Chi Ching Goh

Ultrabright Organic Dots with Aggregation‐Induced Emission Characteristics for Real‐Time Two‐Photon Intravital Vasculature Imaging

Ultrabright organic dots with aggregation-induced emission characteristics (AIE dots) are prepared and shown to exhibit a high quantum yield, a, large two-photon absorption cross-section, and low in vivo toxicity. Real-time two-photon intravital blood vascular imaging in various tissues substantiates that the AIE dots are effective probes for in vivo vasculature imaging in a deep and high-contrast manner.

Intravital multiphoton imaging of immune responses in the mouse ear skin

Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

The CXCR4 antagonist plerixafor augments frequency of circulating neutrophils via release from the lung and prevents neutrophil homing to the bone marrow.

Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization.

Fluorescent organic dots are emerging as promising bioimaging reagents because of their high brightness, good photostability, excellent biocompatibility, and facile surface functionalization. Organic dots with large two-photon absorption (TPA) cross sections are highly desired for two-photon fluorescence microscopy. In this work, we report two biocompatible and photostable organic dots fabricated by encapsulating tetraphenylethene derivatives within DSPE-PEG matrix. The two organic dots show absorption maxima at 425 and 483 nm and emit green and red fluorescence at 560 and 645 nm, with high fluorescence quantum yields of 64% and 22%, respectively. Both organic dots exhibit excellent TPA property in the range of 800-960 nm, affording upon excitation at 820 nm remarkably large TPA cross sections of 1.2×10(6) and 2.5×10(6) GM on the basis of dot concentration. The bare fluorophores and their organic dots are biocompatible and have been used to stain living cells for one- and two-photon fluorescence bioimagings. The cRGD-modified organic dots can selectively target integrin αvβ3 overexpressing breast cancer cells for targeted imaging. The organic dots are also applied for real-time two-photon fluorescence in vivo visualization of the blood vasculature of mouse ear, providing the spatiotemporal information about the whole blood vascular network. These results demonstrate that the present fluorescent organic dots are promising candidates for living cell and tissue imaging.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function

Summary Tissue macrophages arise during embryogenesis from yolk‐sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS‐derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS‐hematopoiesis in vitro. We asked whether iPSC‐derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ‐specific cues. Co‐culturing human or murine iMacs with iPSC‐derived neurons promoted differentiation into microglia‐like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro‐inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue‐resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies. Graphical Abstract Figure. No Caption available. HighlightsHuman and mouse iPSCs can recapitulate YS hematopoiesisHuman and mouse iPSCs can differentiate into YS macrophage‐like cells (iMacs)iMacs can further differentiate to tissue macrophage‐like cells with organ‐specific cuesiMacs derived from an FMF patient’s iPSCs exhibit disease‐specific characteristics &NA; Yolk‐sac (YS) embryonic macrophages contribute to tissue‐resident macrophages but remain difficult to study because of their stage‐dependent limited availability. Takata et al. demonstrate that iPSCs can generate YS macrophage‐like cells (iMacs) that differentiate into functional tissue‐resident macrophage‐like cells upon receiving organ‐specific cues, thus providing a platform for modeling tissue‐resident macrophages.

Nanocrystallization: A Unique Approach to Yield Bright Organic Nanocrystals for Biological Applications

A new bottom-up nanocrystallization method is developed to fabricate highly fluorescent organic nanocrystals in aqueous media using an aggregation-induced emission fluorogen (AIEgen) as an example. The nanocrystallization strategy leads to the fabrication of uniform nanocrystals of 110 ± 10 nm size in aqueous media, which shows over 400% increase in brightness as compared to the amorphous nanoaggregates.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

CD41 is a reliable identification and activation marker for murine basophils in the steady state and during helminth and malarial infections

Basophils, a rare leukocyte population in peripheral circulation, are conventionally identified as CD45intCD49b+FcεRI+ cells. Here, we show that basophils from blood and several organs of naïve wild‐type mice express CD41, the α subunit of αIIbβ3 integrin. CD41 expression on basophils is upregulated after in vivo IL‐3 treatment and during infection with Nippostrongylus brasiliensis (Nb). Moreover, CD41 can be used as a reliable marker for basophils, circumventing technical difficulties associated with FcεRI for basophil identification in a Nb infection model. In vitro anti‐IgE cross‐linking and IL‐3 basophil stimulation showed that CD41 upregulation positively correlates with augmented surface expression of CD200R and increased production of IL‐4/IL‐13, indicating that CD41 is a basophil activation marker. Furthermore, we found that infection with Plasmodium yoelii 17X (Py17x) induced a profound basophilia and using Mcpt8DTR reporter mice as a basophil‐specific depletion model, we verified that CD41 can be used as a marker to track basophils in the steady state and during infection. During malarial infection, CD41 expression on basophils is negatively regulated by IFN‐γ and positively correlates with increased basophil IL‐4 production. In conclusion, we provide evidence that CD41 can be used as both an identification and activation marker for basophils during homeostasis and immune challenge.

