RESUMO
CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG(2a) fusion protein (fCCL20-mIgG(2a)) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.
Assuntos
Quimiocina CCL20/metabolismo , Quimiotaxia , Furões/metabolismo , Receptores CCR6/metabolismo , Sequência de Aminoácidos , Animais , Catequina/análogos & derivados , Catequina/farmacologia , Quimiocina CCL20/química , Quimiotaxia/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Cães , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Ligação Proteica/efeitos dos fármacos , Receptores CCR6/química , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Virais/farmacologiaRESUMO
Human immunodeficiency virus-1 (HIV-1)-infected monocytes/macrophages and microglia release increased levels of proinflammatory cytokines and chemokines, including ELR+ (containing glutamic acid-leucine-arginine motif) chemokines. To investigate the role of HIV-1 infection on chemokine regulation, monocyte-derived macrophages (MDMs) from normal donors were infected with HIV-1 and the expression of chemokines and their downstream biological functions were evaluated. Among the tested chemokines, CXCL5 was upregulated significantly both at the mRNA and protein level in the HIV-1-infected MDMs compared with mock-infected cultures. Upregulation of CXCL5 in the HIV-1-infected MDMs is, in part, regulated by increased interleukin-1ß (IL-1ß) production and phosphorylation of ERK1/2. Functional analyses indicate that HIV-1-induced overexpression of CXCL5 has enhanced the ability to attract neutrophils, as observed by chemotaxis assay. However, exposure of NT2, SH-SY5Y cells, and primary neurons to HIV-1-infected MDM supernatants resulted in cell death that was not rescued by anti-CXCL5 antibody suggesting that CXCL5 does not have direct effect on neuronal death. Together, these results suggest that the increased level of CXCL5 in tissue compartments, including the central nervous system of HIV-1-infected individuals might alter the inflammatory response through the infiltration of neutrophils into tissue compartment, thus causing secondary effects on resident cells.
Assuntos
Viroses do Sistema Nervoso Central/genética , Viroses do Sistema Nervoso Central/virologia , Quimiocina CXCL5/genética , Regulação da Expressão Gênica , HIV-1/fisiologia , Macrófagos/metabolismo , Macrófagos/virologia , Transcrição Gênica , Linhagem Celular , Sobrevivência Celular/genética , Viroses do Sistema Nervoso Central/imunologia , Viroses do Sistema Nervoso Central/metabolismo , Quimiocina CXCL5/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Perfilação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Neurônios/metabolismo , Infiltração de Neutrófilos/imunologia , Replicação ViralRESUMO
Abstract LYVE-1 is a marker expressed by lymphatic endothelial cells (LECs) that line the lymphatic endothelium. Through studies designed to examine potential changes in expression of LYVE-1 in cynomolgus macaque colon tissues during the course of simian immunodeficiency virus (SIV) infection, we discovered that LYVE-1 was expressed by heterogenous populations of cells. As revealed by in situ hybridization (ISH), LYVE-1 mRNA levels in colon were decreased in macaques with AIDS compared with acutely infected or uninfected macaques. In the submucosal layer of the colon, approximately half of the LYVE-1-expressing cells co-expressed the dendritic cell (DC) marker, DC-SIGN/CD209, and this percentage did not change appreciably during infection. Subsets of cells expressing LYVE-1 also co-expressed macrophage markers, such as CD68 and the macrophage mannose receptor (MMR)/CD206, in both the colon and lymph nodes. LECs, DCs, and macrophages that co-expressed LYVE-1 were observed in colon and lymph node from uninfected, healthy animals as well as in tissues with SIV-driven inflammation. These findings provide further definition of the phenotypic overlap between LECs and antigen presenting cells, reveal the heterogeneity within the population of cells expressing the lymphatic marker LYVE-1, and show that SIV modulates this population of cells in a mucosal surface across which the virus is acquired.
Assuntos
Moléculas de Adesão Celular/imunologia , Intestino Grosso/imunologia , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Proteínas de Transporte Vesicular/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Colo/imunologia , Colo/metabolismo , Colo/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Endotélio Linfático/citologia , Endotélio Linfático/imunologia , Endotélio Linfático/metabolismo , Imunofluorescência , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Hibridização In Situ , Intestino Grosso/metabolismo , Intestino Grosso/virologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/virologia , Macaca fascicularis , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Microscopia Confocal , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND: Chemokines provide critical immune cell homing and activation signals that if altered could affect the inflammatory milieu and cellular composition of lymphoid tissues. During HIV-1 and simian immunodeficiency virus (SIV)-infection, the virus triggers an increase in inflammation or activation, leading to immunodeficiency and development of opportunistic infections, such as in the lungs-a massive interface between the host and the environment. METHODS: Chemokine, cytokine, and chemokine receptor expression profiles were determined using real-time reverse transcriptase-polymerase chain reaction and in situ hybridization in hilar lymph nodes (HiLNs) from cynomolgus macaques at different stages after infection with SIV/DeltaB670. Immunostaining of tissue sections and flow cytometric analysis of cryopreserved cells were used to examine cellular compositions of lymph nodes. RESULTS: Interferon-gamma, type 1 chemokine, and cognate chemokine receptor mRNAs were upregulated, whereas type 2 and homeostatic chemokine and chemokine receptor mRNAs were down-regulated in HiLNs after SIV infection. Local SIV and interferon-gamma levels were positively correlated with type 1 chemokine levels but negatively correlated with type 2 and homeostatic chemokine levels. Using in situ hybridization, Pneumocystis carinii rRNA was detected in lung-draining lymph nodes from animals with P. carinii pneumonia. Changes in the cellular composition of HiLNs included decreased proportions of CD4 cells and dendritic cells and increased proportions of CD8, CXCR3, and CCR5 cells. CONCLUSIONS: SIV infection of cynomolgus macaques dramatically alters the cellular homing signals of lung-draining lymph nodes, which correlated with changes in the immune cellular composition. These changes could contribute to the loss of immune function that defines AIDS.
Assuntos
Quimiocinas/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Receptores de Quimiocinas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/metabolismo , Pulmão , Macaca fascicularis , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Pneumocystis carinii/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Subpopulações de Linfócitos T/imunologiaRESUMO
CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.