions. Despite this beneficial role their involvement can also be detrimental. In a number of diseases dysregulated neutrophil influx and activation results in significant tissue damage and worsening of the acute inflammatory event as well as long term tissue injury, scarring and fibrosis. One such pulmonary condition is acute respiratory distress syndrome (ARDS) which, despite decades of intensive research and multiple clinical trials, remains without a cure and has an associated mortality rate of approximately 40%. Delineating and understanding the key pathogenic mediators that drive neutrophil recruitment into the lung in the context of both bacterial and sterile injury is therefore vital in the development of novel therapies. Neutrophils migrate towards a variety of agents but amongst such factors a hierarchy exists with bacterial-derived products, including formylated peptides, dominant in this process. In sterile tissue injury where no bacterial factors are present the mediators involved change but a hierarchy still exists. Mitochondrial formylated peptides are released following necrotic cell death and bind to formyl peptide receptor 1 (FPR1) on the neutrophil surface inducing migration and activation. Like bacterial formylated peptides they are powerful chemoattractants and are therefore likely to be important in recruiting neutrophils to sites of injury and inflammation. Hypothesis: The central hypothesis of this thesis is that mitochondrial formylated peptides, as end-target chemoattractants, are elevated in patients with ARDS and drive neutrophil recruitment through binding to FPR1. Inhibition of FPR1 in models of acute lung injury will therefore result in attenuation of this inflammatory response through multiple FPR1-mediated effects implicating both formylated peptides and their cognate receptor in the pathogenesis of sterile ARDS. Results: Free mitochondrial DNA and formylated peptides were elevated in the circulation of patients with ARDS or severe paracetamol-induced hepatic failure relative to healthy controls. In addition, FPR1 receptor number was increased on the surface of neutrophils isolated from critically ill septic patients. Isolated mitochondrial formylated peptides induced FPR1- dependent chemotaxis in primary human neutrophils in vitro. Alongside this, FPR1 ligand binding resulted in increased cell surface β2-integrin expression [integrin alpha M beta 2 (ITGB2); also called CD11b/CD18, Mac-1 or CR3] through intracellular activation of PI-3Kand MAPK-dependent signalling pathways. Indeed, blockade of neutrophil cell-surface integrin alpha M (ITGAM; also known as CD11b)) resulted in a reduction in mitochondrial formylated peptide-induced chemotaxis. To determine the production of human neutrophil IL-1β, a pivotal chemokine within a sterile inflammatory environment, a novel method for the in vitro isolation of ultrapure neutrophils was developed. Neutrophils were isolated by autofluorescence-based flow sorting as determined by intrinsic differences in neutrophil and eosinophil autofluorescence and their size and granularity relative to circulating mononuclear cells. Analysis of this approach demonstrated the ability to rapidly collect a highly pure neutrophil population (99.95 ± 0.03%). Flow sorting did not alter the activation state or functional capacity of these cells relative to unsorted neutrophils with regards to several measures of neutrophil behaviour/function. Cells also remained fully responsive to a variety of neutrophil agonists with no evidence of neutrophil priming. The capacity of highly pure neutrophils to secrete IL-1β was determined to be approximately 160-fold lower than equivalent numbers of circulating peripheral blood mononuclear cells. In the context of an inflammatory environment however this is likely to be of biological significance given the large number of infiltrating neutrophils. In sterile hydrochloric acid-induced acute lung injury pharmacological inhibition of FPR1 with cyclosporin H (CsH), or use of transgenic FPR1-/- mice, resulted in inhibition of neutrophil migration into the alveolar space 24 hours after injury. This was associated with a reduction in pulmonary haemorrhage, extravascular protein leak and pro-inflammatory cytokine expression with improved histological appearances. Furthermore, the HCl acid-induced reduction in alveolar macrophage numbers was inhibited by CsH with interstitial macrophages displaying an alternatively activated phenotype. Importantly, delivery of CsH 12 hours after the onset of injury also reduced acute lung inflammation demonstrating its potential therapeutic relevance in the treatment of human disease. In non-sterile E. coli-mediated acute lung injury partial antagonism of FPR1 with CsH resulted in a reduction in neutrophil migration and vascular leak with no effect on pulmonary bacterial load. A narrow therapeutic window existed however as increased concentrations of CsH, or infection in FPR1-/- mice, resulted in a reduction in alveolar neutrophil number and increase in E. coli at 24 hours. Alongside effects on myeloid cells within the lung FPR1 was found to be expressed on mouse lung epithelial cells. A technique to isolate and culture mouse type 1 alveolar epithelial (AT1) cells was therefore developed. Flow sorting of anti-type 1 alpha (anti-T1α) stained single cell lung homogenates with subsequent culture on transwell membranes resulted in the development of confluent AT1 cell monolayers after 10 days. Formylated peptides appear to induce a reduction in transepithelial resistance and increase in permeability across a monolayer in vitro alongside an increase in release of the neutrophil chemo-attractant mouse CXCL8 (KC). Conclusions: Taken together, mitochondrial formylated peptides released following cell necrosis and FPR1 play a significant role in the pathogenesis of sterile acute lung injury. This is likely to be predominantly through neutrophil-dependent means but data presented here also suggests that their role in macrophage function and alveolar epithelial cell permeability may be important. Inhibition of FPR1 may therefore represent a novel and multi-cellular therapeutic target in the treatment of ARDS.

