Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

iPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Skinny deeping: Uncovering immune cell behavior and function through imaging techniques.

As the largest organ of the body, the skin is a key barrier tissue with specialized structures where ongoing immune surveillance is critical for protecting the body from external insults. The innate immune system acts as first-responders in a coordinated manner to react to injury or infections, and recent developments in intravital imaging techniques have made it possible to delineate dynamic immune cell responses in a spatiotemporal manner. We review here key studies involved in understanding neutrophil, dendritic cell and macrophage behavior in skin and further discuss how this knowledge collectively highlights the importance of interactions and cellular functions in a systems biology manner. Furthermore, we will review emerging imaging technologies such as high-content proteomic screening, spatial transcriptomics and three-dimensional volumetric imaging and how these techniques can be integrated to provide a systems overview of the immune system that will further our current knowledge and lead to potential exciting discoveries in the upcoming decades.

Visualising cancer in 3D: 3-Dimensional Tissue Imaging for management of cutaneous basal cell carcinoma.

Surgical management of basal cell carcinoma (BCC) typically involves surgical excision with post-operative margin assessment using the bread-loafing technique; or gold-standard Mohs micrographic surgery (MMS), where margins are iteratively examined for residual cancer after tumour removal, with additional excisions performed upon detecting residual tumour at margins. There is limited sampling of resection margins with bread loafing, with detection of positive margins 44% of the time using 2 mm intervals. To resolve this, we have developed three-dimensional (3D) Tissue Imaging for: (1) complete examination of cancer margins and (2) detection of tumour proximity to nerves and blood vessels. 3D Tissue optical clearing with a light sheet imaging protocol was developed for margin assessment in two datasets assessed by two independent evaluators: (1) 48 samples from 29 patients with varied BCC subtypes, sizes and pigmentation levels; (2) 32 samples with matching Mohs' surgeon reading of tumour margins using two-dimensional haematoxylin & eosin-stained sections. The 3D Tissue Imaging protocol permits a complete examination of deeper and peripheral margins. Two independent evaluators achieved negative predictive values of 92.3% and 88.24% with 3D Tissue Imaging. Images obtained from 3D Tissue Imaging recapitulates histological features of BCC, such as nuclear crowding, palisading and retraction clefting and provides a 3D context for recognising normal skin adnexal structures. Concurrent immunofluorescence labelling of nerves and blood vessels allows visualisation of structures closer to tumour-positive regions, which may have a higher risk for neural and vascular infiltration. Together, this method provides more information in a 3D spatial context, enabling better cancer management by clinicians.

Silencing siRNA microneedle patches versus silicone sheets in reducing post-surgical scars: a randomized single-blinded intra-individually controlled clinical trial.

BACKGROUND: A common complication of wounds is exuberant growth of fibrotic scar tissue, which can lead to hypertrophic scars or keloids. There are currently no treatments with good evidence for preventing excessive scar tissue formation. In this study, we explored the use of microneedle patches containing siRNA inhibiting SPARC mRNA in reducing the volume of post-surgical scars. OBJECTIVE: We aim to compare the differences in the volume of post-surgical scars between daily application of siRNA-embedded dissolving microneedle patches and silicone sheets. The primary study outcome measure was the 3D volume of scar elevation.Our hypothesis was that scar formation in the half of the wound treated with siRNA microneedle patches will be lesser, as reflected by a smaller 3D volume, as compared to the half treated with silicone sheets. METHODS: This was an 8-week, single-blinded intra-individually controlled randomised trial in a tertiary dermatological centre. Patients with two-week-old post-operative wounds were recruited. Each half of the scar was randomly assigned to the microneedle patch or silicone sheet. Three-dimensional (3D) volumes were obtained from the scars via a high-resolution scanner at day 0, 30 and 60. RESULTS: At day 30, scars treated with the microneedle patches had a lower geometric mean volume of 0.79mm3 when compared to scars treated with silicone sheets, with a difference in mean percentage volume reduction of 10.70%.At day 60, scars treated with the microneedle patches had a statistically significant lower volume (8.88mm3) when compared to the side treated with silicone sheets (12.77mm3, p=0.005), with a difference in mean percentage reduction of 9.66%. Additionally, there was also a statistically significant difference between the percentage reduction in scar volume, compared to baseline, on the side treated with microneedle patches (mean=83.78%) compared to the side treated with silicone sheets (mean=74.11%). CONCLUSIONS: There was a significantly greater reduction in the volume of post-surgical scars on the side treated with microneedle patches compared to the side treated with silicone sheets. This demonstrates the use of transdermal gene silencing technology for scar inhibition and that siRNA microneedle patches can be an effective and safe modality in the reduction of scar tissue formation. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12622000558729, https://www.anzctr.org.au.

Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice.

Tissue-resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity-driven WAT remodeling remains controversial. Here, using a transgenic CD169-DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity-driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed.

Reprint of "Multi-modal image cytometry approach - From dynamic to whole organ imaging".

Optical imaging is a valuable tool to visualise biological processes in the context of the tissue. Each imaging modality provides the biologist with different types of information - cell dynamics and migration over time can be tracked with time-lapse imaging (e.g. intra-vital imaging); an overview of whole tissues can be acquired using optical clearing in conjunction with light sheet microscopy; finer details such as cellular morphology and fine nerve tortuosity can be imaged at higher resolution using the confocal microscope. Multi-modal imaging combined with image cytometry - a form of quantitative analysis of image datasets - provides an objective basis for comparing between sample groups. Here, we provide an overview of technical aspects to look out for in an image cytometry workflow, and discuss issues related to sample preparation, image post-processing and analysis for intra-vital and whole organ imaging.

The impact of ischemia-reperfusion injuries on skin resident murine dendritic cells.

Pressure ulcers are a chronic problem for patients or the elderly who require extended periods of bed rest. The formation of ulcers is due to repeated cycles of ischemia-reperfusion (IR), which initiates an inflammatory response. Advanced ulcers disrupt the skin barrier, resulting in further complications. To date, the immunological aspect of skin IR has been understudied, partly due to the complexity of the skin immune cells. Through a combination of mass cytometry, confocal imaging and intravital multiphoton imaging, this study establishes a workflow for multidimensionality single cell analysis of skin myeloid cell responses in the context of IR injury with high spatiotemporal resolution. The data generated has provided us with previously uncharacterized insights into the distinct cellular behavior of resident dendritic cells (DCs) and recruited neutrophils post IR. Of interest, we observed a drop in DDC numbers in the IR region, which was subsequently replenished 48h post IR. More importantly, in these cells, we observe an attenuated response to repeated injuries, which may have implications in the subsequent wound healing process.

Multi-modal image cytometry approach - From dynamic to whole organ imaging.

Optical imaging is a valuable tool to visualise biological processes in the context of the tissue. Each imaging modality provides the biologist with different types of information - cell dynamics and migration over time can be tracked with time-lapse imaging (e.g. intra-vital imaging); an overview of whole tissues can be acquired using optical clearing in conjunction with light sheet microscopy; finer details such as cellular morphology and fine nerve tortuosity can be imaged at higher resolution using the confocal microscope. Multi-modal imaging combined with image cytometry - a form of quantitative analysis of image datasets - provides an objective basis for comparing between sample groups. Here, we provide an overview of technical aspects to look out for in an image cytometry workflow, and discuss issues related to sample preparation, image post-processing and analysis for intra-vital and whole organ imaging.

The potential of RNA therapeutics in dermatology.

Ribonucleic acid (RNA) therapeutics hold great potential for the advancement of dermatological treatments due to, among other reasons, the possibility of treating previously undruggable targets, high specificity with minimal side effects, and ability to include multiple RNA targets in a single product. Although there have been research relating to RNA therapeutics for decades, there have not been many products translated for clinical use until recently. This may be because of challenges to the application of RNA therapeutics, including the dearth of effective modes of delivery to the target, and rapid degradation of RNA in the human body and environment. This article aims to provide insight on (1) the wide-ranging possibilities of RNA therapeutics in the field of dermatology as well as (2) how key challenges can be addressed, so as to encourage the development of novel dermatological treatments. We also share our experience on how RNA therapeutics have been applied in the management of hypertrophic and keloid scars.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

SNX3-dependent regulation of epidermal growth factor receptor (EGFR) trafficking and degradation by aspirin in epidermoid carcinoma (A-431) cells.

Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.

