RESUMEN
Our previous studies indicated that mouse beta defensin 14 (mBD14, Defb14), a newly identified member of the beta-defensin super family, interacts with the chemokine receptors CCR2 and CCR6. In this study we report that pre-stimulation of primary mouse macrophages with mBD14 results in a synergistic, enhanced expression of pro-inflammatory cytokines and chemokines induced by TLR ligand re-stimulation. Experiments using specific inhibitors of G(i)-protein-coupled receptor signaling provide evidence that this effect seems to be mediated by a G(i)-protein-coupled receptor expressed on bone marrow derived macrophages. However, using primary macrophages derived from CCR6- and CCR2-deficient mice clearly demonstrated that the enhanced pro-inflammatory cytokine and chemokine expression is independent of the chemokine receptors CCR6 and CCR2. Additionally, signaling pathway analysis indicated that mBD14 is capable of inducing MAPK ERK1/2 phosphorylation and the induction of CD86 and F4/80 expression in bone marrow-derived macrophages after mBD14 stimulation. Collectively, our data indicate that ß-defensins activate primary macrophages and enhance pro-inflammatory responses by using G(i)PCRs in order to support inflammatory reactions induced by TLR ligands.
Asunto(s)
Citocinas/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , beta-Defensinas/farmacología , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Citocinas/biosíntesis , Citocinas/genética , Sinergismo Farmacológico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/inmunología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Fosforilación , Cultivo Primario de Células , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores CCR2/inmunología , Receptores CCR6/deficiencia , Receptores CCR6/genética , Receptores CCR6/inmunología , Proteínas Recombinantes/farmacología , Transducción de Señal , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Our previous studies indicated that LTßR activation mainly by T cell derived LTα1ß2 is crucial for the control and down-regulation of intestinal inflammation. In order to dissect the cellular and molecular role of LTßR activation in the experimental model of DSS-induced intestinal inflammation, we have generated cell type-specific LTßR-deficient mice with specific ablation of LTßR expression on macrophages/neutrophils (LTßR((flox/flox))×LysM-Cre). These mice develop an exacerbated intestinal inflammation in our experimental model indicating that LTßR expression on macrophages/neutrophils is responsible for the control and down-regulation of the inflammatory reaction. These results were verified by adoptive transfer experiments of BMDM from wild-type and LTßR-deficient mice. Furthermore, transfer of activated CD4+ T cells derived from wild-type mice, but not from LTßR ligand-deficient mice attenuated the signs of intestinal inflammation. Finally, we demonstrate that LTßR activation on BMDM results in induction of TRIM30α, a negative regulator of NFκB activation. Concordantly, ablation of LTßR signaling results in the inability to induce TRIM30α expression concomitant with an increased expression of pro-inflammatory cytokines in our experimental model. Taken together, our data demonstrate that LTßR activation on macrophages by CD4+ T cell derived LTαß controls the pro-inflammatory response by activation of a TRIM30α-dependent signaling pathway, crucial for the down-regulation of the inflammatory response in this experimental model.
Asunto(s)
Colitis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptor beta de Linfotoxina/metabolismo , Macrófagos/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Técnicas de Cocultivo , Colitis/inducido químicamente , Colitis/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Péptidos y Proteínas de Señalización Intracelular/inmunología , Receptor beta de Linfotoxina/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Our previous studies indicated that lymphotoxin ß receptor (LTßR) activation controls and downregulates inflammatory reactions. In this study, we report that LTßR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTßR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTßR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTßR-dependent induction of TRIM30α and LTßR-mediated TLR cross-tolerance. Concordantly, LTßR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTßR-deficient mice with ablation of LTßR expression on macrophages/neutrophils (LTßR(flox/flox) × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTßR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTßR expression on macrophages (LTßR(flox/flox) × LysM-Cre) are resistant to LTßR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTßR activation on macrophages by T cell-derived lymphotoxin α(1)ß(2) controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.