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.

The complement system is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other complement components in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund’s adjuvant were accompanied by C5a upregulation and were markedly reduced by C5a receptor (C5aR1) knockout (KO) or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal and mechanical hyperalgesia, an effect that was absent in TRPV1 KO mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca2+ mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis (MAFIA) mice abolished C5a-dependent thermal and mechanical hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of nerve growth factor (NGF), a mediator known to sensitize TRPV1. Pre-injection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that C5a induces thermal and mechanical hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF and TRPV1 as key players in this cross-cellular communication.

Das bekapselte, Gram-negative, diplokokkenförmige Bakterium Neisseria meningitidis (Nme) ist ein asymptomatischer Kommensale des oberen Nasenrachenraums im Men-schen. Gerade bei Kindern ist es dem humanspezifischen Pathogen in seltenen Fällen möglich, in den Blutstrom einzuwandern und lebensbedrohliche Krankheitsbilder wie Meningoenzephalitis und Sepsis auszulösen, welche als „Invasive Meningokokkener-krankung“ (IMD) zusammengefasst werden. Jährlich ereignen sich weltweit bis zu 1,2 Mio Fälle von IMD, welche aufgrund des fulminanten Verlaufs und der hohen Letalität gefürchtet sind. In der Bekämpfung der Nme-Sepsis ist das humane Komplementsystem von entscheidender Bedeutung. Vor diesem Hintergrund ist die protektive Rolle des lytischen (Membranangriffskomplex MAK) und opsonisierenden Arms (Opsonine iC3b und C1q) der Komplementkaskade gut dokumentiert. Dagegen ist der Beitrag des in-flammatorischen Arms (Anaphylatoxine C3a und C5a) in der Nme-Sepsis bisher unklar. Aus diesem Grunde wurde mit dieser Arbeit die Rolle des inflammatorischen Arms an-hand des Komplement C5a-Rezeptors 1 (C5aR1) in der Pathophysiologie der Nme-Sepsis am Mausmodell untersucht. Nach Etablierung des murinen, intraperitonealen In-fektionsmodells konnte ein schädlicher Effekt des C5aR1 in der Nme-Sepsis beobachtet werden. Aus der Abwesenheit des C5aR1 resultierte eine höhere Überlebensrate, ein besserer klinischer Zustand, eine niedrigere Bakteriämie und niedrigere Konzentrationen der pro-inflammatorischen Mediatoren IL-6, CXCL-1 und TNF-α. Im Hinblick auf den zellulären Pathomechanismus sprechen Ergebnisse dieser Arbeit dafür, dass der C5aR1 primär eine gesteigerte Freisetzung inflammatorischer Mediatoren durch verschiedene Zellpopulationen triggert (Zytokinsturm), wodurch sekundär Zellparalyse, steigende Bakteriämie und höhere Letalität bedingt sind. Durch Depletionsversuche und Immun-fluoreszenzfärbungen konnte, unabhängig vom C5aR1, eine allgemein protektive Rolle von neutrophilen Granulozyten und Monozyten/Makrophagen in der Nme-Sepsis beo-bachtet werden. Darüber hinaus präsentierte sich der zyklische C5aR1-Antagonist PMX205 als erfolgsversprechende Therapieoption, um Parameter einer murinen Nme-Sepsis zu verbessern. Weitere Untersuchungen sind nötig, um die Wirksamkeit dieser Substanz in der humanen Nme-Sepsis zu erforschen. Zudem könnte das murine, intrape-ritoneale Infektionsmodell zur Klärung der Rolle des C5aR2 in der Nme-Sepsis genutzt werden. / The encapsulated, Gram-negative diplococcus Neisseria meningitidis (Nme) is an asymp-tomatic commensal in the human upper respiratory tract. In rare cases and especially in children, this human-specific pathogen is able to invade into the blood stream and cause life-threatening disorders like meningoencephalitis and septicemia, which are subsumed as „invasive meningococcal disease“ (IMD). The estimated number of cases is about 1.2 mio per year worldwide. IMD is greatly feared because of its fulminant progression and its high lethality. It is very well known, that the human complement system holds an essential role to fight meningococcal sepsis. In this context, the protective effects of the lytic (membrane attack complex MAC) and opsonizing branches (opsonines iC3b and C1q) are well established. On the contrary, very little is known about the contribution of the inflammatory branch (anaphylatoxines C3a and C5a) in meningococcal sepsis. Therefore, this work focused on the role of the C5a-Receptor 1 (C5aR1) in pathophysi-ology of meningococcal sepsis in a murine model. After having established the para-mount role of complement in murine intraperitoneal infection model, we could observe a detrimental effect of C5aR1 in Meningococcal sepsis. The absence of C5aR1 resulted in a higher overall survival, ameliorated clinical status, lower bacteremia and lower levels of the proinflammatory mediators IL-6, CXCL-1, TNF-α. Particularly with regard to results about the cellular pathomechanism, the C5aR1 seems to cause an increased re-lease of proinflammatory mediators (cytokine storm) exerted by various cell populations. As a consequence, cellular paralysis, increasing bacterial burden and higher lethality rate seems to occur. In reference to depletion experiments and immunofluorescence stain-ings, we could observe protective overall effects of neutrophils and mono-cytes/macrophages, uncorrelated to C5aR1 presence. Ultimately, the cyclic C5aR1-antagonist PMX205 appeared to be a promising option to improve parameters in murine meningococcal sepsis. Further experiments are required to examine the potential of this compound in human meningococcal sepsis. Moreover, the murine, intraperitoneal infec-tion model could be used to clarify the role of C5aR2 in meningococcal sepsis.

Coiled-Coil Domain Containing 9 is a novel gene located beside C5aR1/R2 on murine chromosome 7. This proximal relationship is mirrored on human chromosome 19, making this research translational and relevant to the medical field. CCDC9-/- mice were created through a contract research organization utilizing CRISPR/Cas9 gene editing. Necropsy, gross examination, hematology, and clinical chemistry analysis were conducted to ensure that the global knockout of the CCDC9 gene did not induce any abnormalities in the development of the mice. Bone Marrow Derived Macrophages and Thioglycolate Elicited Peritoneal Macrophages were utilized from wild type and CCDC9-/- mice to explore the cytokine response from these macrophages when stimulated with various pattern recognition receptor agonists. Poly (I:C) and LPS were the two agonists that led to the most significant difference in cytokine release. Interactions with Poly (I:C), a synthetic double-stranded RNA that activates anti-viral RNA sensing immune responses, were targeted to elucidate how CCDC9 may be interacting with RNA. Meanwhile, interactions with LPS demonstrated that the role of CCDC9 is not specific to RNA-sensing but plays a modulatory role in the TLR4 and LPS pathway. These agonists were then paired with complement anaphylatoxin, C5a, and stimulated TEPM to explore the proximal relationship between CCDC9 and C5aR1/R2. The pairing with Poly (I:C) did not reveal any significant changes, but the pairing with LPS lead to a cytokine decrease that was not mirrored in CCDC9-/- macrophages. In-vivo applications were established in which wild type and CCDC9 -/- mice were injected intraperitoneally with LPS and Poly (I:C) to investigate the cytokine release. CCDC9-/- mice and cells consistently displayed a greater inflammatory cytokine induction when encountering stimulators of viral and bacterial pathogen sensing, suggesting that CCDC9 plays a role in immune function. / 2025-03-07T00:00:00Z

<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.

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