Legionella longbeachae é uma espécie da família Legionellaceae que é comumente presente no solo em diversas regiões do globo. Uma infecção por L. longbeachae em indivíduos imunocomprometidos causa uma pneumonia severa, frequentemente levando a hospitalização e à morte. A prevalência destas bactérias como causa de pneumonia é grande, e certamente sub-estimada, uma vez que os métodos de diagnóstico convencionais detectam apenas as espécies de Legionella pneumophila. A anafilatoxina C5a è uma proteína inflamatória ativada pelo complemento, a qual é envolvida no recrutamento de células inflamatórias, um processo induzido pelas células da imunidade inata que leva a dano tecidual. Dados recentes gerados no nosso laboratório sugerem que a mortalidade de camundongos após a infecção por L. longbeachae é causada por uma falência pulmonar, associada a indução de um intenso processo inflamatório nos pulmões dos animais infectados. Nesse trabalho, nós investigamos papel do receptor de C5a (C5aR) na replicação bacteriana e na resistência de camundongos diante de uma infecção letal por L. longbeachae. Experimentos realizados com animais deficientes no receptor C5a indicam que os animais são protegidos durante uma infecção letal por L. longbeachae em comparação com animais selvagens, da linhagem BALB/c. De acordo com esses resultados, foi detectada uma menor carga bacteriana nos pulmões dos animais C5a-/- em comparação com animais selvagens. Experimentos realizados com animais controles da mesma linhagem demonstraram que C5a-/- diferem de animais C5a+/-, o que suporta o papel desse receptor durante a infecção por L. longbeachae. Dessa forma, nossos dados sugerem que a sinalização via C5aR contribui para a patogênese da doença em modelo murino da infecção por L. longbeachae. Os mecanismos envolvidos na patogênese mediada pelo receptor C5a encontram-se sob investigação. / Legionella longbeachae is a species of the Legionellaceae family that is commonly present in the soil in various regions of the globe. Infections by L. longbeachae in immunocompromised individuals cause severe pneumonia, often leading to hospitalization and death. The prevalence of L. longbeachae as a cause of pneumonia is large, and certainly under-estimated, mainly because the conventional diagnostic methods only detect Legionella pneumophila species. The anaphylatoxin C5a is an inflammatory protein activated by the complement system, which is involved in the recruitment of inflammatory cells, a process induced by cells of the innate immunity, which leads to tissue damage. Recent data generated in our laboratory suggest that the mortality of mice after infection with L. longbeachae is caused by a lung failure, associated with the induction of an intense inflammatory process in the lungs of infected animals. In this study, we investigated the role of C5a receptor (C5aR) in bacterial replication and mice resistance on a lethal infection by L. longbeachae. Experiments with animals deficient in the C5a receptor indicate that the animals are protected during a lethal infection by L. longbeachae as compared with-wild type strain, BALB/c. According to these results, a lower bacterial load was detected in the lungs of C5a-/- animals compared with BALB/c animals. Experiments performed with control animals of the same strain demonstrated that C5a-/- differ from C5a+/- animals, which supports the role of this receptor during infection by L. longbeachae. Thus, our data suggest that C5aR signalling pathway contributes to the pathogenesis of the disease in a murine model of infection by L. longbeachae. The mechanisms involved in the pathogenesis mediated by C5a receptor are under investigation.

Atherosclerosis is a slow-developing disease of large and medium sized arteries, and is the premier cardiovascular disease that underlies myocardial and cerebral infarction, aneurysm, stroke and gangrene of the extremities. At least 17 million people die of atherosclerotic complications each year worldwide, with another 15 million surviving unstable events. Despite therapeutic advances such as drug-eluting stents and statins, which reduce cardiovascular events by around 25%, there is an urgent need for additional strategies to complement these treatments and further reduce morbidity and mortality. Inflammation plays a fundamental role in mediating all stages of atherogenesis. The innate immune response has long been implicated in atherogenesis, and activation of the complement system has been associated with all stages of disease. In particular, C5b-9 (membrane attack complex) has been detected in human plaques and may be pathogenic. Since C5b-9 is produced in plaques then the complement activation product 5a (C5a) must also be generated. However, very little is known about the role of C5a in atherogenesis. Indeed, elevated levels of serum C5a have been detected in patients with advanced atherosclerosis and recently the classical C5a receptor, CD88, has been detected on most of the cells found in human atherosclerotic plaques. To date, no studies examining specific C5a receptor antagonism in an animal model of atherosclerosis have been performed. This thesis explored the potential therapeutic benefits of inhibiting C5a, using the C5a receptor antagonist, PMX53, in the ApoE knockout (ApoE-/-) mouse model of atherosclerosis. In Chapter 2, expression of both receptors to C5a, CD88 and C5L2, in aortae of ApoE -/- mice was explored. CD88 and C5L2 mRNA expression was detected in the aorta of ApoE -/- mice at 3, 5, 9,12, 17 and 25 weeks of age. CD88 expression in ApoE -/- mice increased with time, and with macrophage accumulation within the plaque, as indicated by an increase in expression of the macrophage marker, F4/80. Expresssion of CD88 was significantly increased at 17 and 26 weeks of age, compared with age-matched wild-type controls. C5L2 was also expressed albeit at much lower levels compared with wild-type controls. Having established the presence of C5a receptors in