New articles published July 1, 2009

R ecombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8⁺ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.

T he mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the α subunit of the epithelial sodium channel (αENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding αENaC in the presence or absence of aldosterone. Furthermore, expression of αENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal αENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance.

N eural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Focal adhesion kinase (FAK) mediates signal transduction by integrin and growth factor receptors, each of which is important for normal cardiovascular development. To investigate the role of FAK in NCC morphogenesis, we deleted it in murine NCCs using Wnt1cre, yielding craniofacial and cardiovascular malformations resembling those observed in individuals with DiGeorge syndrome. In these mice, we observed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and impaired cardiac outflow tract rotation, which resulted in a dextroposed aortic root. Moreover, within the conotruncal cushions, Fak-deficient NCCs formed a less organized mesenchyme, with reduced expression of perlecan and semaphorin 3C, and exhibited disorganized F-actin stress fibers within the aorticopulmonary septum. Additionally, absence of Fak resulted in reduced in vivo phosphorylation of Crkl and Erk1/2, components of a signaling pathway essential for NCC development. Consistent with this, both TGF-β and FGF induced FAK and Crkl phosphorylation in control but not Fak-deficient NCCs in vitro. Our results indicate that FAK plays an essential role in cardiac outflow tract development by promoting the activation of molecules such as Crkl and Erk1/2.

T ubular damage following ischemic renal injury is often reversible, and tubular epithelial cell (TEC) proliferation is a hallmark of tubular repair. Macrophages have been implicated in tissue repair, and CSF-1, the principal macrophage growth factor, is expressed by TECs. We therefore tested the hypothesis that CSF-1 is central to tubular repair using an acute renal injury and repair model, ischemia/reperfusion (I/R). Mice injected with CSF-1 following I/R exhibited hastened healing, as evidenced by decreased tubular pathology, reduced fibrosis, and improved renal function. Notably, CSF-1 treatment increased TEC proliferation and reduced TEC apoptosis. Moreover, administration of a CSF-1 receptor–specific (CSF-1R–specific) antibody after I/R increased tubular pathology and fibrosis, suppressed TEC proliferation, and heightened TEC apoptosis. To determine the contribution of macrophages to CSF-1–dependent renal repair, we assessed the effect of CSF-1 on I/R in mice in which CD11b⁺ cells were genetically ablated and determined that macrophages only partially accounted for CSF-1–dependent tubular repair. We found that TECs expressed the CSF-1R and that this receptor was upregulated and coexpressed with CSF-1 in TECs following renal injury in mice and humans. Furthermore, signaling via the CSF-1R stimulated proliferation and reduced apoptosis in human and mouse TECs. Taken together, these data suggest that CSF-1 mediates renal repair by both a macrophage-dependent mechanism and direct autocrine/paracrine action on TECs.