RESUMEN
OBJECTIVE: AS is a systemic inflammatory disease of the SpA family. Polymorphisms at loci including HLA-B27, IL-23R and ERAP-1 directly implicate immune mechanisms in AS pathogenesis. Previously, in an SpA model, we identified HLA-B27-mediated effects on dendritic cells that promoted disease-associated Th17 cells. Here we extend these studies to AS patients using deep immunophenotyping of candidate pathogenic cell populations. The aim of our study was to functionally characterize the immune populations mediating AS pathology. METHODS: Using 11-parameter flow cytometry, we characterized the phenotype and functions of lymphocyte and myeloid cells from peripheral blood, and the synovial phenotype of AS patients and age-matched healthy controls. RESULTS: Significantly fewer circulating CD1c-expressing dendritic cells were observed in AS patients, offset by an increase in CD14(-) CD16(+) mononuclear cells. Ex vivo functional analysis revealed that this latter population induced CCR6 expression and promoted secretion of IL-1ß and IL-6 when co-cultured with naive CD4(+) T cells. Additionally, systemic inflammation in AS patients significantly correlated with increased proportions of activated CCR9(+) CD4(+) T cells. CONCLUSION: CD14(-) CD16(+) mononuclear cells may contribute to AS by promoting Th17 responses, and antigen-presenting cells of mucosal origin are likely to contribute to systemic inflammation in AS.
Asunto(s)
Células Dendríticas/inmunología , Inmunidad Celular , Inflamación/inmunología , Interleucina-23/genética , Polimorfismo Genético , Espondilitis Anquilosante/inmunología , Linfocitos T/inmunología , Adulto , Animales , ADN/genética , Femenino , Citometría de Flujo , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-23/metabolismo , Masculino , Persona de Mediana Edad , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/metabolismoRESUMEN
OBJECTIVES: Macrophages are central to the inflammatory processes driving rheumatoid arthritis (RA) synovitis. The molecular pathways that are induced in synovial macrophages and thereby promote RA disease pathology remain poorly understood. METHODS: We used microarray to characterise the transcriptome of synovial fluid (SF) macrophages compared with matched peripheral blood monocytes from patients with RA (n=8). RESULTS: Using in silico pathway mapping, we found that pathways downstream of the cholesterol activated liver X receptors (LXRs) and those associated with Toll-like receptor (TLR) signalling were upregulated in SF macrophages. Macrophage differentiation and tumour necrosis factor α promoted the expression of LXRα. Furthermore, in functional studies we demonstrated that activation of LXRs significantly augmented TLR-driven cytokine and chemokine secretion. CONCLUSIONS: The LXR pathway is the most upregulated pathway in RA synovial macrophages and activation of LXRs by ligands present within SF augments TLR-driven cytokine secretion. Since the natural agonists of LXRs arise from cholesterol metabolism, this provides a novel mechanism that can promote RA synovitis.
Asunto(s)
Artritis Reumatoide/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/biosíntesis , Líquido Sinovial/metabolismo , Receptores Toll-Like/fisiología , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Diferenciación Celular/fisiología , Citocinas/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/fisiología , Transducción de Señal/fisiología , Sinovitis/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
To understand the contribution of mononuclear phagocytes (MNP), which include monocyte-derived intestinal macrophages, to the pathogenesis of inflammatory bowel disease (IBD), it is necessary to identify functionally-different MNP populations. We aimed to characterise intestinal macrophage populations in patients with IBD. We developed 12-parameter flow cytometry protocols to identify and human intestinal MNPs. We used these protocols to purify and characterize colonic macrophages from colonic tissue from patients with Crohn's disease (CD), ulcerative colitis (UC), or non-inflamed controls, in a cross-sectional study. We identify macrophage populations (CD45+CD64+ HLA-DR+) and describe two distinct subsets, differentiated by their expression of the mannose receptor, CD206. CD206+ macrophages expressed markers consistent with a mature phenotype: high levels of CD68 and CD163, higher transcription of IL-10 and lower expression of TREM1. CD206- macrophages appear to be less mature, with features more similar to their monocytic precursors. We identified and purified macrophage populations from human colon. These appear to be derived from a monocytic precursor with high CCR2 and low CD206 expression. As these cells mature, they acquire expression of IL-10, CD206, CD63, and CD168. Targeting the newly recruited monocyte-derived cells may represent a fruitful avenue to ameliorate chronic inflammation in IBD.
Asunto(s)
Susceptibilidad a Enfermedades , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Biomarcadores , Susceptibilidad a Enfermedades/inmunología , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Inmunidad Mucosa , Inmunofenotipificación , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/genética , Interleucina-10/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Glicoproteínas de Membrana/genética , Receptores Inmunológicos/genética , TranscriptomaRESUMEN
The identification of conventional dendritic cells (cDCs) in the intestinal mucosa has been hampered by the difficulties associated with isolating cells from the intestine and by the fact that overlapping markers have made it complicated to discriminate them accurately from other intestinal mononuclear phagocytes such as macrophages (MFs). Here we detail the protocols we have developed to isolate live leukocytes from both murine and human small and large intestines and describe reliable strategies which can be used to identify bona fide cDCs in such preparations.