ApoE -/- mice, in Chapter 3, the effects PMX53-treatment on ApoE -/- mice on a normal chow diet was examined. PMX53 treatment (3 mg/kg; tri-weekly s.c., plus ~1mg/kg/day p.o. for 20 weeks) resulted in a significant reduction in neointimal area and therefore the intima:media ratio in the brachiocephalic artery compared to untreated controls (P < 0.05; n = 6-8). PMX53 treatment also reduced collagen content and outward remodelling of the brachiocephalic artery. In Chapter 4, studies exploring the effects of PMX53-treatment in the more inflammatory environment created by a high fat (or Western-type diet) were explored. Male ApoE -/- mice were treated with PMX53 from 5 – 25 weeks of age (3 mg/kg; tri-weekly s.c., plus v ~1mg/kg/day p.o.). Mice were placed on a high fat diet from 10 weeks of age. While PMX53- treated did not affect neo-intimal area, it did result in a significant increase in cell density (P<0.01; n=12) and a significant reduction in buried caps (P < 0.05; n = 12) in the brachiocephalic artery compared with untreated animals. Interestingly, PMX53-treated mice also had significantly reduced total cholesterol compared with untreated controls (P < 0.05; n = 12). These results provide the first evidence for a role for C5a in plaque destabilisation and cholesterol metabolism. Finally, Chapter 5 described the expression of CD88 and C5L2 in cultured primary rat vascular SMC was explored. Expression of CD88 and C5L2 was detected by Western blot; immunocytochemical analysis demonstrated intracellular expression of both C5L2 and CD88. Conversely, radioligand binding experiments suggested the presence of ~25000 cell surface receptors with a high affinity to C5a (KD = 0.3 nM). After establishing the presence of receptors to C5a, experiments were conducted to determine whether C5a has any functional effects on these cells. C5a induced a moderate increase in TNF-α release after 4 hours of treatment (P < 0.05, n = 3), but did not affect SMC proliferation (n = 3). In summary, this study is the first to demonstrate the benefits of specifically inhibiting C5a in a mouse model of atherosclerosis. These findings suggest that C5a plays a role in atherogenesis in ApoE -/- mice and that the C5a receptor antagonist PMX53 may have therapeutic potential in human atherosclerotic disease.

Legionella longbeachae é uma espécie da família Legionellaceae que é comumente presente no solo em diversas regiões do globo. Uma infecção por L. longbeachae em indivíduos imunocomprometidos causa uma pneumonia severa, frequentemente levando a hospitalização e à morte. A prevalência destas bactérias como causa de pneumonia é grande, e certamente sub-estimada, uma vez que os métodos de diagnóstico convencionais detectam apenas as espécies de Legionella pneumophila. A anafilatoxina C5a è uma proteína inflamatória ativada pelo complemento, a qual é envolvida no recrutamento de células inflamatórias, um processo induzido pelas células da imunidade inata que leva a dano tecidual. Dados recentes gerados no nosso laboratório sugerem que a mortalidade de camundongos após a infecção por L. longbeachae é causada por uma falência pulmonar, associada a indução de um intenso processo inflamatório nos pulmões dos animais infectados. Nesse trabalho, nós investigamos papel do receptor de C5a (C5aR) na replicação bacteriana e na resistência de camundongos diante de uma infecção letal por L. longbeachae. Experimentos realizados com animais deficientes no receptor C5a indicam que os animais são protegidos durante uma infecção letal por L. longbeachae em comparação com animais selvagens, da linhagem BALB/c. De acordo com esses resultados, foi detectada uma menor carga bacteriana nos pulmões dos animais C5a-/- em comparação com animais selvagens. Experimentos realizados com animais controles da mesma linhagem demonstraram que C5a-/- diferem de animais C5a+/-, o que suporta o papel desse receptor durante a infecção por L. longbeachae. Dessa forma, nossos dados sugerem que a sinalização via C5aR contribui para a patogênese da doença em modelo murino da infecção por L. longbeachae. Os mecanismos envolvidos na patogênese mediada pelo receptor C5a encontram-se sob investigação. / Legionella longbeachae is a species of the Legionellaceae family that is commonly present in the soil in various regions of the globe. Infections by L. longbeachae in immunocompromised individuals cause severe pneumonia, often leading to hospitalization and death. The prevalence of L. longbeachae as a cause of pneumonia is large, and certainly under-estimated, mainly because the conventional diagnostic methods only detect Legionella pneumophila species. The anaphylatoxin C5a is an inflammatory protein activated by the complement system, which is involved in the recruitment of inflammatory cells, a process induced by cells of the innate immunity, which leads to tissue damage. Recent data generated in our laboratory suggest that the mortality of mice after infection with L. longbeachae is caused by a lung failure, associated with the induction of an intense inflammatory process in the lungs of infected animals. In this study, we investigated the role of C5a receptor (C5aR) in bacterial replication and mice resistance on a lethal infection by L. longbeachae. Experiments with animals deficient in the C5a receptor indicate that the animals are protected during a lethal infection by L. longbeachae as compared with-wild type strain, BALB/c. According to these results, a lower bacterial load was detected in the lungs of C5a-/- animals compared with BALB/c animals. Experiments performed with control animals of the same strain demonstrated that C5a-/- differ from C5a+/- animals, which supports the role of this receptor during infection by L. longbeachae. Thus, our data suggest that C5aR signalling pathway contributes to the pathogenesis of the disease in a murine model of infection by L. longbeachae. The mechanisms involved in the pathogenesis mediated by C5a receptor are under investigation.

