Single-cell analysis of human skin identifies CD14+ type 3 dendritic cells co-producing IL1B and IL23A in psoriasis

Inflammatory skin diseases including atopic dermatitis (AD) and psoriasis (PSO) are underpinned by dendritic cell (DC)–mediated T cell responses. Currently, the heterogeneous human cutaneous DC population is incompletely characterized, and its contribution to these diseases remains unclear. Here, we performed index-sorted single-cell flow cytometry and RNA sequencing of lesional and nonlesional AD and PSO skin to identify macrophages and all DC subsets, including the newly described mature LAMP3+BIRC3+ DCs enriched in immunoregulatory molecules (mregDC) and CD14+ DC3. By integrating our indexed data with published skin datasets, we generated a myeloid cell universe of DC and macrophage subsets in healthy and diseased skin. Importantly, we found that CD14+ DC3s increased in PSO lesional skin and co-produced IL1B and IL23A, which are pathological in PSO. Our study comprehensively describes the molecular characteristics of macrophages and DC subsets in AD and PSO at single-cell resolution, and identifies CD14+ DC3s as potential promoters of inflammation in PSO.

A rapid method for identifying ploidy level in Isatis indigotica Fortune and Isatis tinctoria Linnaeus

The genus Isatis is widely distributed throughout the world. In this work, thirty-two Isatis indigotica Fortune germplasms, collected from different regions and geographical locations in China, were analyzed the ploidy levels by flow cytometry. I. indigotica Fort. and Isatis tinctoria Linnaeus distinguished with root tip chromosome compression staining and cell flow cytometry. Microscopic observation showed that the chromosome numbers of I. indigotica Fort. and I. tinctoria L. were 2n = 14 and 2n = 28, respectively. In order to establish a flow cytometric nuclear experiment system suitable for I. indigotica Fort., the leaves of I. indigotica Fort. were used to prepare nuclear suspension with LB01, OTTO, Tris·MgCl2, patent of Luochang and Galbraith extraction buffer. It was found that the histogram generated by LB01, OTTO, Tris·MgCl2 and Patent of Luochang dissociation solution has poor peak shapes and large CV values, and Galbraith dissociation solution extraction buffer was suitable for extracting nuclei from most germplasms. Flow cytometry proved to be a simple, rapid, and highly accurate method for identifying ploidy levels Isatis species.

Assessment of Protocol Impact on Subjectivity Uncertainty When Analyzing Peripheral Blood Mononuclear Cell Flow Cytometry Data Files

Measured variability of product within Cell and Gene Therapy (CGT) manufacturing arises from numerous sources across pre-analytical to post-analytical phases of testing. Operators are a function of the manufacturing process and are an important source of variability as a result of personal differences impacted by numerous factors. This research uses measurement uncertainty in comparison to Coefficient of Variation to quantify variation of participants when they complete Flow Cytometry data analysis through a 5-step gating sequence. Two study stages captured participants applying gates using their own judgement, and then following a diagrammatical protocol, respectively. Measurement uncertainty was quantified for each participant (and analysis phase) by following Guide to the Expression of Uncertainty in Measurement protocols, combining their standard deviations in quadrature from each gating step in the respective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57%, increasing confidence in a more uniform reported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytometry gating process is from the very first gate, where isolating target cells from dead or dying cells is required. This work has demonstrated the potential for greater usage of measurement uncertainty within CGT manufacturing scenarios, due to the resolution it provides for root cause analysis and continuous improvement.

High-Dimensional Analysis of Single-Cell Flow Cytometry Data Predicts Relapse in Childhood Acute Lymphoblastic Leukaemia

Artificial intelligence methods may help in unveiling information that is hidden in high-dimensional oncological data. Flow cytometry studies of haematological malignancies provide quantitative data with the potential to be used for the construction of response biomarkers. Many computational methods from the bioinformatics toolbox can be applied to these data, but they have not been exploited in their full potential in leukaemias, specifically for the case of childhood B-cell Acute Lymphoblastic Leukaemia. In this paper, we analysed flow cytometry data that were obtained at diagnosis from 56 paediatric B-cell Acute Lymphoblastic Leukaemia patients from two local institutions. Our aim was to assess the prognostic potential of immunophenotypical marker expression intensity. We constructed classifiers that are based on the Fisher’s Ratio to quantify differences between patients with relapsing and non-relapsing disease. We also correlated this with genetic information. The main result that arises from the data was the association between subexpression of marker CD38 and the probability of relapse.

