RESUMO
The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar-/- mice, whereas C5ar1-/- and C5ar2-/- mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1-/- mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8Δ71-73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.
Assuntos
Infecções Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de Complemento/imunologia , Anafilatoxinas/imunologia , Animais , Quimiocinas/imunologia , Citocinas/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neisseria meningitidis/patogenicidade , Neutrófilos/imunologia , Neutrófilos/microbiologia , Receptor da Anafilatoxina C5a/genética , Receptores de Complemento/genética , SepseRESUMO
The single greatest barrier to studying the lifestyle of Neisseria meningitidis stems from its exquisite adaptation to life in humans, a specialization which prevents it from infecting other animals. This barrier to modeling meningococcal infection has been overcome by the provision of factors that allow the meningococci to overcome one or more aspects of host restriction, including the use of mice expressing receptors that allow mucosal colonization and/or the inclusion of serum factors that facilitate meningococcal replication during disseminated meningococcal disease. Here we discuss these advances, consider variables that influence the outcome of infection, and detail the technical requirements to establish robust and reproducible nasal colonization or sepsis. Once established, these models can then be used to study the meningococcal lifestyle and the immune response during infection, and to facilitate development of novel drug or vaccine-based approaches to intervene in meningococcal carriage and disease.
Assuntos
Modelos Animais de Doenças , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/patogenicidade , Nariz/microbiologia , Sepse/microbiologia , Animais , Antígenos CD/fisiologia , Moléculas de Adesão Celular/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Although usually asymptomatically colonizing the human nasopharynx, the Gram-negative bacterium Neisseria meningitidis (meningococcus) can spread to the blood stream and cause invasive disease. For survival in blood, N. meningitidis evades the complement system by expression of a polysaccharide capsule and surface proteins sequestering the complement regulator factor H (fH). Meningococcal strains belonging to the sequence type (ST-) 41/44 clonal complex (cc41/44) cause a major proportion of serogroup B meningococcal disease worldwide, but they are also common in asymptomatic carriers. Proteome analysis comparing cc41/44 isolates from invasive disease versus carriage revealed differential expression levels of the outer membrane protein NspA, which binds fH. Deletion of nspA reduced serum resistance and NspA expression correlated with fH sequestration. Expression levels of NspA depended on the length of a homopolymeric tract in the nspA promoter: A 5-adenosine tract dictated low NspA expression, whereas a 6-adenosine motif guided high NspA expression. Screening German cc41/44 strain collections revealed the 6-adenosine motif in 39% of disease isolates, but only in 3.4% of carriage isolates. Thus, high NspA expression is associated with disease, but not strictly required. The 6-adenosine nspA promoter is most common to the cc41/44, but is also found in other hypervirulent clonal complexes.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Meningite Meningocócica/metabolismo , Neisseria meningitidis Sorogrupo B/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Fator H do Complemento/metabolismo , Deleção de Genes , Interações Hospedeiro-Patógeno , Humanos , Neisseria meningitidis Sorogrupo B/genética , Poli A/genética , Regiões Promotoras Genéticas , TranscriptomaRESUMO
The complement system is the primary innate immune determinant protecting against invasive diseases caused by the Gram-negative bacterium Neisseria meningitidis (Nme, meningococcus), as evidenced by the extreme susceptibility of individuals with complement deficiencies. In contrast, the role of phagocytes such as neutrophils is much less well understood, although they are recruited in great numbers to the cerebrospinal fluid during meningococcal meningitis. Here, we consider the interaction of Nme with primary human neutrophils using either purified cells or a whole blood model of infection. We found that neutrophils are capable of non-opsonic uptake and killing of different Nme strains. However, in the presence of immune serum featuring active complement, Nme association is strongly increased, whereas this is not the case in heat-inactivated immune serum. Blockade of complement at the level of C3 using the inhibitor compstatin Cp20 reduces the uptake dramatically. In addition, purified neutrophils did not mount an oxidative burst towards Nme unless complement was added and, vice versa, the oxidative burst was strongly reduced in whole blood upon complement inhibition. In contrast, there was no significant impact of complement on neutrophil degranulation or IL-8 secretion. Taken together, neutrophils require complement activation in order to mount a full response towards Nme.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Fatores Imunológicos/metabolismo , Meningite Meningocócica/fisiopatologia , Neisseria meningitidis/imunologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Adulto , Voluntários Saudáveis , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Explosão Respiratória , Adulto JovemRESUMO
Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA.
