Orientador: Erick José Ramo da Silva / Resumo: O epidídimo é responsável por transformar os espermatozoides imaturos em células funcionalmente competentes, capazes de adquirir motilidade progressiva e de reconhecer e fertilizar o oócito. Além disso, o epidídimo é primordial para proteger e armazenar o gameta masculino antes da ejaculação. Alterações da fisiologia epididimária podem afetar a sua função, causando infertilidade masculina. A inflamação do epidídimo, conhecida como epididimite, é uma das doenças mais prevalentes do trato urogenital masculino, sendo um fator relevante de infertilidade masculina. Sua principal etiologia envolve a invasão de bactérias, como Escherichia coli, via ascensão retrógrada pela uretra. O epidídimo expressa constitutivamente diversos componentes do sistema imunológico inato, incluindo os receptores do tipo Toll (TLRs), como o TLR4, que é ativado pelo lipopolissacarídeo (LPS) de bactérias Gram-negativas, além de proteínas com atividade antimicrobiana, como os inibidores da protease do tipo WFDC (Whey-acidic protein four disufide core), indicando que este órgão está em constante estado de alerta para combater infecções bacterianas. Além da ação antimicrobiana e inibidora de protease, as proteínas WFDC apresentam ação anti-inflamatória, imunomoduladora e sobre a função espermática, sendo proteínas multifuncionais no trato reprodutor masculino. Neste estudo, testamos a hipótese de que a expressão de genes Wfdc no epidídimo é modificada por estímulos inflamatórios, como parte da resposta tecid... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The epididymis is responsible for transforming immature sperm into functionally competent cells capable of acquiring progressive motility and recognizing and fertilizing the oocyte. In addition, the epididymis is paramount for protecting and storing the male gamete before ejaculation. Changes in epididymal physiology may affect sperm maturation, transport or storage, causing male infertility. Epididymal inflammation, known as epididymitis, is one of the most prevalent diseases of the male urogenital tract, being a relevant factor of male infertility. Its main etiology involves the invasion of bacteria, such as E. coli, through retrograde ascension through the urethra. The epididymis constitutively expresses several elements of the innate immune system, including Toll-like receptors (TLRs), like the TLR4, which is activated by the lipopolysaccharide (LPS) of Gram-negative bacteria, as well as proteins with antimicrobial activity, like the WFDC (Whey-acidic difulfide core)-type protease inhibitors, indicating that this organ is constantly alert to fight bacterial infections. In addition to antimicrobial and protease inhibitory activities, WFDC proteins show anti-inflammatory, immunomodulatory and reproductive functions, being multifunctional proteins in the male reproductive tract. In this study, we tested the hypothesis that Wfdc gene expression in the epididymis is modified by inflammatory stimuli as part of tissue defense mechanisms to microbial aggression. For that, we char... (Complete abstract click electronic access below) / Mestre

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

As phosphorylation represents the pivotal mechanism for regulation of biological processes, kinases belong to one of the most biologically significant enzyme classes. The development of analytical techniques for characterization of kinase activity, in particular at a global scale, is a central priority for proteomic and cell biology researchers. In order to facilitate global analysis of cellular phosphorylation, a new paradigm of microarray technology which focuses on analysis of total cellular kinase activity, kinome, has emerged in the past few years. As the specificity of many kinases is dictated primarily by recognition of residues immediately surrounding the site of phosphorylation a logical methodology is to employ peptides representing these immediate sequences as experimental substrates. Microarray chips carrying hundreds of such substrate targets have been developed for human kinome analysis, however, lack of similar tools for species outside research mainstream has limited kinome analysis in these species.<p> Based on sequence alignment of orthologous phosphoproteins from mammalian species, conservation of amino acid identity is reported to be 80 %. Accordingly, the potential exists to utilize phosphorylation sequence databases to extrapolate phosphorylation sites in other species based on their genomic sequence information. Peptides representing these proposed phosphorylation sites can then be utilized as substrates to quantify the activity of the corresponding kinase. Based on these principles, a bovine microarray of 300 unique peptide targets was constructed. The bovine phosphorylation targets were selected to represent a spectrum of cellular events but with focus on processes related to innate immunity. Initial application and validation of the bovine peptide arrays was carried out for kinome analysis of bovine blood monocytes stimulated with either lipopolysaccharide (LPS) or CpG-ODNs; ligands for Toll-like receptors (TLR) 4 and 9, respectively. The arrays confirmed activation of the known TLR signaling pathway as well as identifying receptor-specific phosphorylation events. Phosphorylation events not previously attributed to TLR activation were also identified and validated by independent bioassays. This investigation offers insight into the complexity of TLR signaling and more importantly verifies the potential to use bioinformatics approaches to create tools for species-specific kinome analysis based on genomic information.

