Jackson angYao Li

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.

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.

Platelets as autonomous drones for hemostatic and immune surveillance

Despite the lack of nuclei and regulated transcription, platelets actively participate in multiple physiological processes, including hemostasis and immunity. Li et al. discuss aspects of platelet design that optimize its functions and argue that platelets may be best conceived as automated, fully equipped surveillance vehicles.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions

Summary Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half‐life. Although it is established that neutrophils are derived from the granulocyte‐macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell‐cycle‐based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non‐proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBP&egr; transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor‐bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses. Graphical Abstract Figure. No Caption available. HighlightsProliferation activity identifies committed neutrophil precursor in mice and humansNeutrophil subsets possess distinct transcriptomic and functional signaturesDefect in neutrophil development leads to impaired neutrophil‐mediated responsesIncreased circulating immature neutrophils are associated with cancer progression &NA; The neutrophil differentiation pathway is poorly defined. Evrard et. al. demonstrate a workflow of characterizing bone marrow neutrophil subsets on the basis of their proliferative capacity and molecular signatures and thereby define the developmental trajectory and functional properties of neutrophils.

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.

Identification of a novel lymphoid population in the murine epidermis

T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαβ/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified 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

Imaging of Inflammatory Responses in the Mouse Ear Skin

The skin is one of the most physiologically important organs where the organism comes into contact with the external environment and is often a site where pathogen entry first occurs. Thus, a better understanding of the specialized cellular behavior of the immune system in the skin may be important for the improved treatment of diseases. Here, we describe in detail a procedure to image the dorsal mouse ear skin, using a customized ear stage and its associated coverslip holder, with an upright multiphoton microscope. As a demonstrative example, we describe the specific protocol for visualizing robust neutrophil trafficking in albino lysozyme-EGFP mice in response to zymosan particles. Instructive sections are provided for the mouse ear preparation, intradermal delivery of zymosan, design and use of the custom ear stage, as well as a solution for the uninterrupted live imaging of mice during prolonged sessions within a dark box. The mouse ear is easily accessible for imaging, and unlike most other organs, does not require any invasive surgery to be performed.

Neutrophils instruct homeostatic and pathological states in naive tissues

Immune protection relies on the capacity of neutrophils to infiltrate challenged tissues. Naive tissues, in contrast, are believed to remain free of these cells and protected from their toxic cargo. Here, we show that neutrophils are endowed with the capacity to infiltrate multiple tissues in the steady-state, a process that follows tissue-specific dynamics. By focusing in two particular tissues, the intestine and the lungs, we find that neutrophils infiltrating the intestine are engulfed by resident macrophages, resulting in repression of Il23 transcription, reduced G-CSF in plasma, and reinforced activity of distant bone marrow niches. In contrast, diurnal accumulation of neutrophils within the pulmonary vasculature influenced circadian transcription in the lungs. Neutrophil-influenced transcripts in this organ were associated with carcinogenesis and migration. Consistently, we found that neutrophils dictated the diurnal patterns of lung invasion by melanoma cells. Homeostatic infiltration of tissues unveils a facet of neutrophil biology that supports organ function, but can also instigate pathological states.