Assuntos
Anticoagulantes/farmacologia , Imunidade Inata/efeitos dos fármacos , Infecções Meningocócicas/imunologia , Neisseria meningitidis/crescimento & desenvolvimento , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Células Sanguíneas/imunologia , Células Sanguíneas/microbiologia , Proteínas de Transporte/metabolismo , Ácido Cítrico/farmacologia , Ativação do Complemento/efeitos dos fármacos , Heparina/farmacologia , Hirudinas/farmacologia , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/imunologiaRESUMO
Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1-/- mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1-/- mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy.IMPORTANCE The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease.
Assuntos
Neisseria meningitidis/imunologia , Neisseria meningitidis/patogenicidade , Receptor da Anafilatoxina C5a/metabolismo , Sepse/microbiologia , Sepse/fisiopatologia , Animais , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiência , Análise de SobrevidaRESUMO
Neisseria meningitidis (Nme) can cause meningitis and sepsis, diseases which are characterised by an overwhelming inflammatory response. Inflammation is triggered by host pattern recognition receptors (PRRs) which are activated by pathogen-associated molecular patterns (PAMPs). Nme contains multiple PAMPs including lipooligosaccharide, peptidoglycan, proteins and metabolites. Various classes of PRRs including Toll-like receptors, NOD-like receptors, C-type lectins, scavenger receptors, pentraxins and others are expressed by the host to respond to any given microbe. While Toll-like receptors and NOD-like receptors are pivotal in triggering inflammation, other PRRs act as modulators of inflammation or aid in functional antimicrobial responses such as phagocytosis or complement activation. This review aims to give an overview of the various Nme PAMPs reported to date, the PRRs they activate and their implications during the inflammatory response to infection.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/metabolismo , Neisseria meningitidis/imunologia , Animais , Humanos , Ligação Proteica , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
Neisseria meningitidis, the meningococcus, bears the potential to cause life-threatening invasive diseases, but it usually colonizes the nasopharynx without causing any symptoms. Within the nasopharynx, Neisseria meningitidis must face temperature changes depending on the ambient air temperature. Indeed, the nasopharyngeal temperature can be substantially lower than 37°C, the temperature commonly used in experimental settings. Here, we compared the levels of meningococcal biofilm formation, autoaggregation, and cellular adherence at 32°C and 37°C and found a clear increase in all these phenotypes at 32°C suggestive of a stronger in vivo colonization capability at this temperature. A comparative proteome analysis approach revealed differential protein expression levels between 32°C and 37°C, predominantly affecting the bacterial envelope. A total of 375 proteins were detected. Use of database annotation or the PSORTb algorithm predicted 49 of those proteins to be localized in the outer membrane, 21 in either the inner or outer membrane, 35 in the periplasm, 56 in the inner membrane, and 208 in the cytosol; for 6 proteins, no annotation or prediction was available. Temperature-dependent regulation of protein expression was seen particularly in the periplasm as well as in the outer and inner membranes. Neisserial heparin binding antigen (NHBA), NMB1030, and adhesin complex protein (ACP) showed the strongest upregulation at 32°C and were partially responsible for the observed temperature-dependent phenotypes. Screening of different global regulators of Neisseria meningitidis suggested that the extracytoplasmic sigma factor σE might be involved in temperature-dependent biofilm formation. In conclusion, subtle temperature changes trigger adaptation events promoting mucosal colonization by meningococci.