Cholesterol Modification of p40-Specific Small Interfering RNA Enables Therapeutic Targeting of Dendritic Cells.

Small interfering RNA (siRNA)-based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10-producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.

Sulforaphane protects from T cell-mediated autoimmune disease by inhibition of IL-23 and IL-12 in dendritic cells.

Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with anti-inflammatory properties. The published reports are best conceivable with an inhibition of T cell function, but the mode of action remains unknown. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis. Disease amelioration was associated with reduced IL-17 and IFN-γ expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DCs) inhibited TLR4-induced IL-12 and IL-23 production, and severely suppressed Th1 and Th17 development of T cells primed by SFN-treated DCs. SFN regulated the activity of the TLR4-induced transcription factor NF-κB, without affecting the degradation of its inhibitor IκB-α. Instead, SFN treatment of DCs resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with and thereby inhibits NF-κB p65. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 activity at the IL23a and IL12b promoters. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS. Thus, our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting NF-κB p65-regulated IL-23 and IL-12 expression.

Genesis of rods in the zebrafish retina occurs in a microenvironment provided by polysialic acid-expressing Müller glia.

Polysialic acid (polySia) is a posttranslational modification of the neural cell adhesion molecule NCAM, which in the vertebrate brain is dynamically regulated during development and crucially involved in developmental and adult neurogenesis. In the fish retina, new neurons are persistently generated, but the possible contribution of polySia has not yet been addressed. Here we used immunohistochemistry with NCAM- and polySia-specific antibodies to study spatiotemporal expression patterns of NCAM and polySia in the developing and mature zebrafish retina. As early as 2.3 days postfertilization (dpf), NCAM but not polySia was detected on cell somata and fibers of the developing retina. At 4.3 dpf polySia immunoreactivity first appeared in the ventral retina and was localized to the nascent outer nuclear layer (ONL). In mature zebrafish, polySia immunoreactivity in the ONL extended to the entire retina. Colocalization with rhodopsin-EGFP in transgenic zebrafish or the Müller glia-specific protein cellular retinaldehyde-binding protein (CRALBP) revealed that polySia immunoreactivity was confined to the compartment of radial Müller glia processes crossing the ONL and to a small band of processes positioned proximal to the horizontal cell layer of the mature retina. As shown by 5-bromo-2-deoxyuridine (BrdU) labeling, both newly generated rod precursors within the mature ONL and precursors of the marginal zone were polySia-negative. Thus, polySia-negative rod precursors of the mature zebrafish retina face a polySia-NCAM-positive microenvironment presented by radial Müller glia. In view of the prominent role of polySia in other neurogenic systems, this pattern indicates that polySia provides environmental cues that are relevant for the generation of new rods.

cGMP-dependent cone photoreceptor degeneration in the cpfl1 mouse retina.

Inherited retinal degeneration affecting both rod and cone photoreceptors constitutes one of the leading causes of blindness in the developed world. Such degeneration is at present untreatable, and the underlying neurodegenerative mechanisms are unknown, even though certain genetic causes have been established. The rd1 mouse is one of the best characterized animal models for rod photoreceptor degeneration, whereas the cpfl1 mouse is a recently discovered model for cone cell death. Because both animal models are affected by functionally similar mutations in the rod and cone phosphodiesterase 6 genes, respectively, we asked whether the mechanisms of photoreceptor degeneration in these two mouse lines share common pathways. In the present study, we followed the temporal progression of photoreceptor degeneration in the cpfl1 retina, correlated it with specific metabolic markers, and compared it with the wild-type and the rd1 situation. Similar to corresponding rd1 observations, cpfl1 cone photoreceptor cell death was associated with an accumulation of cyclic guanosine monophosphate (cGMP), activity of calpains, and phosphorylation of vasodilator-stimulated protein (VASP). Cone degeneration progressed rapidly, with a peak in cell death around postnatal day 24. Furthermore, cpfl1 cone photoreceptor migration during early postnatal development was delayed significantly compared with the corresponding wild-type retina. The finding that rod and cone photoreceptor degeneration was associated with the same metabolic markers suggests that in both cell types similar degenerative mechanisms are active. This raises the possibility that equivalent neuroprotective strategies may be used to prevent both rod and cone photoreceptor degeneration.