Dual Modal Ultra-bright Nanodots with Aggregation-induced Emission and Gadolinium-chelation for Vascular Integrity and Leakage Detection

The study of blood brain barrier (BBB) functions is important for neurological disorder research. However, the lack of suitable tools and methods has hampered the progress of this field. Herein, we present a hybrid nanodot strategy, termed AIE-Gd dots, comprising of a fluorogen with aggregation-induced emission (AIE) characteristics as the core to provide bright and stable fluorescence for optical imaging, and gadolinium (Gd) for accurate quantification of vascular leakage via inductively-coupled plasma mass spectrometry (ICP-MS). In this report, we demonstrate that AIE-Gd dots enable direct visualization of brain vascular networks under resting condition, and that they form localized punctate aggregates and accumulate in the brain tissue during experimental cerebral malaria, indicative of hemorrhage and BBB malfunction. With its superior detection sensitivity and multimodality, we hereby propose that AIE-Gd dots can serve as a better alternative to Evans blue for visualization and quantification of changes in brain barrier functions.

Real-Time Imaging of Dendritic Cell Responses to Sterile Tissue Injury

TO THE EDITOR Dermal dendritic cells (DDCs) are immunological sentinels that continuously scan the dermal microenvironment for the presence of danger signals (Ng et al., 2008). Upon encounter of such stress signals––for example, following pathogenic insults–– DDCs are thought to exit the skin via lymphatics and subsequently travel to skin-draining lymph nodes where they activate the adaptive immune response. Little is known, however, how DDCs respond to sterile tissue injury. Despite literature describing the use of lasers for assisting vaccine uptake and the activation of DDCs (Chen et al., 2010, 2012, 2014; Wang et al., 2014), a detailed, real-time characterization of how DDCs respond to sterile tissue injury has not been performed. In recent years, intravital multiphoton microscopy (IV-MPM) has been employed to study dendritic cell function within their native microenvironment. These studies have revealed the dynamic behavior of dendritic cells under homeostasis and how they orchestrate immune responses during inflammation/infection (Garside and Brewer, 2008; Lammermann and Germain, 2014; Weninger et al., 2014). In this study, we employed our previously established mouse ear skin intravitalimaging model (Li et al., 2012) to monitor in real time how DDCs respond to sterile injury imposed by a small laser-induced lesion in the dermis. These experiments should provide insights into the earliest steps of DDC responses during non-microbial triggered inflammation commonly associated with conditions such as ischaemia-reperfusion injury, metabolic, and autoimmune diseases (Shen et al., 2013). DDC behavior was visualized in CD11c-EYFP mice, in which dendritic cells express EYFP (Lindquist et al., 2004; studies were performed with the approval of the IACUC, A*STAR.) A sterile injury was induced by multiphoton laser ablation using a Tunable Coherent Chameleon Ultra II One Box Titanium:sapphire laser source (Coherent, Santa Clara, CA; 800 nm at B25 mW; pulse length of 140 femtoseconds) for 5–8 s, with the burn area measuring 75 75mm, focused 20mm below the dermal–epidermal junction. Time-lapse images were then obtained by IV-MPM post injury to observe how DDCs respond to laser-induced injury in real time (Figure 1a–c). In the absence of laser injury, and as reported previously (Ng et al., 2008), DDCs displayed a relatively slow motility (B1.49mm min ) with a constant probing behavior, taking random and seldom repeated paths (Figure 1a–d). Immediately after injury, an increase in DDC motility was observed (Figure 1d), which gradually translated into a highly directed migratory behavior toward the site of laser injury B50 min post injury (Figure 1b), as indicated by an increase in their meandering index (Figure 1e). Such migratory behavior (Figure 1f) was sustained until DDCs successfully reached the injury site, followed by a cessation of migration. It is well established that dendritic cell migration is dependent on pertussis toxin (PTX)-sensitive Gai proteincoupled receptor signals (Itano et al., 2003; Ng et al., 2008). In order to determine whether similar signals are involved in the migration of DDCs toward the sterile injury site, we intradermally injected PTX (2ml of 50 ngml ; List Biologicals, Campbell, CA) prior to laser ablation. Similar to previous reports, we found that DDCs lost their mobility within minutes following PTX treatment (data not shown; Ng et al., 2008). Consequently, DDC migration toward the injury site was abolished (Figure 1g; Supplementary Movie 2 online), whereas in control phosphate-buffered saline-treated ears (Figure 1h; Supplementary Movie 3 online), DDC migratory behavior was comparable with untreated ears (Figure 1f). Together, these results suggest that DDCs migrate toward a chemoattractant source originating from the sterile injury site in a PTXsensitive manner. Conceivably, these migratory cues may arise from factors synthesized de novo from resident or recruited inflammatory cells at the site of injury. It is well established that neutrophils are among the first cells to be recruited to sites of injury (Amulic et al., 2012). We and others have previously shown that initial rare neutrophils accumulating at the injury site were capable of triggering waves of additional neutrophils (Ng et al., 2011; Lammermann et al., 2013). Thus, we next examined the migratory behavior of the neutrophils as compared with DDCs. We utilized LysM-eGFP-CD11c-EYFP mice, in which dendritic cells and neutrophils are YFPþ and GFPþ , respectively (Faust et al., 2000). Of note, dermal monocyte–derived dendritic cells and macrophages have been shown to express LysM at steady state (Tamoutounour et al., 2013). However, in the context of our current imaging setting, only high levels of GFP expression in neutrophils could be visualized in the ear skin of the LysM-eGFP mice used in our studies. Our data showed that in response to laser injury, GFPþ neutrophils started to roll along the Accepted article preview online 28 November 2014; published online 8 January 2015 Abbreviations: DDC, dermal dendritic cell; IV-MPM, intravital multiphoton microscopy; PTX, pertussis toxin CC Goh et al. Dendritic Cell Responses to Sterile Inflammation