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Neisseria meningitidis/fisiologia , Proteoma/metabolismo , Temperatura , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: The effect of protein-based meningococcal vaccines on prevention of nasopharyngeal colonization has been difficult to investigate experimentally because a reliable animal colonization model did not exist. METHODS: Human CEACAM1 transgenic mice, which can be colonized by meningococci, were immunized IP with one of two meningococcal native outer membrane vesicle (NOMV) vaccines prepared from mutants with attenuated endotoxin (lpxL1 knockout) and over-expressed sub-family B Factor H-binding proteins (FHbp). Animals were challenged intranasally two weeks after the third dose with wild-type strain H44/76, or were treated IP with anti-NOMV serum before and during the bacterial challenge. RESULTS: The NOMV-1 vaccine, prepared from the serogroup B H44/76 mutant, elicited â¼40-fold higher serum bactericidal antibody titers against the wild-type H44/76 challenge strain than the NOMV-2 vaccine prepared from a heterologous serogroup W mutant strain with different PorA and FHbp amino acid sequence variants. Compared to aluminum hydroxide-immunized control mice, the efficacy for prevention of any H44/76 colonization was 93% (95% confidence interval, 52-99, P<0.0001) for the NOMV-1 vaccine, and 19% (-3-36, P=0.23) for NOMV-2. NOMV-2-vaccinated mice had a 5.6-fold decrease in geometric mean CFU of bacteria per animal in tracheal washes compared to control mice (P=0.007). The efficacy of passive administration of serum from NOMV-1-vaccinated mice to immunologically naïve mice against colonization was 44% (17-61; P=0.002). CONCLUSIONS: Both NOMV vaccines protected against meningococcal colonization but there was greater protection by the NOMV-1 vaccine with antigens matched with the challenge strain. Meningococcal vaccines that target protein antigens have potential to decrease colonization.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Meningocócicas/imunologia , Nasofaringe/microbiologia , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis/crescimento & desenvolvimento , Animais , Antígenos de Bactérias/genética , Antígenos CD/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Moléculas de Adesão Celular/genética , Endotoxinas/imunologia , Humanos , Imunização Passiva , Injeções Intraperitoneais , Infecções Meningocócicas/prevenção & controle , Camundongos , Camundongos Transgênicos , Mutação , Neisseria meningitidis/imunologia , Neisseria meningitidis Sorogrupo B/crescimento & desenvolvimento , Ensaios de Anticorpos Bactericidas Séricos , VacinaçãoRESUMO
Neisseria meningitidis asymptomatically colonizes the human upper respiratory tract but is also the cause of meningitis and severe septicemia. Carriage or disease evokes an immune response against the infecting strain. Hitherto, we have known little about the breadth of immunity induced by natural carriage of a single strain or its implications for subsequent infectious challenge. In this study, we establish that transgenic mice expressing human CEACAM1 support nasal colonization by a variety of strains of different capsular types. Next, we nasally challenged these mice with either of the N. meningitidis strains H44/76 (serogroup B, ST-32) and 90/18311 (serogroup C, ST-11), while following the induction of strain-specific immunoglobulin. When these antisera were tested for reactivity with a diverse panel of N. meningitidis strains, very low levels of antibody were detected against all meningococcal strains, yet a mutually exclusive "fingerprint" of high-level cross-reactivity toward certain strains became apparent. To test the efficacy of these responses for protection against subsequent challenge, CEACAM1-humanized mice exposed to strain 90/18311 were then rechallenged with different N. meningitidis strains. As expected, the mice were immune to challenge with the same strain and with a closely related ST-11 strain, 38VI, while H44/76 (ST-32) could still colonize these animals. Notably, however, despite the paucity of detectable humoral response against strain 196/87 (ST-32), this strain was unable to colonize the 90/18311-exposed mice. Combined, our data suggest that current approaches may underestimate the actual breadth of mucosal protection gained through natural exposure to N. meningitidis strains.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos CD/imunologia , Portador Sadio/imunologia , Moléculas de Adesão Celular/imunologia , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/imunologia , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Humanos , Camundongos TransgênicosRESUMO
Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.