<p>In patients with sepsis, the activation of the cascade systems, for example the complement system with the generation of C5a, is followed by a state of immunosuppression with impaired bactericidal capacity caused by suppression of the neutrophil granulocytes. To inhibit the C5a-induced systemic inflammatory and the following anti-inflammatory responses, different anti-C5a strategies have been successful in experimental models of sepsis. In animals and in healthy volunteers after injection of lipopolysaccharide (LPS), an up-regulation of the C5a receptor (C5aR) has been reported. Before designing clinical studies, it was of importance to increase the knowledge of C5a and C5aR regulation in humans. </p><p>At the time when the diagnosis of severe sepsis or septic shock can be established clinically, granulocyte C5aR expression, analysed by flow cytometer, was shown to be reduced, whereas monocyte C5aR expression was unchanged. There was a correlation between granulocyte C5aR expression and the severity of disease, as measured by the APACHE II score. </p><p><i>Ex vivo</i> incubation of whole blood with LPS resulted in a reduction in granulocyte C5aR expression. Such a reduction was not found in isolated cells, indicating that the effect was mediated via plasma factors, such as C5a, IL-8 and TNF-α which all were shown to reduce C5aR expression <i>ex vivo</i>.</p><p>Although there was a trend between chemotaxis, as measured by migration in a modified Boyden chamber, and C5aR expression on granulocytes from patients with severe sepsis or septic shock or from healthy individuals, the correlation failed to reach statistical significance.</p><p>It is concluded that granulocyte C5aR expression is affected by several plasma factors and that a reduction is clinically evident at the time of the sepsis diagnosis. Reduced granulocyte C5aR expression is associated with an impaired chemotaxis but does not alone limit the chemotactic response.</p>

In patients with sepsis, the activation of the cascade systems, for example the complement system with the generation of C5a, is followed by a state of immunosuppression with impaired bactericidal capacity caused by suppression of the neutrophil granulocytes. To inhibit the C5a-induced systemic inflammatory and the following anti-inflammatory responses, different anti-C5a strategies have been successful in experimental models of sepsis. In animals and in healthy volunteers after injection of lipopolysaccharide (LPS), an up-regulation of the C5a receptor (C5aR) has been reported. Before designing clinical studies, it was of importance to increase the knowledge of C5a and C5aR regulation in humans. At the time when the diagnosis of severe sepsis or septic shock can be established clinically, granulocyte C5aR expression, analysed by flow cytometer, was shown to be reduced, whereas monocyte C5aR expression was unchanged. There was a correlation between granulocyte C5aR expression and the severity of disease, as measured by the APACHE II score. Ex vivo incubation of whole blood with LPS resulted in a reduction in granulocyte C5aR expression. Such a reduction was not found in isolated cells, indicating that the effect was mediated via plasma factors, such as C5a, IL-8 and TNF-α which all were shown to reduce C5aR expression ex vivo. Although there was a trend between chemotaxis, as measured by migration in a modified Boyden chamber, and C5aR expression on granulocytes from patients with severe sepsis or septic shock or from healthy individuals, the correlation failed to reach statistical significance. It is concluded that granulocyte C5aR expression is affected by several plasma factors and that a reduction is clinically evident at the time of the sepsis diagnosis. Reduced granulocyte C5aR expression is associated with an impaired chemotaxis but does not alone limit the chemotactic response.