IBEX – A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an ‘open’ system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues.Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

Harnessing lipid signaling pathways to target specialized pro-angiogenic neutrophil subsets for regenerative immunotherapy

To gain insights into neutrophil heterogeneity dynamics in the context of sterile inflammation and wound healing, we performed a pseudotime analysis of single-cell flow cytometry data using the spanning-tree progression analysis of density-normalized events algorithm. This enables us to view neutrophil transitional subsets along a pseudotime trajectory and identify distinct VEGFR1, VEGFR2, and CXCR4 high-expressing pro-angiogenic neutrophils. While the proresolving lipid mediator aspirin-triggered resolvin D1 (AT-RvD1) has a known ability to limit neutrophil infiltration, our analysis uncovers a mode of action in which AT-RvD1 leads to inflammation resolution through the selective reprogramming toward a therapeutic neutrophil subset. This accumulation leads to enhanced vascular remodeling in the skinfold window chamber and a proregenerative shift in macrophage and dendritic cell phenotype, resulting in improved wound closure after skin transplantation. As the targeting of functional immune subsets becomes the key to regenerative immunotherapies, single-cell pseudotime analysis tools will be vital in this field.

Direct relationship between increased expression and mistrafficking of the Charcot–Marie–Tooth–associated protein PMP22

Charcot–Marie–Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ∼1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third WT PMP22 allele. In rodent models overexpressing the PMP22 (peripheral myelin protein 22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum quality control system, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of WT or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells and PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mistrafficking of PMP22, which is likely a contributor to disease pathology and progression.

Zymosan exerts radiation protection on AHH-1 and HIEC cells through TLR2/4-MyD88-G-CSF/GM-CSF/IL-12/IL-6 pathway

Background: This study was to confirm the radiation protective effect of different doses of zymosan on AHH-1 and HIEC cells irradiated at different times and different doses, and further to explore whether zymosan exerts a radiation protection mechanism by targeting TLR2/4.Methods: AHH-1 and HIEC cells were respectively administered to Zymosan at 0, 20, 40, 80 and 160 μg/ml. CCK-8 and cell flow cytometry were used to detect the cell activity and apoptosis at 24 h, 48 h, and 72 h after administration to determine the dose-limiting toxicities of zymosan. Twelve hours before irradiation, cells were treated with zymosan at 0, 5, 10, and 20 μg/ml, and then irradiated with 4Gy X-rays. The cell activity and apoptosis were measured by CCK-8 and cell flow cytometry at 24 h to determine the optimal dose of zymosan. LPS was used as a positive control to compare the protective effect of zymosan. The cells were treated with MyD88 inhibitors to explore the protective mechanism of zymosan.Results: The activity of AHH-1 and HIEC cells treated with different concentration of zymosan at different time was not affected and the apoptosis of cells was not promoted. The radiation protection effect of Zymosan pretreated cells on cells is dose-dependent. After zymosan pre-treated the cells, its radiation protection effect on the cells was dose-dependent. The higher zymosan’s concentration was, the stronger the activities of AHH-1 cells and HIEC cells were, and the lower the apoptosis rate was. The activity of cells pretreated with zymosan was higher than that pretreated with LPS at the same dose (20 μg/ml), and the cell apoptosis rate was lower than that pretreated with LPS. After zymosan pretreated AHH-1 and HIEC cells, TLR2/4-MyD88-G-CSF/GM-CSF/IL-12/IL-6 pathway was activated.Conclusion: Zymosan is nontoxic to cells and has better radiation protection effect than LPS. Its mechanism of action is related to the activation of the TLR2/4-MyD88/G-CSF/GM-CSF/IL-12/IL-6 pathway.