Assuntos
Adaptação Fisiológica , Infecções Meningocócicas/microbiologia , Nasofaringe/microbiologia , Neisseria meningitidis/crescimento & desenvolvimento , Mucosa Respiratória/microbiologia , Infecções Respiratórias/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Aderência Bacteriana , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Escherichia coli/metabolismo , Células HeLa , Humanos , Imunidade nas Mucosas , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/metabolismo , Camundongos , Camundongos Transgênicos , Viabilidade Microbiana , Mutação , Nasofaringe/imunologia , Nasofaringe/metabolismo , Nasofaringe/patologia , Neisseria meningitidis/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Neutrófilos/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologiaRESUMO
The capsule of Neisseria meningitidis is the major virulence factor that enables this bacterium to overcome host immunity elicited by complement and phagocytes, rendering it capable of surviving in blood. As such, nonencapsulated N. meningitidis isolates are generally considered nonpathogenic. Here, we consider the inherent virulence of two nonencapsulated N. meningitidis isolates obtained from our national surveillance of infected blood cultures in Canada. Capsule deficiency of both strains was confirmed by serology and PCR for the ctrA to ctrD genes and siaA to siaC genes, as well as siaD genes specific to serogroups B, C, Y, and W135. In both strains, the capsule synthesis genes were replaced by the capsule null locus, cnl-2. In accordance with a lack of capsule, both strains were fully susceptible to killing by both human and baby rabbit complement. However, in the presence of cytidine-5' monophospho-N-acetylneuraminic acid (CMP-NANA), allowing for lipooligosaccharide (LOS) sialylation, a significant increase of resistance to complement killing was observed. Mass spectrometry of purified LOS did not reveal any uncommon modifications that would explain their invasive phenotype. Finally, in a mouse intraperitoneal challenge model, these nonencapsulated isolates displayed enhanced virulence relative to an isogenic mutant of serogroup B strain MC58 lacking capsule (MC58ΔsiaD). Virulence of all nonencapsulated isolates tested was below that of encapsulated serogroup B strains MC58 and B16B6. However, whereas no mortality was observed with MC58ΔsiaD, 5/10 mice succumbed to infection with strain 2275 and 2/11 mice succumbed to strain 2274. Our results suggest the acquisition of a new virulence phenotype by these nonencapsulated strains.
Assuntos
Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/patogenicidade , Fatores de Virulência/deficiência , Fatores de Virulência/metabolismo , Animais , Cápsulas Bacterianas/imunologia , Canadá , Proteínas do Sistema Complemento/imunologia , Modelos Animais de Doenças , Feminino , Genes Bacterianos , Humanos , Masculino , Infecções Meningocócicas/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Neisseria meningitidis/imunologia , Neisseria meningitidis/isolamento & purificação , Peritonite/microbiologia , Peritonite/mortalidade , Reação em Cadeia da Polimerase , Coelhos , Sorotipagem , Análise de Sobrevida , Fatores de Virulência/imunologiaRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is a critical public health issue; more and more people are affected, but treatment options remain limited. Complement activation and the anaphylatoxin C5a have been shown to play a role in IBD. In this study, mouse models of acute and chronic dextran sulfate-induced colitis were used to further elucidate the impact of C5a and its receptor (C5aR) on disease development. METHODS: In C57BL/6J wildtype and C5aR(-/-) mice the extent of complement activation, changes in weight, and water/food consumption were determined. Disease severity was evaluated via a clinical score, histology, cytokine- and myeloperoxidase-determination as well as real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of anaphylatoxin receptors and inflammatory mediators. RESULTS: C5aR(-/-) mice showed milder disease symptoms, less histological damage, and a lower expression of inflammatory mediators in acute colitis, a setting where complement was activated. In chronic colitis the knockout mice exhibited aggravated weight loss, a higher degree of histological damage and granulocyte infiltration. Intriguingly, increases in C3a-receptor and C5L2 mRNA were dependent on C5aR. Compared to wildtype mice, C5aR(-/-) animals displayed smaller lymph nodes in acute colitis, but extensive swelling and diminished IL-4 and IFN-γ responses in the chronic disease, demonstrating that C5aR modifies T-helper cell polarization. CONCLUSIONS: C5aR exerts detrimental functions in acute colitis, strongly supporting the idea that a C5aR-antagonist might be useful for IBD treatment. However, since the absence of C5aR was no longer protective and in some regards disadvantageous in chronic IBD, future studies should address the efficacy and the possible side effects of a sustained antagonist treatment.
