RESUMO
Paired immunoglobulin-like type 2 receptors (PILRs) inhibitory PILRalpha and activating PILRbeta are predominantly expressed on myeloid cells. Their functions in host defense and inflammation are largely unknown, and in this study, we evaluated their roles in an acute Staphylococcus aureus pneumonia model. Compared to their respective controls, Pilrb(-/-) mice or mice in which PILRalpha was activated with an agonistic antibody showed improved clearance of pulmonary staphylococci and improved survival. These mice had reduced serum or bronchoalveolar lavage fluid levels of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 and elevated levels of gamma interferon (IFN-gamma), IL-12, and IL-10. In contrast, mice in which PILRbeta was activated had increased lung bacterial burdens and higher mortality coupled with an intense proinflammatory response with highly elevated levels of IL-1beta, TNF-alpha, and IL-6. Treatment groups with reduced bacterial burdens had higher levels of Keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), and MIP-1alpha in bronchoalveolar lavage fluid and an increased influx of neutrophils and macrophages to the lungs. Consistent with our in vivo findings, bone marrow-derived macrophages from Pilrb(-/-) mice released significantly less IL-1beta and TNF-alpha and more IFN-gamma and IL-12 than did the wild-type macrophages when directly stimulated with heat-killed S. aureus. To our knowledge, this is the first evidence that S. aureus directly interacts with PILRbeta. It provides a mechanism by which manipulating the balance in favor of an inhibitory PILR signal, by activation of PILRalpha or deletion of PILRbeta, helps to control acute S. aureus-mediated pneumonia and attenuate the inflammatory response. These results highlight the importance of PILRs in innate immunity and the control of inflammation.
Assuntos
Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/patologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Staphylococcus aureus/imunologia , Animais , Análise Química do Sangue , Líquido da Lavagem Broncoalveolar/química , Contagem de Colônia Microbiana , Citocinas/análise , Citocinas/sangue , Feminino , Pulmão/microbiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/deficiência , Receptores Imunológicos/imunologia , Análise de SobrevidaRESUMO
Role of viral genes in modulating T helper 1 (Th1) and T helper 2 (Th2) balance is of principal interest in the study of cytomegalovirus (CMV) immunity. Murine CMV (MCMV) mutants were used to explore a possible mechanism for the ability of virus to induce a predominant Th1 response and to suppress Th2 response by examining the production of Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-10) cytokines by the splenocytes of mice infected with wild type (WT) and MCMV mutants. Results (n=6) show that as compared with WT, the MCMV mutant with specific disruption of M43 gene upregulates the production of IL-4 (P=0.0002) and to a lesser extent IL-10 (P=0.015) at 14 days post infection. This indicates that M43 gene may play a role in suppressing Th2 (IL-4) production, especially in the later stage of infection. The IL-4 and IL-10 production during infection with M43 mutant occurs in the presence of a strong IFN-gamma (Th1) response, overriding the cross-regulatory effects of these cytokines within the Th1/Th2 paradigm and suggesting that the predominant response during CMV infection is still a Th1 type response.
Assuntos
Citocinas/biossíntese , Muromegalovirus/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Muromegalovirus/patogenicidadeRESUMO
Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of typhoid fever, is an exclusively human pathogen. Little is known about specific factors that may confer to this bacterium its unique pathogenic features. One of these determinants is CdtB, a homologue of the active subunit of the cytolethal distending toxin, which causes DNA damage leading to cell cycle arrest and distension of intoxicated cells. A unique property of S. Typhi CdtB is that it is only synthesized when this bacterium is within an intracellular compartment. Through a genetic screen, we have identified a transcriptional regulatory protein that controls the intracellular expression of cdtB. This regulator, which we have named IgeR, is a member of the DeoR family of transcriptional regulatory proteins and is highly conserved in all S. enterica serovars. IgeR directly binds the cdtB promoter and represses its expression in the extracellular environment. Microarray analysis identified additional IgeR-regulated genes that are involved in virulence. Constitutive expression of igeR resulted in the reduction of intracellular expression of cdtB by S. Typhi and in significant impairment of the virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mice. We propose that IgeR may co-ordinate gene expression during Salmonella's transition from an extracellular to an intracellular environment.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras , Salmonella typhi/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Linhagem Celular , Células Epiteliais , Feminino , Perfilação da Expressão Gênica , Humanos , Intestinos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhi/genética , Salmonella typhi/metabolismo , Salmonella typhimurium/patogenicidade , Transcrição Gênica , Febre Tifoide/microbiologia , VirulênciaRESUMO
Many bacterial pathogens encode the cytolethal distending toxin (CDT), which causes host cells to arrest during their cell cycle by inflicting DNA damage. CDT is composed of three proteins, CdtA, CdtB, and CdtC. CdtB is the enzymatically active or A subunit, which possesses DNase I-like activity, whereas CdtA and CdtC function as heteromeric B subunits that mediate the delivery of CdtB into host cells. We show here that Salmonella enterica serovar Typhi encodes CDT activity, which depends on the function of a CdtB homologous protein. Remarkably, S. enterica serovar Typhi does not encode apparent homologs of CdtA or CdtC. Instead, we found that toxicity, as well as cdtB expression, requires bacterial internalization into host cells. We propose a pathway of toxin delivery in which bacterial internalization relieves the requirement for the functional equivalent of the B subunit of the CDT toxin.
Assuntos
Toxinas Bacterianas/farmacocinética , Salmonella typhi/crescimento & desenvolvimento , Salmonella typhi/patogenicidade , Animais , Transporte Biológico , Ciclo Celular , Linhagem Celular , Células Cultivadas , Cloranfenicol/farmacologia , Genes Reporter , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Luciferases/análise , Inibidores da Síntese de Proteínas/farmacologia , Salmonella typhi/efeitos dos fármacosRESUMO
A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.
Assuntos
Elementos de DNA Transponíveis , Infecções por Herpesviridae/mortalidade , Muromegalovirus/crescimento & desenvolvimento , Muromegalovirus/patogenicidade , Mutagênese Insercional , Fases de Leitura Aberta/genética , Animais , Infecções por Herpesviridae/virologia , Camundongos , Camundongos SCID , Muromegalovirus/genética , Mutação , Células NIH 3T3 , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência , Replicação ViralRESUMO
We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.