Yes / The formylpeptide receptor-1 (FPR1) is a member of the chemotactic GPCR-7TM formyl peptide receptor family, whose principle function is in trafficking of various leukocytes into sites of bacterial infection and inflammation. More recently, FPR1 has been shown to be expressed in different types of cancer and in this context, plays a significant role in their expansion, resistance and recurrence. ICT12035 is a selective and potent (30 nM in calcium mobilisation assay) small molecule FPR1 antagonist. Here, we demonstrate the efficacy of ICT12035, in a number of 2D and 3D proliferation and invasion in vitro assays and an in vivo model. Our results demonstrate that targeting FPR1 by a selective small molecule antagonist, such as ICT12035, can provide a new avenue for the treatment of cancers.

Submitted by NATHÁLIA MACIEL MANIEZZO STUCHI null (nmmbiomedica@hotmail.com) on 2016-07-28T14:13:21Z No. of bitstreams: 1 Tese capa dura.pdf: 5464428 bytes, checksum: f53c801765e2d85e4f507ebaaebe4625 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-07-28T15:52:03Z (GMT) No. of bitstreams: 1 stuchi_nmm_dr_sjrp.pdf: 5464428 bytes, checksum: f53c801765e2d85e4f507ebaaebe4625 (MD5) / Made available in DSpace on 2016-07-28T15:52:03Z (GMT). No. of bitstreams: 1 stuchi_nmm_dr_sjrp.pdf: 5464428 bytes, checksum: f53c801765e2d85e4f507ebaaebe4625 (MD5) Previous issue date: 2016-05-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Apesar do declínio da incidência, o câncer gástrico ocupa ainda a terceira posição em causa de morte por câncer no mundo, tendo como principal fator de risco a bactéria Helicobacter pylori. Esta bactéria pode levar a uma inflamação persistente através da produção de citocinas pró-inflamatórias e de espécies reativas de oxigênio e nitrogênio, estimulando a proliferação celular bem como outros processos envolvidos na carcinogênese. Ainda envolvidos nestes processos tem sido observada a participação de microRNAs, que exercem papel importante na regulação pós-transcricional, influenciando processos fisiológicos normais da célula bem como aqueles ligados às doenças, como por exemplo o câncer gástrico. Alguns miRNAs podem atuar como oncogenes, genes supressores de tumor e biomarcadores para diversas patologias, podendo alvejar genes relacionados com inflamação e câncer como o gene ANXA1 (Anexina-A1). A Anexina-A1 é uma proteína anti-inflamatória e com ação anti-proliferativa, que se liga à receptores do tipo formil peptídeo como por exemplo FPR1 e FPR2, ambos sabidamente relacionados com a progressão de doenças como o câncer. Desta forma o presente trabalho teve como objetivos avaliar a expressão da proteína Anexina A1 e seus receptores FPR1 e FPR2, bem como avaliar a expressão do RNAm da ANXA1 e de miRNAs que possam modular a expressão desse gene (hsa-mir-27a, hsa-mir-196a e hsa-mir-222) em adenocarcinoma gástrico e correlacionar estes resultados com os aspectos clínico-patológicos. Foram avaliadas 31 amostras de adenocarcinoma gástrico, assim como as regiões metaplásica ou normal adjacentes ao tumor. A quantificação relativa (RQ) do RNAm da ANXA1 e miRNAs foi realizada por PCR quantitativo em tempo real utilizando ensaio TaqMan, e a expressão proteica da AnxA1, FPR1 e FPR2 por imuno-histoquímica e análise densitométrica. Em relação à expressão gênica relativa foram observados o aumento da expressão da ANXA1 nos tumores (RQ=1,374; p<0,001), assim como do miR-196a que apresentou aumento de expressão tanto no tecido metaplásico (RQ= 4,784; p=0,0016) e tumoral (RQ=16,99; p< 0,001) em relação ao tecido normal. O miR-27a não se apresentou diferencialmente expresso nos diferentes tecidos e houve diminuição da expressão do miR-222 (RQ=0,687; p=0,01) no tecido tumoral em relação ao tecido normal. Apenas o miR-196a e a ANXA1 apresentaram correlação significantemente inversa (r= -0,55; p=0,003), e este miRNA foi o único que apresentou associação com o sexo feminino, devido aumento de expressão em mulheres. Quando se comparou a expressão relativa aos parâmetros clínico-patológicos e tumorais não foram encontradas diferenças significativas. Quanto à expressão proteica o FPR2 não apresentou marcação no tecido normal, metaplásico e tumoral. Contudo a AnxA1 e FPR1 apresentaram imunomarcação