Assuntos
Doenças Inflamatórias Intestinais/imunologia , Receptor da Anafilatoxina C5a/imunologia , Doença Aguda , Animais , Polaridade Celular/imunologia , Ativação do Complemento/imunologia , Sulfato de Dextrana/imunologia , Sulfato de Dextrana/toxicidade , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Interferon gama/imunologia , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Índice de Gravidade de Doença , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
The anaphylatoxin (AT) C3a, C5a and C5a-desArg are generally considered pro-inflammatory polypeptides generated after proteolytic cleavage of C3 and C5 in response to complement activation. Their well-appreciated effector functions include chemotaxis and activation of granulocytes, mast cells and macrophages. Recent evidence suggests that ATs are also generated locally within tissues by pathogen-, cell-, or contact system-derived proteases. This local generation of ATs is important for their pleiotropic biologic effects beyond inflammation. The ATs exert most of the biologic activities through ligation of three cognate receptors, i.e. the C3a receptor, the C5a receptor and the C5a receptor-like, C5L2. Here, we will discuss recent findings suggesting that ATs regulate cell apoptosis, lipid metabolism as well as innate and adaptive immune responses through their impact on antigen-presenting cells and T cells. As we will outline, such regulatory functions of ATs and their receptors play important roles in the pathogenesis of allergy, autoimmunity, neurodegenerative diseases, cancer and infections with intracellular pathogens.
Assuntos
Anafilatoxinas/imunologia , Hipersensibilidade/imunologia , Inflamação/imunologia , Neoplasias/imunologia , Sepse/imunologia , Linfócitos T/imunologia , Anafilatoxinas/metabolismo , Animais , Apoptose/imunologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Ativação do Complemento/imunologia , Humanos , Hipersensibilidade/metabolismo , Imunidade Ativa/imunologia , Imunidade Inata/imunologia , Inflamação/metabolismo , Camundongos , Neoplasias/metabolismo , Neurogênese/imunologia , Sepse/metabolismo , Linfócitos T/metabolismoRESUMO
C5L2 is a 7 transmembrane domain receptor for complement fragment C5a that, unlike the classical C5a receptor, C5aR, does not couple to G proteins. However, in mice where C5L2 has been deleted, the response to C5a is altered, suggesting that C5L2 may have a signaling function. In order to investigate whether human C5L2 also has some capacity to transduce signals, we have attempted to produce a signaling competent form of human C5L2 by inserting C5aR sequences at three key G protein activation motifs. However, we detected neither an intracellular Ca(2+) response nor beta-arrestin redistribution in mutated C5L2, suggesting that the potential for G protein coupling is completely absent in this receptor and that, in humans, C5L2 may have functions that are unrelated to signaling. In confirmation of this, we detected constitutive ligand-independent internalization of C5L2 that resulted in the rapid accumulation of C5a and its stable metabolite, C5a des Arg, within the cell with only a small net change in cell surface receptor levels. Internalization was found to be through a clathrin-dependent mechanism that led to the retention and, in cells natively expressing C5L2, the degradation of the ligand within an intracellular compartment. In contrast, the classical C5a receptor, C5aR, internalized ligand much more slowly and a majority of this ligand was released back into the extracellular environment in an apparently undegraded form. These data suggest that a major function of human C5L2 is to remove active complement fragments from the extracellular environment.
