RESUMEN
Chronic kidney disease (CKD) results from the development of fibrosis, ultimately leading to end-stage renal disease (ESRD). Although human bone marrow-derived mesenchymal stem cells (MSCs) can accelerate renal repair following acute injury, the establishment of fibrosis during CKD may affect their potential to influence regeneration capacity. Here we tested the novel combination of MSCs with the antifibrotic serelaxin to repair and protect the kidney 7 d post-unilateral ureteral obstruction (UUO), when fibrosis is established. Male C57BL6 mice were sham-operated or UUO-inured (n = 4-6) and received vehicle, MSCs (1 × 10(6)), serelaxin (0.5 mg/kg per d), or the combination of both. In vivo tracing studies with luciferin/enhanced green fluorescent protein (eGFP)-tagged MSCs showed specific localization in the obstructed kidney where they remained for 36 h. Combination therapy conferred significant protection from UUO-induced fibrosis, as indicated by hydroxyproline analysis (P < 0.001 vs. vehicle, P < 0.05 vs. MSC or serelaxin alone). This was accompanied by preserved structural architecture, decreased tubular epithelial injury (P < 0.01 vs. MSCs alone), macrophage infiltration, and myofibroblast localization in the kidney (both P < 0.01 vs. vehicle). Combination therapy also stimulated matrix metalloproteinase (MMP)-2 activity over either treatment alone (P < 0.05 vs. either treatment alone). These results suggest that the presence of an antifibrotic in conjunction with MSCs ameliorates established kidney fibrosis and augments tissue repair to a greater extent than either treatment alone.
Asunto(s)
Fibrosis/fisiopatología , Fallo Renal Crónico/fisiopatología , Riñón/fisiopatología , Células Madre Mesenquimatosas/citología , Relaxina/uso terapéutico , Insuficiencia Renal Crónica/terapia , Animales , Diferenciación Celular , Proliferación Celular , Colágeno/metabolismo , Gelatinasas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Riñón/lesiones , Riñón/metabolismo , Macrófagos/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/citología , Miofibroblastos/metabolismo , Proteínas Recombinantes/uso terapéutico , Regeneración , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Serum amyloid A (SAA) is expressed locally in chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), where macrophages that do not accord with the classic M1/M2 paradigm also accumulate. In this study, the role of SAA in regulating macrophage differentiation was investigated in vitro using human blood monocytes from healthy subjects and patients with COPD and in vivo using an airway SAA challenge model in BALB/c mice. Differentiation of human monocytes with SAA stimulated the proinflammatory monokines IL-6 and IL-1ß concurrently with the M2 markers CD163 and IL-10. Furthermore, SAA-differentiated macrophages stimulated with lipopolysaccharide (LPS) expressed markedly higher levels of IL-6 and IL-1ß. The ALX/FPR2 antagonist WRW4 reduced IL-6 and IL-1ß expression but did not significantly inhibit phagocytic and efferocytic activity. In vivo, SAA administration induced the development of a CD11c(high)CD11b(high) macrophage population that generated higher levels of IL-6, IL-1ß, and G-CSF following ex vivo LPS challenge. Blocking CSF-1R signaling effectively reduced the number of CD11c(high)CD11b(high) macrophages by 71% and also markedly inhibited neutrophilic inflammation by 80%. In conclusion, our findings suggest that SAA can promote a distinct CD11c(high)CD11b(high) macrophage phenotype, and targeting this population may provide a novel approach to treating chronic inflammatory conditions associated with persistent SAA expression.
Asunto(s)
Diferenciación Celular , Pulmón/citología , Macrófagos/citología , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animales , Western Blotting , Estudios de Casos y Controles , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Hematopoyesis , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fagocitosis/fisiología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Amiloide A Sérica/genética , Transducción de SeñalRESUMEN
The chemokine receptor CCR6 marks subsets of T cells and innate lymphoid cells that produce IL-17 and IL-22, and as such may play a role in the recruitment of these cells to certain inflammatory sites. However, the precise role of CCR6 has been controversial, in part because no effective monoclonal antibody (mAb) inhibitors against this receptor exist for use in mouse models of inflammation. We circumvented this problem using transgenic mice expressing human CCR6 (hCCR6) under control of its native promoter (hCCR6-Tg/mCCR6-/-). We also developed a fully humanized mAb against hCCR6 with antagonistic activity. The expression pattern of hCCR6 in hCCR6-Tg/mCCR6-/- mice was consistent with the pattern observed in humans. In mouse models of experimental autoimmune encephalomyelitis (EAE) and psoriasis, treatment with anti-hCCR6 mAb was remarkably effective in both preventive and therapeutic regimens. For instance, in the imiquimod model of psoriasis, anti-CCR6 completely abolished all signs of inflammation. Moreover, anti-hCCR6 attenuated clinical symptoms of myelin oligodendrocyte glycoprotein-induced (MOG-induced) EAE and reduced infiltration of inflammatory cells in the central nervous system. CCR6 plays a critical role in Th17 type inflammatory reactions, and CCR6 inhibition may offer an alternative approach for the treatment of these lesions.
RESUMEN
Chemokines and their receptors are pivotal for the trafficking of leukocytes during immune responses, and host defense. However, immune cell migration also contributes to a wide variety of autoimmune and chronic inflammatory diseases. Compelling evidence suggests that both CXCR3 and CCR6 chemokine receptors play crucial roles in the migration of pathological Th1 and Th17 cells during the course of certain inflammatory diseases. The use of two or more receptors by pathogenic cells may explain why targeting of individual receptors has proven disappointing in the clinic. We therefore hypothesized that simultaneous targeting of both CXCR3 and CCR6 with a bispecific antibody (BsAb) might result in decreased chemotaxis and/or specific depletion of pro-inflammatory T cell subsets. In this study, we designed and characterized a fully humanized BsAb. We show that the BsAb binds to both chemokine receptors, as demonstrated by Flow Cytometry and Surface Plasmon Resonance analysis. Furthermore, we demonstrate that the BsAb effectively blocks cell chemotaxis and induces specific antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Therefore, we propose that dual targeting of CXCR3 and CCR6 with a fully humanized BsAb may display a potent interventional approach for the treatment of inflammatory and autoimmune diseases.