Implementing High Dimensional Reduction Analysis on Histocytometric Data.

In a previous protocol article, we demonstrated construction of a histocytometry pipeline that is capable of both segmenting highly aggregated cell populations and retaining the original intensity data range of the input microscopy images. In the protocol presented here, using the output from the aforementioned article, we demonstrate how to phenotype the data using the high dimensional reduction analysis technique optimized t-distributed stochastic neighbor embedding (opt-t-SNE) and compare it to traditional manual gating. Additionally, we present a protocol illustrating the advantage of the inclusion of cell junction/membrane markers for accurately segmenting highly aggregated cell populations in ilastik. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping lymph node populations using manual gating Basic Protocol 2: Phenotyping lymph node populations using t-SNE dimensional reduction Support Protocol: ilastik segmentation using a pan marker.

Creation of High-Dimensional Reduction Analysis-Compatible Histocytometry Files from Images of Densely-Packed Cells and/or Variable Stain Intensity.

The power of high-dimensional reduction techniques using multiparameter images has been demonstrated across a variety of different publications. Recently, we published an end-to-end low-cost GUI-based protocol for performing histocytometric spatial analysis on images derived from the most common microscope image formats. However, this protocol is limited by the normalized marker intensity outputs and the difficulty in processing images of highly aggregated and/or exceptionally heterogenous cell populations. Here we present the basic protocols required to construct an advanced histocytometric data file using only freeware. This data file is compatible with images containing cell nuclei clusters that are difficult to segment, and results in histocytometry files retaining the original marker intensity values of the microscopic images they were derived from. This is especially useful in cells that are phenotyped based on relative marker expression levels. Histocytometry data files produced by these protocols are compatible with high-dimensional reduction analysis using marker intensity data, such as tSNEs. This methodology is showcased using stitched microscopic images of murine lymph nodes, complex organs with highly aggregated heterogenous cell populations, that are typically difficult to segment. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Image preprocessing and generation of nuclei marker probability maps Basic Protocol 2: Cell segmentation using ilastik-derived probability maps Basic Protocol 3: Generation of histocytometric .fcs files.

How to Build an Image-Processing Pipeline for Automating Multiparameter Histocytometry Analysis.

Until relatively recently, analysis of imaging data has been primarily quantitative and limited to 3-4 markers. The advancement of various technologies overcoming this marker limitation provided the capability of analyzing multiparameter imaging data down to the single cell level, termed histocytometry. Currently, most published end-to-end histocytometric analysis of imaging data is performed using expensive commercial programs or freely available analysis packages that require significant knowledge of programming languages for execution. Here we present a protocol that performs cell segmentation, phenotyping and spatial analysis, using software with easy-to-use GUIs (graphical user interfaces). These protocols allow the user to derive spatial and phenotypical data for the analysis of multiparameter microscopic images from most imaging platforms in a low-cost manner. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cell Segmentation and generation of histocytometric .csv file Basic Protocol 2: Phenotyping of cell populations Basic Protocol 3: Spatial relationship analyses of phenotyped populations Support Protocol 1: Nuclei Segmentation Accuracy Test Support Protocol 2: Correcting y-axis Inversion of Histocytometry Data Relative to Original Image File.

Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

High-Dimensional Image Analysis using Histocytometry.

Histocytometry is a technique for processing multiparameter microscopy images using computational approaches to identify and quantify cellular phenotypes. It allows for spatial analyses of cellular phenotypes in relation to each other and within defined spatial regions. The benefit of this technique over manual annotation and characterization of cells is a high degree of automation/throughput, significantly decreased user bias, and increased reproducibility. Recently, an increase in freely available software amenable to or deliberately designed for histocytometry has resulted in these complex analyses being available to a broader base of users who have amassed multi-component microscopic imaging data. This article provides an overview of a histocytometry pipeline, focusing on the strategic planning and software requirements to allow readers to perform cell segmentation, phenotyping, and spatial analyses to advance their research outputs. © 2021 Wiley Periodicals LLC.

Dendritic cells in Th2 immune responses and allergic sensitization.

Allergic responses are characterized by the activation of a specific subset of effector CD4+ T cells, the T-helper type 2 (Th2) cells, that respond to harmless environmental antigens causing inflammation and pathology. Th2 cells are also found in the context of parasite infections, where they can mediate parasite clearance and expulsion, and support tissue repair. The process that leads to the activation of Th2 cells in vivo is incompletely understood: while it has become clear that "conventional" dendritic cells are essential antigen-presenting cells for the initiation of Th2 immune responses, the molecules that are expressed by dendritic cells exposed to allergens, and the mediators that are produced as a consequence and signal to naïve CD4+ T cells to promote their development into effector Th2, remain to be defined. Here we summarize recent developments in the identification of the dendritic cell subsets involved in Th2 responses, review potential mechanisms proposed to explain the generation of these immune responses, and discuss the direct and indirect signals that condition dendritic cells to drive the development of Th2 responses during allergen or parasite exposure.

Tertiary lymphoid structures in cancer - considerations for patient prognosis.

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that form within nonlymphoid tissue. They share structural and functional characteristics with secondary lymphoid structures such as lymph nodes and can contain B-cell follicles and germinal centers surrounded by a T-cell region. TLS have been described in several types of cancers and are usually associated with positive patient outcomes. However, TLS differ vastly in cellular composition and location within tissue types. In this review, we discuss factors confounding the interpretation of the evidence for a prognostic role for TLS in cancer and frame these factors in the context of translation to regular clinical use.

