Immune modulation and tolerance induction by RelB-silenced dendritic cells through RNA interference

Dendritic cells (DC), the most potent APCs, can initiate the immune response or help induce immune tolerance, depending upon their level of maturation. DC maturation is associated with activation of the NF-kappaB pathway, and the primary NF-kappaB protein involved in DC maturation is RelB, which coordinates RelA/p50-mediated DC differentiation. In this study, we show that silencing RelB using small interfering RNA results in arrest of DC maturation with reduced expression of the MHC class II, CD80, and CD86. Functionally, RelB-silenced DC inhibited MLR, and inhibitory effects on alloreactive immune responses were in an Ag-specific fashion. RelB-silenced DC also displayed strong in vivo immune regulation. An inhibited Ag-specific response was seen after immunization with keyhole limpet hemocyanin-pulsed and RelB-silenced DC, due to the expansion of T regulatory cells. Administration of donor-derived RelB-silenced DC significantly prevented allograft rejection in murine heart transplantation. This study demonstrates for the first time that transplant tolerance can be induced by means of RNA interference using in vitro-generated tolerogenic DC.     

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappaB

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.     

Abnormal NF-kappa B function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-kappaB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in approximately 60% of patients. The monocyte and DC NF-kappaB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-kappaB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.     

Leptin induces CD40 expression through the activation of Akt in murine dendritic cells

Increasing evidence suggests a regulatory role for leptin, an adipocyte-derived hormone, in immunity. Although recent studies indicated an essential role of leptin signaling in dendritic cell (DC) maturation, the molecular mechanisms by which leptin modulates DC functional maturation remained unclear. In this study, we showed that leptin induced CD40 expression in murine DC and significantly up-regulated their immunostimulatory function in driving T cell proliferation. Moreover, leptin markedly enhanced lipopolysaccharide-mediated DC activation. Using pharmacological inhibitors for Akt, STAT-1alpha, or NF-kappaB and the dominant negative forms of Akt and IkappaB kinase alpha/beta/gamma, as well as small interfering RNA for STAT-1alpha, we showed that Akt, STAT-1alpha, and NF-kappaB were important for the leptinor lipopolysaccharide-induced CD40 expression. Coimmunoprecipitation analysis revealed that leptin promoted immune complex formation between Akt and the IkappaB kinase subunits as well as STAT-1alpha. Blocking the activity of Akt demonstrated a crucial role for Akt in translocation of STAT-1alpha and NF-kappaB to the nucleus and activation of the CD40 promoter. Further analysis with chromatin immunoprecipitation assay confirmed that leptin recruited STAT-1alpha, NF-kappaBp65, and RNA polymerase II to the CD40 promoter and enhanced histone 4 acetylation in a time-dependent manner. Thus, our results have elucidated the molecular mechanisms underlying leptin-induced CD40 expression and DC maturation.     

Dendritic cell-intrinsic expression of NF-kappa B1 is required to promote optimal Th2 cell differentiation

A number of receptors and signaling pathways can influence the ability of dendritic cells (DC) to promote CD4(+) Th type 1 (Th1) responses. In contrast, the regulatory pathways and signaling events that govern the ability of DC to instruct Th2 cell differentiation remain poorly defined. In this report, we demonstrate that NF-kappaB1 expression within DC is required to promote optimal Th2 responses following exposure to Schistosoma mansoni eggs, a potent and natural Th2-inducing stimulus. Although injection of S. mansoni eggs induced production of IL-4, IL-5, and IL-13 in the draining lymph node of wild-type (WT) mice, NF-kappaB1(-/-) hosts failed to express Th2 cytokines and developed a polarized Ag-specific IFN-gamma response. In an in vivo adoptive transfer model in which NF-kappaB-sufficient OVA-specific DO11.10 TCR transgenic T cells were injected into OVA-immunized WT or NF-kappaB1(-/-) hosts, NF-kappaB1(-/-) APCs efficiently promoted CD4(+) T cell proliferation and IFN-gamma responses, but failed to promote Ag-specific IL-4 production. Further, bone marrow-derived DC from NF-kappaB1(-/-) mice failed to promote OVA-specific Th2 cell differentiation in in vitro coculture studies. Last, S. mansoni egg Ag-pulsed NF-kappaB1(-/-) DC failed to prime for Th2 cytokine responses following injection into syngeneic WT hosts. Impaired Th2 priming by NF-kappaB1(-/-) DC was accompanied by a reduction in MAPK phosphorylation in Ag-pulsed DC. Taken together, these studies identify a novel requirement for DC-intrinsic expression of NF-kappaB1 in regulating the MAPK pathway and governing the competence of DC to instruct Th2 cell differentiation.     

Reovirus activates human dendritic cells to promote innate antitumor immunity

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.     

Inhibition of microRNA let-7i depresses maturation and functional state of dendritic cells in response to lipopolysaccharide stimulation via targeting suppressor of cytokine signaling 1

Dendritic cells (DCs) can initiate immune responses or confer immune tolerance depending on functional status. LPS-induced DC maturation is defined by enhanced surface expression of CD80 and CD86. MicroRNAs are critical for the regulation of DC function and immunity, and the microRNA let-7i was upregulated during LPS-induced DC maturation. Downregulation of let-7i significantly impeded DC maturation as evidenced by reduced CD80 and CD86 expression. DCs stimulated by LPS promoted T cell proliferation in coculture, whereas LPS-stimulated DCs with downregulated let-7i were not effective at stimulating T cell proliferation but promoted expansion of the regulatory T cell (Treg) population. There were two subpopulations of LPS-stimulated DCs with downregulated let-7i, CD86(-) and CD86(+), and it was the CD86(-) DCs that were more effective in inducing T cell hyporesponsiveness and enhancing Treg numbers, indicating that this DC population had tolerogenic properties. Furthermore, Tregs with upregulated IL-10 underscored the tolerogenic effect of CD86(-) DCs. Suppressor of cytokine signaling 1 (SOCS1), a crucial mediator of DC maturation, was confirmed as a let-7i target gene by luciferase construct assay. Suppression or overexpression of let-7i caused reciprocal alterations in SOCS1 protein expression, but had no significant effects on SOCS1 mRNA levels, indicating that let-7i regulated SOCS1 expression by translational suppression. The modulation of SOCS1 protein by let-7i was mainly restricted to CD86(-) DCs. Our study demonstrates that let-7i regulation of SOCS1 is critical for LPS-induced DC maturation and immune function. Dynamic regulation of let-7i may fine-tune immune responses by inducing Ag-specific immune tolerance.