In vivo imaging of patients with chronic pruritus of unknown origin reveals partial sweat duct obstruction with partial itch resolution upon retinoid treatment.

Background: Chronic pruritus of unknown origin (CPUO) is poorly understood and lacks effective treatment options. Objectives: We aimed to elucidate abnormalities in the sweat apparatus of patients with CPUO, and to assess efficacy and safety of treatment with systemic retinoids. Methods: An initial case-control study included 20 affected patients and five healthy controls, for whom heat and sweating were induced, either through a standardized exercise protocol or ingestion of hot water. In vivo high-definition optical coherence tomography, whole-body starch-iodine testing, and skin biopsy for immunofluorescence staining were done to evaluate for sweat duct obstruction. A subsequent retrospective cohort analysis included 56 patients with CPUO, seen at an Itch subspecialty clinic of a single tertiary referral centre, who failed conventional treatments and were treated with isotretinoin and/or acitretin from May 2014 to November 2020. Treatment response to retinoids was defined as a sustained reduction in itch score of ≥2/10. Safety was assessed by proportion stopping treatment due to side effects. Results: In vivo imaging in 19 (95%) patients revealed features of partial keratinaceous sweat duct obstruction with statistically significant luminal dilatation compared to controls. Immunofluorescence studies of three patients' paired lesional/non-lesional biopsies revealed dermcidin accumulation within sweat glands coupled with dermcidin leakage in itchy skin. Fifty-six patients (mean [SD] age 55.2 [17.5] years, 69.6% male) were treated with systemic retinoids. Mean (SD) duration of itch was 116.3 (140.4) months and mean (SD) itch score was 8.2 (1.8). Forty-one (73.2%) initially received isotretinoin, and 15 (26.8%) acitretin. At three months, mean itch score reduced by 2.38 (95% CI -3.2 to -1.6, p < 0.0001). Thirty-eight (67.9%) had a sustained response. Eight (14.81%) achieved an itch score of 0 or 1, with four stopping treatment for a mean (SD) of 318.5 (291.2) days without relapse. Eight (14.3%) stopped or switched retinoid due to adverse effects, with similar incidences between both retinoids, the commonest being dryness. Conclusion: Based on novel findings from physiological imaging studies identifying partial keratinaceous sweat duct obstruction in CPUO, we instituted systemic retinoid treatment to address the underlying pathology. In patients who failed conventional therapies, the treatment appears effective and safe.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

Intravital Imaging of Bone Marrow Microenvironment in the Mouse Calvaria and Tibia.

The complex bone marrow microenvironment or niche is an important anatomical structure responsible for hematopoiesis and providing support to the immune cells function. Being the source of immune and blood cells, the interaction of these hematopoietic stem and progenitor cells with the cellular niches regulates their ability for self-renewal, proliferation, and differentiation. Dynamic imaging not only provides spatiotemporal information of cell motility but also the morphological changes due to cell-cell interactions in the bone marrow, providing insights into the ongoing physiological activities within the tissue. Here, we describe customized stages with compatible equipment best suited for the upright two-photon microscope, accompanied by detailed methods for both calvarial and tibial intravital imaging. We demonstrate a general protocol for calvarial imaging using a minimally invasive surgical approach, and introduce a bone shaving-based tibial imaging as a complementary method. To demonstrate the applicability of our method we used Lyz2-EGFP transgenic mice to track bone marrow neutrophil activities as an example.

Research Techniques Made Simple: Optical Clearing and Three-Dimensional Volumetric Imaging of Skin Biopsies.

Skin histology is traditionally carried out using two-dimensional tissue sections, which allows for rapid staining, but these sections cannot accurately represent three-dimensional structures in skin such as nerves, vasculature, hair follicles, and sebaceous glands. Although it may be ideal to image skin in a three-dimensional manner, it is technically challenging to image deep into tissue because of light scattering from collagen fibrils in the dermis and refractive index mismatch owing to the presence of differing biological materials such as cytoplasm, and lipids in the skin. Different optical clearing methods have been developed recently, making it possible to render tissues transparent using different approaches. Here, we discuss the steps involved in tissue preparation for three-dimensional volumetric imaging and provide a brief overview of the different optical clearing methods as well as different imaging modalities for three-dimensional imaging.