Interaction of Dendritic Cells with Engineered Ligand Nanostructures
Yang
1
Liu ,
Kang-Hsin
1
Wang ,
Ming
1
Zhang ,
Jie-Ren
1
Li ,
Huan-Yuan
2,
3
Chen
, Shailise S.
1
Ross
, Fu-Tong
2,
3
Liu
and Gang-yu
1
Liu *
1Department
of Chemistry, University of California, Davis, CA 95616, USA
2Department of Dermatology, School of Medicine, Sacramento, University of California, Davis, California, 95817, USA
3Institute of Biomedical Sciences, Academia Sinica, Taiwan, ROC
Abstract
Dendritic cells (DCs) are unique antigen-presenting cells that elicit specific T-cell
responses, which in principle, can be used for immune-based effective antitumor
therapy. The signal transduction of DCs’ activation is dictated by the local
arrangement of the ligand-receptor complex at molecular level. This poster
presents our preliminary investigation on the impact of engineered ligands
nanostructures on the activation status of DCs. Lipopolysaccharide (LPS) is widely
used for in vitro stimulation of DCs, thus providing a good ligand for
nanoengineering. A series of arrays of LPS nanodots with designed size and
geometry are successfully fabricated via particle lithography and used for studying
DCs’ activation. The heights of the LPS nanodots are 9.1 ± 0.5 nm and the
diameters are 240 ± 10 nm, with the periodicities set at 200, 300, 500, 700 and
1000 nm. Upon exposure to bone marrow-derived dendritic cells (BMDCs) from
C57BL/6 mice, under culture conditions, cellular morphologies of BMDCs were
characterized by scanning electron microscopy (SEM) and atomic force
microscopy (AFM). Characteristic morphologies corresponding to immature and
mature BMDCs were observed. Under given times, the population of mature
BMDCs depends on the local arrangement of ligand nanostructures. In addition,
both a highly-dendritized and a hyper-branched morphology were found upon 30
min contact with LPS nanostructures with a periodicity of 500 nm. To the best of
our knowledge, the morphologies are new, and the level of dendritization and
branching is the highest yet found. Work is in progress toward understanding the
biological status and immune functions of the two new morphology types of
BMDCs. These preliminary results indicate that varying the size and geometry of
these ligand nanostructures provides a potent means to regulate cellular
morphologies and functions of BMDCs. We hope this approach provides a new
platform for design and fabrication of engineered extracellular matrices (ECM) for
effective regulation of signal transduction of DCs and cells in general.
Fabrication of LPS nanostructures
Two new morphologies of DC were observed
when activated with LPS nanostructures
Figure 3. Schematic diagram illustrates fabrication of LPS nanostructures via particle lithography.
Characterization of LPS Nanostructures
Figure 6. The SEM images reveal the cellular morphology of (A) higly-dendritized BMDCs and (B) hyper-branched
BMDCs after cultured on array of LPS nanodots with a periodicity of 500 nm for 30 min. To the best of our
knowledge, the morphologies are new, and the level of dendritization and branching is the highest yet found.
3D Information of two New DC
Morphologies
Local Assembling of Ligand-Receptor Complex
Dictate DCs Activation
Figure 7. The AFM images reveal the fine structures of (A) highly-dendritized BMDCs and (B) hyper-branched BMDCs.
The zoom-in view (C) revealed that the protrusions were stretching out of the cell body; (D) showed the hyper-branched
structure.
Conclusion
•
Figure 1. (A) Molecular structures of lipopolysaccharide (LPS) ligand used as ligand in this investigation. (B) LPS binds
to a protein complex composed of Toll-like receptor 4 (TLR-4) and myeloid factor 2 (MD-2), then triggers down-stream
cascades of activation and maturation of DCs. The molecular level arrangement of LPS-TLR-4-MD-2 complex dictates
subsequent signaling processes. (C) Using nanoengineering approach to dictate arrangement of signaling complex
on molecular level.
Anticipated Result
Figure 4. Sequence of chemical steps for selective immobilization of LPS on nanopatterns of organosilanes produced on silicon substrate using LPS nanostructures
with a periodicity of 500 nm as an example. (A) Schematic diagram of pore-shaped OEG-SAM nanopatterns, (B) topography of OEG-silane produced particle
lithography with 500 nm silica sphere; (C) corresponding cursory files for (B); (D) Schematic diagram of dot-shaped OTS/OEG nanopatterns, (E) topography of a
surface nanopatterned with OEG and OTS, (F) corresponding cursory files for (E); (G) Schematic diagram of dot-shaped OTS/OEG nanopatterns, (H) topography
of arrays of LPS nanodots; (I) corresponding cursory files for (H).
•
•
We Successfully Activate DCs with Soluble LPS
•
•
A series of arrays of LPS nanodots with different periodicities are
successfully fabricated via particle lithorgraphy.
In addition to the morphologies reported by previous study, two new types of
morphologies of DCs are observed upon interacting with LPS nanostructures.
To the best of our knowledge, such highly dendritic phenotype and
activation has not been reported previously, yet occurred easily within a
short time of 30 min, in contrast to 24 h activation using soluble LPS.
The data support an important aspect of our hypothesis: the controlled
and programmed spatial arrangements of ligand-receptor complexes
exhibit superior performance in forming “super” activated DCs to other
methods such as soluble LPS.
This strategy provides a new platform for design and fabrication of
engineered extracellular matrices (ECM) for effective regulation of signal
transduction of DCs and cells in general.
Acknowledgement
Figure 2. A schematic showing strategy for this research. Engineered LPS nanostructures with designed geometry have
been produced and used to activate immature DCs. The preliminary results indicate that LPS nanostructures exhibit
potent regulation on DCs’ activation.
Figure 5. Soluble LPS induces maturation of BMDC. SEM images reveal cellular morphologies of (A) immature BMDCs and (C) soluble LPS-induced mature
BMDCs. Compared with previous SEM images of immature (B) and mature (D) BMDCs, BMDCs became mature after cultured in LPS solution for 24 h.
We thank Ying Xin Liu, Drs. Arpad Kasrsai, ad Jianli Zhao at UC Davis for many helpful discussions.