positiva amplamente distribuída no tecido tumoral e positivamente correlacionados tanto no epitélio (r=0,87; p<0,0001) como estroma (r=0,62; p=0,004). Portanto, nossos resultados sugerem que a ANXA1 é modulada pelo miR-196a em câncer gástrico, e evidenciam que tanto a AnxA1 como o FPR1 também estão envolvidos no processo de carcinogênese gástrica. Tais achados podem abrir possibilidades para futuros estudos sobre novos alvos terapêuticos já que AnxA1 e FPR1 são possíveis alvos farmacológicos, e as terapias baseadas em microRNAs tem sido amplamente pesquisadas. / Gastric cancer still ranks third in cause of cancer death worldwide, despite the decline in its incidence, being Helicobacter pylori the main risk factor for this disease. This bacterium can lead to persistent inflammation via the production of proinflammatory cytokines and reactive oxygen and nitrogen species, stimulating cell proliferation and other processes involved in carcinogenesis. Still involved in this process, it has been observed the participation of miRNAs, which play an important role in post- transcriptional regulation, influencing normal physiological processes of the cell as well as those linked to diseases such as gastric cancer. Some miRNAs can act as oncogenes, tumor suppressor genes and biomarkers for various diseases, can targetting genes linked to inflammation and cancer such as ANXA1 gene (Annexin A1). Annexin A1 is an anti-inflammatory protein with anti-proliferative action that binds to formyl peptide receptors such as, FPR1 and FPR2, both known to be related to the progression of diseases such as cancer. Thus, the present study aimed to evaluate the expression of Annexin A1, FPR1 and FPR2 receptors, and the expression of mRNA ANXA1 and miRNAs (hsa-mir-27a, hsa-mir-196a and hsa-miR 222) in gastric adenocarcinoma, also correlate these results to the clinicopathological features. 31 adenocarcinoma samples were evaluated, as well as normal or metaplastic region adjacent to the tumor. Relative quantification (RQ) of miRNA and mRNA ANXA1 was evaluated by TaqMan assay and protein expression AnxA1, FPR1 and FPR2 by immunohistochemistry and densitometry analysis. Regarding the relative expression the following results were observed, increased expression in tumors of ANXA1 (RQ = 1.374; p < 0.001) and miR- 196a that showed increased expression it he metaplastic tissue (RQ = 4.784; p = 0.0016) and tumor (RQ = 16.99; p < 0.001) compared to normal tissue. The miR- 27a did not appear differentially expressed in different tissues and there was a decrease of miR -222 expression (RQ = 0.687; p = 0.01) in tumor tissue compared to normal tissue. Only the miR-196a and ANXA1 presented a significant inverse correlation (r = -0.55; p = 0.003), and this miRNA was the only one that showed association to female gender, due to increased expression in women when comparing the relative expression to clinicopathological parameters and tumor features. The FPR2 was not expressed in normal, metaplasic and tumor tissue. However, the AnxA1 and FPR1 were widely distributed positive immunostaining in tumor tissue and positively correlated both in the epithelium (r = 0.87 ; p < 0.0001) and stromal (r = 0.62 ; p = 0.004). Thus, our results suggest that ANXA1 is modulated by miR-196a in gastric cancer, and evidencing that both AnxA1 and FPR1 are also involved in gastric carcinogenesis process. These data open the possibility for future studies on new therapeutic targets since AnxA1 and FPR1 are adjustable pharmacological targets, and microRNAs ` therapies have been widely researched. / FAPESP: 2012/15036-8 / CNPq: 474776/2013-1

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-02-13T17:19:32Z No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-28T12:19:20Z (GMT) No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / Made available in DSpace on 2017-06-28T12:19:20Z (GMT). No. of bitstreams: 1 Dissertação Ivonete Queiroz.pdf: 1073414 bytes, checksum: af4071808ba3af365237d21558158bf4 (MD5) / CAPES / A hanseníase é uma doença infectocontagiosa crônica, causada pelo Mycobacterium leprae, caracterizada por lesões na pele e ampla apresentação clínica, a qual depende principalmente da resposta imunológica do hospedeiro frente à infecção pelo Mycobacterium leprae. Esta infecção está distribuída mundialmente persistindo como grave problema de saúde pública no Brasil. As incapacidades e deformidades duradouras decorrentes dos quadros reacionais podem ocorrer antes, durante ou após a instituição do tratamento específico. Os episódios reacionais inflamatórios são decorrentes da exacerbação abrupta da resposta imune mediada por célula contra antígenos do M. leprae. O episódio reacional tipo 1 ou reação reversa é uma ocorrência inflamatória aguda que acomete a pele e nervos periféricos, sendo causa comum de incapacidade física. A reação tipo 2 ou eritema nodoso hansênico acomete somente pacientes virchowianos (LL) e borderline-virchowianos (BL) com presença de muitos bacilos e pouca ou nenhuma imunidade celular ao M. leprae. O objetivo principal deste estudo foi avaliar a moléculas MARCO Scavenger, Receptor peptídeo formil 1(FPR1) e Receptor peptídeo N-formil 2 (FPR2) em monócitos de pacientes com hanseníase em associação ao desenvolvimento de reações hansênicas, para alcançar este objetivo células mononucleares do sangue periférico foram obtidas de pacientes com diferentes formas clínicas da hanseníase. A marcação de superfície celular foi realizada por citometria de fluxo e os dados analisados com FlowJo em 35 pacientes com hanseníase, divididos em 19 paucibacilar e 16 multibacilar. Os nossos resultados mostraram que os pacientes com a forma paucibacilar apresentam a frequência de monócitos intermediários aumentada. Não houve diferença estatística na média de intensidade de fluorescência (MFI) de CD80, CD86 e MHC II entre monócitos de pacientes com a forma paucibacilar e multibacilar da hanseníase. No entanto, monócitos intermediários de pacientes com a forma multibacilar apresentaram média de intensidade de fluorescência maior em CD86 e MCHII. Os nossos dados sugerem que o receptor FPR1 pode ter um papel relevante para detecção na amplificação da resposta inflamatória de indivíduos com hanseníase quando comparado com as outras moléculas aplicadas neste estudo. / Leprosy is a chronic infectious disease caused by Mycobacterium leprae, characterized by skin lesions and extensive clinical presentation which depends mainly on the immune front of the host response to infection by Mycobacterium leprae. This infection is distributed worldwide persist as a serious public health problem in Brazil. Disability and lasting deformities resulting from reaction symptoms can occur before, during or after the institution of specific treatment. Reactional inflammatory episodes are due to the abrupt exacerbation of immune cell-mediated response against M. leprae antigens. The episode reaction type 1 or reverse reaction is an acute inflammatory occurrence that affects the skin and peripheral nerves, and common cause of disability. Reaction type 2 or ENL only affects lepromatous patients (LL) and borderline-lepromatous (BL) with the presence of many bacilli and little or no cellular immunity to M. leprae. To assess the association of biomarkers MARCO Scavenger, Formyl peptide receptor (FPR1) and N-formyl peptide receptor 2 (FPR2) in monocytes of patients with leprosy associated with the development of leprosy reactions, mononuclear cells from peripheral blood were obtained from patients with different clinical forms of leprosy. To achieve this goal peripheral blood mononuclear cells were obtained from patients with different clinical forms of leprosy. The cell surface stain was performed by flow cytometry and the data analyzed with FlowJo in 35 patients with leprosy, divided into 19 paucibacillary and 16 multibacillary patients. Our results showed that patients with the paucibacillary form exhibit increased monocyte frequency. There was no statistical difference in the mean fluorescence intensity (MFI) of CD80, CD86 and MHC II between monocytes of paucibacillary and multibacillary leprosy patients. However, intermediate monocytes from patients with the multibacillary form had higher mean fluorescence intensity in CD86 and MCHII. Our data suggest that the FPR1 receptor may play a relevant role in detecting the amplification of the inflammatory response of individuals with leprosy when compared with the other molecules applied in this study.