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

As phosphorylation represents the pivotal mechanism for regulation of biological processes, kinases belong to one of the most biologically significant enzyme classes. The development of analytical techniques for characterization of kinase activity, in particular at a global scale, is a central priority for proteomic and cell biology researchers. In order to facilitate global analysis of cellular phosphorylation, a new paradigm of microarray technology which focuses on analysis of total cellular kinase activity, kinome, has emerged in the past few years. As the specificity of many kinases is dictated primarily by recognition of residues immediately surrounding the site of phosphorylation a logical methodology is to employ peptides representing these immediate sequences as experimental substrates. Microarray chips carrying hundreds of such substrate targets have been developed for human kinome analysis, however, lack of similar tools for species outside research mainstream has limited kinome analysis in these species.<p> Based on sequence alignment of orthologous phosphoproteins from mammalian species, conservation of amino acid identity is reported to be 80 %. Accordingly, the potential exists to utilize phosphorylation sequence databases to extrapolate phosphorylation sites in other species based on their genomic sequence information. Peptides representing these proposed phosphorylation sites can then be utilized as substrates to quantify the activity of the corresponding kinase. Based on these principles, a bovine microarray of 300 unique peptide targets was constructed. The bovine phosphorylation targets were selected to represent a spectrum of cellular events but with focus on processes related to innate immunity. Initial application and validation of the bovine peptide arrays was carried out for kinome analysis of bovine blood monocytes stimulated with either lipopolysaccharide (LPS) or CpG-ODNs; ligands for Toll-like receptors (TLR) 4 and 9, respectively. The arrays confirmed activation of the known TLR signaling pathway as well as identifying receptor-specific phosphorylation events. Phosphorylation events not previously attributed to TLR activation were also identified and validated by independent bioassays. This investigation offers insight into the complexity of TLR signaling and more importantly verifies the potential to use bioinformatics approaches to create tools for species-specific kinome analysis based on genomic information.

Mikroglia sind residente Makrophagen-artige Zellen des Zentralnervensystems (ZNS), die das Gewebe kontinuierlich auf Anzeichen homöostatischer Störungen überwachen. Als die wesentlichen immunkompetenten Effektorzellen im Hirnparenchym exprimieren sie eine Vielzahl von Rezeptoren für pathogen-assoziierte molekulare Strukturmuster (pathogen-associated molecular patterns, PAMPs). Zu diesen Rezeptoren zählt der Toll-like receptor (TLR) 4, der nicht nur Reaktionen der Mikroglia auf bakterielle Infektionen, sondern auch auf Gewebe Schädigungen ermöglicht. Stimulation des TLR4 mit bakteriellem Lipopolysaccharid (LPS) und endogenen schädigung-sassoziierten molekularen Strukturen (damage-associated molecular patterns, DAMPs), die durch Gewebebeeinträchtigung freigesetzt werden, löst sowohl TRIF- als auch MyD88-abhängige Signalkaskaden aus. Die damit induzierte Freisetzung von Zytokinen und Chemokinen rekrutiert und instruiert periphere Immunzellen für eine Protektion und unterstützende Geweberegeneration des ZNS. Wir zeigen hier, dass der TLR4-Korezeptor CD14 ein essenzieller gate keeper für die Generierung von Immunantworten im ZNS ist, die durch LPS oder E. coli-Verabreichung, aber auch durch mechanisches Trauma und ischämischen Schlaganfall ausgelöst werden. In gewissem Gegensatz zu extraneuralen Makrophagen nutzen Mikroglia CD14 zur Erlangung einer extremen Sensitivität gegenüber sehr geringen LPS-Mengen. Gleichzeitig schützt CD14 Mikroglia vor überschießenden Reaktionen auf hohe LPS-Dosen und verhindert dabei insbesondere die exzessive Produktion von CXCL1, eines chemoattraktiven Signals für neutrophile Granulozyten. Entsprechend unterstützt CD14 die ZNS-Rekrutierung von Monozyten und Neutrophilen durch niedrige LPS-Dosen, während es die verstärkte Einwanderung von Neutrophilen durch hohe Dosen von LPS oder E. coli verhindert. Als eine besonders wichtige Funktion beschreiben wir dabei die absolute CD14-Abhängigkeit DAMP-ausgelöster und TLR4-vermittelter Immunreaktionen. CD14-Defizienz (unter cd14-/--Bedingungen) oder CD14 Blockade (durch Antikörper) löschen mikrogliale Reaktionen, die durch Plasma-Fibronektin (als repräsentatives DAMP-Molekül) ausgelöst werden können, komplett aus und beeinträchtigen die Leukozyten-Infiltration nach ZNS-Trauma. Bei einer ischämischen ZNS-Schädigung weisen cd14-/--Mäuse im Gehirn nicht nur weniger Monozyten auf, sondern gleichzeitig ein vergrößertes Infarktvolumen. Wir konnten für Interferon (IFN) b eine Schlüsselfunktion in der CD14-vermittelten Eindämmung der CXCL1-Synthese darstellen, die auf eine negative CD14/TLR4-TRIF-IFNβ-INAR1-Jak- Rückkopplung für MyD88-getriebene Chemokine schließen lässt. Obwohl CD14 somit TLR4-vermittelte Reaktionen auf infektiöse und nicht-infektiöse Agenzien orchestriert, wird seine Expression durch verschiedene TLR-Liganden und Zytokine reguliert. Letztlich unterliegen damit CD14-kontrollierte Funktionen selbst einer komplexen Kontrolle durch ZNS-residente und eingewanderte periphere Zellen. Diese Regulationen können über die Einbeziehung oder den Ausschluss der Kapazitäten des TLR4-Komplexes für eine Schadenserkennung während der ZNS-Reaktionen in unterschiedlichsten pathologischen Szenarien entscheiden.

Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A-/-), TLR4-/- and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4-/- or SR-A-/- macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.

Recent studies have shown that Toll-like receptors (TLRs) are involved in cerebral ischemia/reperfusion (I/R) injury. This study was to investigate the role of TLR2 and TLR4 in acute focal cerebral I/R injury. Cerebral infarct size, neurological function and mortality were evaluated. NFk{cyrillic}B binding activity, phosphorylation of Ik{cyrillic}Bα, Akt and ERK1/2 were examined in ischemic cerebral tissue by EMSA and Western blots. Compared to wild type (WT) mice, in TLR4 knockout (TLR4KO) mice, brain infarct size was decreased (2.6 ± 1.18% vs 11.6 ± 1.97% of whole cerebral volume, p < 0.05) and neurological function was maintained (7.3 ± 0.79 vs 4.7 ± 0.68, p < 0.05). However, compared to TLR4KO mice, TLR2 knockout (TLR2KO) mice showed higher mortality (38.2% vs 13.0%, p < 0.05), decreased neurological function (2.9 ± 0.53 vs 7.3 ± 0.79, p < 0.05) and increased brain infarct size (19.1 ± 1.33% vs 2.6 ± 1.18%, p < 0.05). NFk{cyrillic}B activation and Ik{cyrillic}Bα phosphorylation were attenuated in TLR4KO mice (1.09 ± 0.02 and 1.2 ± 0.04) compared to TLR2KO mice (1.31 ± 0.02 and 2.2 ± 0.32) after cerebral ischemia. Compared to TLR4KO mice, in TLR2KO mice, the phosphorylation of Akt (0.2 ± 0.03 vs 0.9 ± 0.16, p < 0.05) and ERK1/2 (0.8 ± 0.06 vs 1.3 ± 0.17) evoked by cerebral I/R was attenuated. The present study demonstrates that TLR2 and TLR4 play differential roles in acute cerebral I/R injury. Specifically, TLR4 contributes to cerebral I/R injury, while TLR2 appears to be neuroprotective by enhancing the activation of protective signaling in response to cerebral I/R.

Activation of TLRs mediated the NF-κB signaling pathway plays an important pathophysiological role in cardiac hypertrophy. Triad3A, a ubiquitin E3 ligase, has been reported to negatively regulate NF-κB activation pathway via promoting ubiquitination and degradation of TLR4 and TLR9 in innate immune cells. The role of Triad3A in cardiac hypertrophic development remains unknown. The present study investigated whether there is a link between Triad3A and TLR4 and TLR9 in pressure overload induced cardiac hypertrophy. We observed that Triad3A levels were markedly reduced following transverse aortic constriction (TAC) induced cardiac hypertrophy. Similarly, stimulation of neonatal rat cardiac myocytes (NRCMs) with angiotensin-II (Ang II) significantly decreased Triad3A expression. To determine the role of Triad3A in TAC-induced cardiac hypertrophy, we transduced the myocardium with adenovirus expressing Triad3A followed by induction of TAC. We observed that increased expression of Triad3A significantly attenuated cardiac hypertrophy and improved cardiac function. To investigate the mechanisms by which Triad3A attenuated cardiac hypertrophy, we examined the Triad3A E3 ubiquitination on TLR4 and TLR9. We found that Triad3A promoted TLR4 and TLR9 degradation through ubiquitination. Triad3A mediated TLR4 and TLR9 degradation resulted in suppression of NF-κB activation. Our data suggest that Triad3A plays a protective role in the development of cardiac hypertrophy, at least through catalyzing ubiquitination-mediated degradation of TLR4 and TLR9, thus negatively regulating NF-κB activation.