Assuntos
Cálcio/fisiologia , Receptores de Quimiocinas/imunologia , Sequência de Aminoácidos , Animais , Arrestinas/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Endocitose , Humanos , Dados de Sequência Molecular , Mutação , Transporte Proteico/fisiologia , Receptor da Anafilatoxina C5a , Receptores de Quimiocinas/genética , Transdução de Sinais , beta-ArrestinasAssuntos
Anti-Inflamatórios/imunologia , Complemento C3a/imunologia , Receptores de Quimiocinas/imunologia , Anafilatoxinas/genética , Anafilatoxinas/imunologia , Animais , Metabolismo dos Carboidratos , Fator D do Complemento/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Metabolismo dos Lipídeos , Ligação Proteica , Receptor da Anafilatoxina C5a , Receptores de Quimiocinas/genética , Transdução de Sinais/fisiologiaRESUMO
During complement activation the pro-inflammatory anaphylatoxins C3a and C5a are generated, which interact with the C3a receptor and C5a receptor (CD88), respectively. C5a and its degradation product C5a-des-Arg(74) also bind to the C5a receptor-like 2 (C5L2). C3a and C3a-des-Arg(77), also called acylation-stimulating protein, augment triglyceride synthesis and glucose uptake in adipocytes and skin fibroblasts. Based on data obtained using transfected HEK293 and RBL cells, C5L2 is additionally proposed as a functional receptor for C3a and C3a-des-Arg(77). Here we use (125)I-ligand binding assays and flow cytometry with fluorescently labeled ligands to demonstrate that neither C3a nor C3a-des-Arg(77) binds to C5L2. C5L2 expression and its regulation are investigated on various cell lines by a novel C5L2-restricted binding assay and quantitative real time PCR. Dibutyryl cAMP and interferon-gamma induce up-regulation of this receptor on myeloblastic cell lines (U937 and HL-60), whereas tumor necrosis factor-alpha (TNF-alpha) has no effect. In contrast, epithelial HeLa cells are found to constitutively express C5L2 but not the C5a receptor. In HeLa cells, interferon-gamma and TNF-alpha drastically reduce C5L2 expression. No C5a-dependent Ca(2+) signaling is observed even in these cells endogenously expressing C5L2. Taken together, C5L2 is not a receptor for C3a or C3a-des-Arg(77). Thus, this receptor is unlikely to be directly involved in lipid metabolism. Instead, the identification of stimuli modifying C5L2 expression indicates that C5L2 is a highly regulated scavenger receptor for C5a and C5a-des-Arg(74).
Assuntos
Células Epiteliais/citologia , Células Mieloides/metabolismo , Receptores de Quimiocinas/fisiologia , Anafilatoxinas/química , Animais , Complemento C3a/química , Complemento C5a/química , Células HL-60 , Células HeLa , Humanos , Ligantes , Lipídeos/química , Ratos , Receptor da Anafilatoxina C5a , Receptores de Quimiocinas/química , Transfecção , Células U937RESUMO
The anaphylatoxin C3a is a proinflammatory mediator generated during complement activation. The tight control of C3a receptor (C3aR) expression is crucial for the regulation of anaphylatoxin-mediated effects. Key factors regulating constitutive expression of the C3aR in the mast cell line HMC-1 and receptor induction by dibutyryl-cAMP in monomyeloblastic U937 cells were determined by functional characterization of the C3aR promoter. Nucleotides -18 to -285 upstream of the translational start site proved to be critical for promoter activity in HMC-1 cells. Binding sites for the transcription factors AP-1 and Ets could be located. Overexpressed c-Jun/c-Fos (AP-1) and Ets-1 led synergistically to increased promoter activity that was substantially reduced by site-directed mutagenesis of the corresponding elements within the C3aR promoter. In HMC-1 cells, Ets interacted directly with the predicted binding motif of the C3aR promoter as determined by electromobility shift assays. AP-1 binding to the C3aR promoter was augmented during C3aR induction in U937 cells. A retroviral gene transfer system was used to express a dominant negative mutant of Ets-1 in these cells. The resulting cells failed to up-regulate the C3aR after stimulation with dibutyryl-cAMP and showed decreased AP-1 binding, suggesting that Ets acts here indirectly. Thus, it was established that Ets and the AP-1 element mediates dibutyryl-cAMP induction of C3aR promoter activity, hence providing a mechanistic explanation of dibutyryl-cAMP-dependent up-regulation of C3aR expression. In conclusion, this study demonstrates an important role of AP-1 and a member of the Ets family in the transcriptional regulation of C3aR expression, a prerequisite for the ability of C3a to participate in immunomodulation and inflammation.