Negative regulation of bacterial killing and inflammation by two novel CD16 ligands.

Sepsis, a leading cause of death worldwide, involves exacerbated proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here, we used a phage display library to identify two peptides in Escherichia coli that interact with host innate receptors. One of these peptides, encoded by the wzxE gene of E. coli K-12, was involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. Peptide-receptor interactions induced CD16-mediated inhibitory immunoreceptor tyrosine-based activating motif signaling, blocking the production of ROS and bacterial killing. This CD16-mediated inhibitory signaling was abrogated in a WzxE(-/-) mutant of E. coli K-12, restoring the production of ROS and bacterial killing. Taken together, the two novel CD16 ligands identified negatively regulate bacterial killing and inflammation. Our findings may contribute toward the development of new immunotherapies for E. coli-mediated infectious diseases and inflammation.

Reduced frequency of two activating KIR genes in patients with sepsis.

Natural killer (NK) cell activity is regulated by activating and inhibitory signals transduced by killer cell immunoglobulin-like receptors (KIR). Diversity in KIR gene repertoire among individuals may affect disease outcome. Sepsis development and severity may be influenced by genetic factors affecting the immune response. Here, we examined sixteen KIR genes and their human leucocyte antigen (HLA) class I ligands in critical patients, aiming to identify patterns that could be associated with sepsis. Male and female patients (ages ranging between 14 and 94years-old) were included. DNA samples from 211 patients with sepsis and 60 controls (critical care patients with no sepsis) collected between 2004 and 2010 were included and genotyped for KIR genes using the polymerase chain reaction method with sequence-specific oligonucleotide (PCR-SSO), and for HLA genes using the polymerase chain reaction method with sequence-specific primers (PCR-SSP). The frequencies of activating KIR2DS1 and KIR3DS1 in sepsis patients when compared to controls were 41.23% versus 55.00% and 36.49% versus 51.67% (p=0.077 and 0.037 respectively before Bonferroni correction). These results indicate that activating KIR genes 2DS1 and 3DS1 may more prevalent in critical patients without sepsis than in patients with sepsis, suggesting a potential protective role of activating KIR genes in sepsis.

Fc Gamma Receptor IIA (CD32A) R131 Polymorphism as a Marker of Genetic Susceptibility to Sepsis.

Sepsis is a devastating disease that can affect humans at any time between neonates and the elderly and is associated with mortality rates that range from 30 to 80%. Despite intensive efforts, its treatment has remained the same over the last few decades. Fc receptors regulate multiple immune responses and have been investigated in diverse complex diseases. FcγRIIA (CD32A) is an immunoreceptor, tyrosine-based activation motif-bearing receptor that binds immunoglobulin G and C-reactive protein, important opsonins in host defense. We conducted a study of 702 patients (184 healthy individuals, 171 non-infected critically ill patients, and 347 sepsis patients) to investigate if genetic polymorphisms in the CD32A coding region affect the risk of septic shock. All individuals were genotyped for a variant at position 131 of the FcγRIIA gene. We found that allele G, associated with the R131 genotype, was significantly more frequent in septic patients than in the other groups (p = 0.05). Our data indicate that FcγRIIA genotyping can be used as a marker of genetic susceptibility to sepsis.

Identificação de peptídeos de Escherichia coli capazes de inibir a própria fagocitose em sepse / Identification of Escherichia coli peptides that can inhibit its own phagocytosis in sepsis

Introdução: Sepse é uma síndrome complexa definida por resposta inflamatória sistêmica, de origem infecciosa e caracterizada por manifestações múltiplas que podem determinar disfunção ou falência de um ou mais órgãos ou sistemas. É a principal causa de morte em unidades de terapia intensiva em pacientes críticos e tem representado uma fonte constante de preocupação para os sistemas de saúde em todo o mundo, devido, principalmente, às taxas elevadas de morbimortalidade. O tratamento da sepse é um desafio e continua a ser uma tarefa difícil devido a inúmeros fatores interferentes. Um estudo do nosso grupo demonstrou que a Escherichia coli (E. coli) é capaz de se ligar CD16 de um modo independente de opsonina, levando a um aumento na resposta inflamatória e a inibição da sua própria fagocitose, por conseguinte, procurou-se identificar os peptídeos no proteoma da E. coli envolvidos neste cenário. Metodologia: Utilizando a metodologia de Phage Display, que consiste numa técnica de clonagem, que permite a expressão de diversas sequências de peptídeos na superfície de bacteriófagos, nós identificamos 2 peptídeos que obtiveram interação com CD16. Após a seleção dos peptídeos identificamos uma proteína de membrana de E.coli que possui alta similaridade com um de nossos peptídeos selecionados. Nós acreditamos que esta proteína de membrana possa estar envolvida no processo de evasão imune desenvolvida pela E.coli e parece ser um forte candidato como uma nova opção terapêutica para controlar infecções por E. coli. Conclusão: A identificação de proteínas capazes de induzir inibição de fagocitose, através do receptor CD16, pode ser usada como uma nova forma de tratamento da sepse, assim como explorada no tratamento de doenças autoimunes.

Introduction: Sepsis is a complex syndrome defined by a systemic inflammatory response of infectious origin and characterized by multiple manifestations that can determine dysfunction/failure of one or more organs and systems. It is the leading cause of death in intensive care units and represents a major health problem around the world, mainly due to its high mortality and morbidity rates. The treatment of sepsis is challenging and remains a difficult task due to numerous interfering factors. A study from our group demonstrated that Escherichia coli (E. coli) is able to bind CD16 in an opsoninindependent manner, leading to an increase in the inflammatory response and inhibition of its own phagocytosis, therefore we sought to identify the peptides in the E. coli proteome involved in this scenario. Methods and Results: Using the Phage Display technique, which is a cloning technique that allows the expression of various peptide sequences on the surface of bacteriophages (phages) and selecting these on the basis of affinity for a target molecule, we identified two peptides that interact with CD16. Next, using bioinformatic tools, we found an E. coli membrane protein that has high similarity with one of our selected peptides. We believe this membrane protein is involved in the process of immune evasion developed by E. coli and it is a strong candidate as a new therapeutic option to control E. coli infections. Conclusion: The identification of proteins capable of inducing inhibition of phagocytosis through the CD16 receptor, can be used as a new treatment of sepsis, as well as exploited in the treatment of autoimmune diseases.