Density of conjugated antibody determines the extent of Fc receptor dependent capture of nanoparticles by liver sinusoidal endothelial cells

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.Drug Delivery Technolog

Transforming growth factor β induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis

BACKGROUND AND AIMS: The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25− T cells by treatment with transforming growth factor β (TGF‐β). Here, we analysed the in vivo function of TGF‐β induced regulatory T (Ti‐Treg) cells in experimental colitis. METHODS: Ti‐Treg cells were generated in cell culture in the presence or absence of TGF‐β and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. RESULTS: Ti‐Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti‐Treg cells showed that exogenous interleukin 2 (IL‐2) was crucial for survival and expansion of these cells. CONCLUSION: Our data suggest that regulatory Ti‐Treg cells expand by TGF‐β and exogenous IL‐2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti‐Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation

EBV-induced gene 3 transcription is induced by TLR signaling in primary dendritic cells via NF-kappa B activation

The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8-, CD8+, and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footpriniing and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-κB and Ets binding sites at -90 and -73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF-κB p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBD-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF-κB p50-deficient mice. In summary, these data suggest that EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF-κB. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell development