RESUMEN
Viruses, particularly the Epstein-Barr virus (EBV) has long been suspected to exacerbate acute arthritic symptoms. However, the cell populations that contribute to import viruses into the inflamed tissues remain to be identified. In the present study, we have investigated the role of monocytes in the transport of Murid herpesvirus 68 (MHV-68), a mouse virus closely related to EBV, using the serum transfer-induced arthritis (STIA) model. We found compelling evidence that MHV-68 infection markedly increased disease severity in NR4A1-/- mice, which are deficient for Ly6Clow monocytes. In contrast, the MHV-68-induced enhancement of joint inflammation was lessened in CCR2-/- mice, suggesting the involvement of inflammatory Ly6Chigh monocytes in viral transport. We also observed that following selective depletion of monocyte subsets by administration of clodronate, MHV-68 transport into the synovium occurs only in the presence of Ly6Chigh monocytes. Tracking of adoptively transferred Ly6Chigh GFP infected monocytes into arthritic CCR2-/- mice by two-photon intravital microscopy showed that this monocyte subset has the capacity to deliver viruses to inflamed AR joints, as confirmed by the detection of viral DNA in inflamed tissues of recipient mice. We thus conclude that Ly6Chigh monocytes import MHV-68 when they are mobilized to the inflamed arthritic joint.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/fisiología , Monocitos/inmunología , Rhadinovirus/fisiología , Infecciones Tumorales por Virus/inmunología , Traslado Adoptivo , Animales , Antígenos Ly/metabolismo , Artritis Experimental/virología , Artritis Reumatoide/virología , Células Cultivadas , ADN Viral/análisis , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/virología , Femenino , Infecciones por Herpesviridae/virología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/trasplante , Muridae , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Receptores CCR2/genética , Rhadinovirus/patogenicidad , Infecciones Tumorales por Virus/virologíaRESUMEN
Pattern-recognition receptors such as Toll-like receptors (TLRs) are essential sensors implicated in the early and efficient innate immune response against pathogens. We have previously demonstrated that leukotriene B(4)(LTB(4)) has the capacity to enhance leukocyte responses to TLR9 ligands and to control viral infection. In this report, we provide evidence that LTB(4) treatment of human neutrophils leads to a potentiation in proinflammatory cytokine secretion induced by various myeloid differentiation factor 88-dependent TLR agonists. LTB(4) failed to enhance TLR mRNA levels as well as expression of TLR2 and TLR4 receptors, suggesting that LTB(4) acts through intracellular mechanism(s) to potentiate neutrophil responses to TLR ligands. We found that while IRAK can be activated by LTB(4), this process is dispensable to LTB(4) to potentiate neutrophil responses to TLR ligands since pretreatment of neutrophils with IRAK1/4 inhibitor did not affect its potentiating effects. However, our data clearly show that LTB(4) treatment of neutrophils led to the phosphorylation of downstream signaling molecules, TAK1 and p38, a process found essential to observe an increased secretion of cytokines by neutrophils activated with TLR ligands. Pretreatment of neutrophils with TAK1 or p38 kinase inhibitors strongly repressed the effect of LTB(4) on cytokine synthesis by neutrophils stimulated with LTA, LPS or CpG. The same pattern was observed in agonist-treated human embryonic kidney 293 cells transfected with TAK1-targeting siRNA where secretion of IL-8 was significantly reduced to basal levels. These results indicate that TAK1 and p38 kinases appear to be central in the 'priming effect' of LTB(4) on neutrophils to enhance response to TLR ligands.
Asunto(s)
Leucotrieno B4/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , Neutrófilos/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Citometría de Flujo , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Interleucina-8/inmunología , Interleucina-8/metabolismo , Leucotrieno B4/farmacología , Ligandos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Inhibidores de Proteínas Quinasas/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácidos Teicoicos/inmunología , Ácidos Teicoicos/farmacología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/agonistas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Synovial infiltration of monocytes is commonly associated with inflammation in rheumatoid arthritis (RA). Toll-like receptors (TLRs) are innate sensors that recognize cell debris and microbial components in host, a process contributing to maintain chronic inflammation in RA. We assessed the expression levels of TLR2 and TLR9 in monocyte subsets of active RA patients and characterized their cytokine profiles in response to synthetic and viral TLR2 and TLR9 agonists, including Epstein-Barr virus (EBV) which is suspected to contribute to RA symptoms. METHODS: Prevalence of monocyte subsets CD14(++) CD16(-), CD14(+) CD16(+) and CD14(low) CD16(++) was evaluated in blood and synovial fluids of active RA patients and levels of TLR2 and TLR9 in monocyte subsets were measured by flow cytometry. Enriched monocytes derived from RA patients and healthy donors were stimulated in vitro with synthetic TLR2 and TLR9 agonists and with EBV particles or viral DNA. Intracellular cytokine profiles were determined in respective monocyte subsets. Finally, the presence of EBV genome was evaluated by real-time PCR in blood and synovial monocytes of RA patients. RESULTS: Numbers of CD14(+) CD16(+) and CD14(low) CD16(++) were found to increase in blood of RA patients compared to healthy controls, while all three subsets were detected in synovial fluids. TLR2 is abundantly expressed on blood and synovial CD14(++) CD16(-) and CD14(+) CD16(+) monocytes from RA patients. Levels of TLR9 were increased on all three subsets of blood monocytes but markedly enhanced in monocytes isolated from synovial fluids. Compared to healthy controls, CD14(++) CD16(-) monocytes of RA patients displayed an enlarged capacity to produce proinflammatory cytokines after stimulation with synthetic TLR2 and TLR9 agonists while both CD14(++) CD16(-) and CD14(+) CD16(+) monocytes showed increased response to EBV stimulation. The presence of EBV genome was also detected in monocytes and neutrophils of a significant proportion of patients. CONCLUSION: Patients with active RA show an increased expression of TLR2 and TLR9 on monocyte subsets and display higher production of inflammatory cytokines in response to TLR agonists. The presence of EBV genome in monocytes and neutrophils reinforces the suspected role of the virus in the exacerbation of RA symptoms.