In vivo multi-photon molecular imaging reveals inflammatory
immune cell cross-talks in adult common diseases
Satoshi Nishimura(1) Department of Cardiovascular Medicine, (2) Translational Systems Biology and Medicine
Initiative, The University of Tokyo, Tokyo, Japansnishi-tky@umin.ac.jp
Keywords: multi-photon, in vivo imaging, inflammation, metabolic syndrome, thrombus
Metabolic syndrome is a major risk factor of cardiovascular events, and adipose tissue
remodeling based on chronic inflammation play a central role in obesity. To assess dynamic
inflammatory cellular interplay between multiple cell-types in adult common diseases,
including metabolic syndrome, obesity, and cardiovascular diseases, “in vivo molecularimaging technique” based on single- and multi-photon microscopy was developed. We first
applied this technique to metabolic disease field. Our imaging visualized precise adipose
tissue structures, and revealed close spatial and temporal interrelationships between
angiogenesis and adipogenesis in obese adipose tissue (Figure A) [1]. In addition, increased
leukocyte- platelet- endothelial cell interactions in the microcirculation of obese adipose wereobserved, a hallmark of inflammation [2]. Inflammatory cell kinetics, such as rolling,adhesion and extravasation was observed, clearly indicating the inflammatory status of obese
adipose tissue. We also found that large numbers of CD8+ effector T cells infiltrated into
obese adipose [3]. Infiltration by CD8+ T cells is essential for the initiation and developmentof adipose inflammation, showing that the CD8+ T cells are the key regulator of obese
adipose tissue inflammation. These CD8+ T cells contribute to macrophage recruitment andadipose tissue inflammation associated with obesity. In addition, we recently found that Bcells are major component of stromal vascular fraction of adipose tissue, and we clarified
their function in vivo. These B cells secrete high amount of IL-10 in unstimulated conditions,and have new phenotype with anti-inflammatory properties against diet-induced-obesity
inflammation. These B cells survive and secrete IL-10 under the stimulation of fatty acid,through TLR4/Myd88 pathways. Genetic and immunological B cell depletion ameliorated theadipose tissue inflammation and insulin resistance in obesity, indicating that these B cells canbe the new therapeutic target against diabetic and obese conditions.
Our imaging also visualized multiple cell types specifically in real time and in vivo settings(Figure B), and single platelet kinetics can be visualized by this technique. We applied thistechnique to another field, cardiovascular diseases, to elucidate the multi-cellular process ofdeveloping thrombus in vivo. As for the thrombus formation, inflammations are recently
considered to play a central role in pathogenesis of cardiovascular diseases, and contribute tothe thrombotic response via cellular and humoral modulations. However, the directcontribution to thrombosis formation in vivo was not clear due to the lack of a real-timevisualization technique of developing thrombus. Recently, we developed new animal models
of developing thrombus by modifying in vivo imaging technique, and we visualized single
platelet kinetics in developing thrombus (Figure C) [4].
In our models, discoid platelet aggregation on intact endothelium was triggered by ROS
production within vessels, where such ROS signal was maintained with inflammatory

cytokines including TNF-alpha or IL-1. Pharmacological examination and kinetic analysis ofthrombi revealed that initiation was regulated by the complex of GPIb-alpha with exposed
von Willebrand factor through TNF-alpha/TNF-R1 axis in ECs. Moreover, integrin activation
through and the actin cytoskeleton organization through Rac1 signaling is required forthrombus stability in late phase.
Using this technique, we found Lnk (an adapter protein) contribute to stabilization ofdeveloping thrombus in vivo via out-side-in integrin signaling [4]. In addition, we havesucceeded in efficient production of human iPS-derived platelets by regulating the c-Myc
reactivation process [5]. And to elucidate the clinical usefulness of these artificial platelets,
we visualized that produced platelets have ability of thrombus formation in vivo [5].
Our imaging technique can be applied to bone marrows to visualize immune and stem cellfunctions during bone marrow reconstruction processes (Figure D). We have succeeded invisualizing stem cells, T cells, myeloid cells, megakaryocytes, platelets etc. in bone marrow
with high time and spatial resolution using multi-photon in vivo molecular imaging technique.
We found T cell proliferations have pivotal roles during re-constructive process of bonemarrows after transplantations, closely related to haemtopoietic stem cell differentiation inniches.
Our results clearly demonstrated the power of our imaging technique to analyze complex
cellular interplays in inflammatory diseases: metabolic syndrome, thrombus formation, andbone marrow reconstructive processes. Our imaging elucidate the parenchymal and stromal
cell cross talks in inflammatory diseased conditions. This imaging might contribute todeveloping new therapeutic interventions against them.
References
1.
Nishimura S, et al. Diabetes. (2007),56:1517.
2.
Nishimura S, et al. J Clin Invest. (2008), 118: 710
3.
Nishimura S, et al. Nature Medicine, (2009), 5;8:914.
4.
Nishimura S, et al. J Clin Invest, (2010), 120(1): 179-190.
5.
Takayama N & Nishimura S et al, J Exp Med. (2010), 207(13):2817-2830

Figure. In vivo imaging visualizing inflammatory cell dynamics in diseased conditions. (a)Adipose tissue remodeling in obesity including adipogenesis and angiogenesis (b) Multicellular kinetics visualized by novel in vivo imaging technique (c) Thrombus formation andsingle platelet kinetics revealed by in vivo imaging (d) T cell proliferations in bone marrows
after transplantation