Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging.

A highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.

Specific subfamilies of transposable elements contribute to different domains of T lymphocyte enhancers.

Transposable elements (TEs) compose nearly half of mammalian genomes and provide building blocks for cis-regulatory elements. Using high-throughput sequencing, we show that 84 TE subfamilies are overrepresented, and distributed in a lineage-specific fashion in core and boundary domains of CD8+ T cell enhancers. Endogenous retroviruses are most significantly enriched in core domains with accessible chromatin, and bear recognition motifs for immune-related transcription factors. In contrast, short interspersed elements (SINEs) are preferentially overrepresented in nucleosome-containing boundaries. A substantial proportion of these SINEs harbor a high density of the enhancer-specific histone mark H3K4me1 and carry sequences that match enhancer boundary nucleotide composition. Motifs with regulatory features are better preserved within enhancer-enriched TE copies compared to their subfamily equivalents located in gene deserts. TE-rich and TE-poor enhancers associate with both shared and unique gene groups and are enriched in overlapping functions related to lymphocyte and leukocyte biology. The majority of T cell enhancers are shared with other immune lineages and are accessible in common hematopoietic progenitors. A higher proportion of immune tissue-specific enhancers are TE-rich compared to enhancers specific to other tissues, correlating with higher TE occurrence in immune gene-associated genomic regions. Our results suggest that during evolution, TEs abundant in these regions and carrying motifs potentially beneficial for enhancer architecture and immune functions were particularly frequently incorporated by evolving enhancers. Their putative selection and regulatory cooption may have accelerated the evolution of immune regulatory networks.

Characterization of reference genes for qRT-PCR normalization in rice-field eel (Monopterus albus) to assess differences in embryonic developmental stages, the early development of immune organs, and cells infected with rhabdovirus.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become a popular technique to assess gene expression. Suitable reference genes are normally identified first to ensure accurate normalization. The aim of the present study was to select the most stable genes in embryonic developmental stages, the early development of immune organs, and cells infected with Chinese rice-field eel rhabdovirus (CrERV) of the rice-field eel (Monopterus albus). Four reference genes, including those encoding 18S ribosomal RNA (18SrRNA), beta actin (ß-actin), elongation factor 1 alpha (EF1ɑ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using geNorm, NormFinder, BestKeeper, and RefFinder software. Analyses indicated the stability ranking was 18SrRNA > ß-actin > GAPDH > EF1α in the embryonic stage, with 18SrRNA as the most stable reference gene. For immunity-related organs at different developmental stages, the order in the thymus was ß-actin > GAPDH > EF1α > 18SrRNA, with ß-actin as the most stable gene. In both spleen and kidney tissues, the rank order was EF1ɑ > GAPDH > ß-actin > 18SrRNA, with EF1α as the most stable gene. Furthermore, in rice-field eel kidney (CrE-K) cells infected with CrERV, the ranking was EF1ɑ > ß-actin > GAPDH > 18SrRNA, with EF1α as the most stable gene. The results for cells infected with CrERV were verified by testing signaling pathway genes catenin beta 1 (CTNNB1) and NOTCH1 based on the above four genes after virus infection in CrE-K cells. This study laid the foundation for choosing suitable reference genes for immunity-related gene expression analysis in rice-field eel.

HMGB1, angel or devil, in ischemic stroke.

INTRODUCTION: High-mobility group box 1 protein (HMGB1) is extensively involved in causing ischemic stroke, pathological damage of ischemic brain injury, and neural tissue repair after ischemic brain injury. However, the precise role of HMGB1 in ischemic stroke remains to be elucidated. METHODS: Comprehensive literature search and narrative review to summarize the current field of HMGB1 in cerebral ischemic based on the basic structure, structural modification, and functional roles of HMGB1 described in the literature. RESULTS: Studies have exhibited the crucial roles of HMGB1 in cell death, immunity and inflammation, thrombosis, and remodeling and repair. HMGB1 released after cerebral infarction is extensively involved in the pathological injury process in the early stage of cerebral infarction, whereas it is involved in the promotion of brain tissue repair and remodeling in the late stage of cerebral infarction. HMGB1 plays a neurotrophic role in acute white matter stroke, whereas it causes sustained activation of inflammation and plays a damaging role in chronic white matter ischemia. CONCLUSIONS: HMGB1 plays a complex role in cerebral infarction, which is related to not only the modification of HMGB1 and bound receptors but also different stages and subtypes of cerebral infarction. future studies on HMGB1 should investigate the spatial and temporal dynamics of HMGB1 after cerebral infarction. Moreover, future studies on HMGB1 should attempt to integrate different stages and infarct subtypes of cerebral infarction.