In-Vitro Effects of Secreted Frizzled-Related Protein 1 (SFRP1) On Human Corneal Epithelial Cells.

PURPOSE: Limbal corneal epithelial cells (LCECs) are responsible for corneal epithelial cell regeneration. However, corneal central epithelial cells (CCECs) are also suggested to display potential for self-renewal. Additionally, a better understanding of molecules that regulate corneal epithelial cell regeneration is important for studying conditions affecting the cornea, for example, keratoconus. Given our previous findings of reduced levels of secreted frizzled-related protein 1 (SFRP1) in tears from keratoconus patients compared to controls, we investigated the effects of SFRP1 on the proliferation and survival of cultured central and limbal human corneal epithelial cells. MATERIAL AND METHODS: Limbal and central corneal explants were established from postmortem human corneas, and cultured in CnT-PR, an epithelial-specific tissue culture media. Subcultured cells from explants were immunostained for the cytokeratins CK3, 12, 19, and the proliferative/oligopotent markers Ki67 and p63. BrdU flow cytometry, Alamar Blue and LDH assays were used to assess effects of SFRP1 treatment on central and LCECs. RESULTS: Primary limbal and central corneal epithelial cells were successfully cultured in vitro to confluence (P6 and P4, respectively). They all expressed varying levels of cytokeratins CK3, CK12 and CK19, and Ki67 and p63. Additionally, they showed significantly increased metabolic activity after SFRP1 treatment (p < 0.05), with a maximum response at 1 µg/mL of SPRF1. No difference in proliferation was detected in SFRP1 treated LCECs; however, a reduction in cell death was noted (p < 0.05). CONCLUSION: Similar to the LCECs, primary human CCECs can be cultured in vitro, and expressed epithelial markers. SFRP1 demonstrated an improvement on the metabolic activity of both CCECs and LCECs, which in LCECs could be resulted from reduced cell death. This may have implications in degenerative corneal disorders, such as keratoconus.

Antiviral macrophage responses in flavivirus encephalitis.

Mosquito-borne flaviviruses are a major current and emerging threat, affecting millions of people worldwide. Global climate change, combined with increasing proximity of humans to animals and mosquito vectors by expansion into natural habitats, coupled with the increase in international travel, have resulted in significant spread and concomitant increase in the incidence of infection and severe disease. Although neuroinvasive disease has been well described for some viral infections such as Japanese Encephalitis virus (JEV) and West Nile virus (WNV), others such as dengue virus (DENV) have recently displayed an emerging pattern of neuroinvasive disease, distinct from the previously observed, systemically-induced encephalomyelopathy. In this setting, the immune response is a crucial component of host defence, in preventing viral dissemination and invasion of the central nervous system (CNS). However, subversion of the anti-viral activities of macrophages by flaviviruses can facilitate viral replication and spread, enhancing the intensity of immune responses, leading to severe immune-mediated disease which may be further exacerbated during the subsequent infection with some flaviviruses. Furthermore, in the CNS myeloid cells may be responsible for inducing specific inflammatory changes, which can lead to significant pathological damage during encephalitis. The interaction of virus and cells of the myeloid lineage is complex, and this interaction is likely responsible at least in part, for crucial differences between viral clearance and pathology. Recent studies on the role of myeloid cells in innate immunity and viral control, and the mechanisms of evasion and subversion used by flaviviruses are rapidly advancing our understanding of the immunopathological mechanisms involved in flavivirus encephalitis and will lead to the development of therapeutic strategies previously not considered.

Microarray analysis of gene expression in West Nile virus-infected human retinal pigment epithelium.

PURPOSE: To identify key genes differentially expressed in the human retinal pigment epithelium (hRPE) following low-level West Nile virus (WNV) infection. METHODS: Primary hRPE and retinal pigment epithelium cell line (ARPE-19) cells were infected with WNV (multiplicity of infection 1). RNA extracted from mock-infected and WNV-infected cells was assessed for differential expression of genes using Affymetrix microarray. Quantitative real-time PCR analysis of 23 genes was used to validate the microarray results. RESULTS: Functional annotation clustering of the microarray data showed that gene clusters involved in immune and antiviral responses ranked highly, involving genes such as chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X-C motif) ligand 10 (CXCL10), and toll like receptor 3 (TLR3). In conjunction with the quantitative real-time PCR analysis, other novel genes regulated by WNV infection included indoleamine 2,3-dioxygenase (IDO1), genes involved in the transforming growth factor-ß pathway (bone morphogenetic protein and activin membrane-bound inhibitor homolog [BAMBI] and activating transcription factor 3 [ATF3]), and genes involved in apoptosis (tumor necrosis factor receptor superfamily, member 10d [TNFRSF10D]). WNV-infected RPE did not produce any interferon-γ, suggesting that IDO1 is induced by other soluble factors, by the virus alone, or both. CONCLUSIONS: Low-level WNV infection of hRPE cells induced expression of genes that are typically associated with the host cell response to virus infection. We also identified other genes, including IDO1 and BAMBI, that may influence the RPE and therefore outer blood-retinal barrier integrity during ocular infection and inflammation, or are associated with degeneration, as seen for example in aging.