Succinate metabolism and its regulation of host-microbe interactions.

Succinate is a circulating metabolite, and the relationship between abnormal changes in the physiological concentration of succinate and inflammatory diseases caused by the overreaction of certain immune cells has become a research focus. Recent investigations have shown that succinate produced by the gut microbiota has the potential to regulate host homeostasis and treat diseases such as inflammation. Gut microbes are important for maintaining intestinal homeostasis. Microbial metabolites serve as nutrients in energy metabolism, and act as signal molecules that stimulate host cell and organ function and affect the structural balance between symbiotic gut microorganisms. This review focuses on succinate as a metabolite of both host cells and gut microbes and its involvement in regulating the gut - immune tissue axis by activating intestinal mucosal cells, including macrophages, dendritic cells, and intestinal epithelial cells. We also examined its role as the mediator of microbiota - host crosstalk and its potential function in regulating intestinal microbiota homeostasis. This review explores feasible ways to moderate succinate levels and provides new insights into succinate as a potential target for microbial therapeutics for humans.

Regulatory T cells require IL6 receptor alpha signaling to control skeletal muscle function and regeneration.

Muscle-residing regulatory T cells (Tregs) control local tissue integrity and function. However, the molecular interface connecting Treg-based regulation with muscle function and regeneration remains largely unexplored. Here, we show that exercise fosters a stable induction of highly functional muscle-residing Tregs with increased expression of amphiregulin (Areg), EGFR, and ST2. Mechanistically, we find that mice lacking IL6Rα on T cells (TKO) harbor significant reductions in muscle Treg functionality and satellite and fibro-adipogenic progenitor cells, which are required for muscle regeneration. Using exercise and sarcopenia models, IL6Rα TKO mice demonstrate deficits in Tregs, their functional maturation, and a more pronounced decline in muscle mass. Muscle injury models indicate that IL6Rα TKO mice have significant disabilities in muscle regeneration. Treg gain of function restores impaired muscle repair in IL6Rα TKO mice. Of note, pharmacological IL6R blockade in WT mice phenocopies deficits in muscle function identified in IL6Rα TKO mice, thereby highlighting the clinical implications of the findings.

Interaction between early in ovo stimulation of the gut microbiota and chicken host - splenic changes in gene expression and methylation.

BACKGROUND: Epigenetic regulation of the gene expression results from interaction between the external environment and transcription of the genetic information encoded in DNA. Methylated CpG regions within the gene promoters lead to silencing of the gene expression in most cases. Factors contributing to epigenetic regulation include intestinal microbiota, which in chicken can be potently modified by in ovo stimulation. The main aim of this study was to determine global and specific methylation patterns of the spleen under the influence of host-microbiome interaction. RESULTS: Fertilized eggs of two genotypes: Ross 308 and Green-legged Partridgelike were in ovo stimulated on d 12 of incubation. The injected compounds were as follows: probiotic - Lactococcus lactis subsp. cremoris IBB477, prebiotic - galactooligosaccharides, and synbiotic - combination of both. Chickens were sacrificed on d 42 post-hatching. Spleen was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. We have proved that negative regulation of gene expression after administration of bioactive substances in ovo might have epigenetic character. Epigenetic changes depend on the genotype and the substance administered in ovo. CONCLUSION: Epigenetic nature of microbial reprogramming in poultry and extension of issues related to host-microbiome interaction is a new direction of this research.

An Examination of the Development and Localization of Key Immune Cells in Developing Pouch Young of the Red-Tailed Phascogale (Phascogale calura).

Cells expressing the surface markers CD3, CD4, CD79b, IgM, MHC class II, and ModoUG (nonclassical MHC class I) were detected in red-tailed phascogale tissues using immunohistochemistry, and the appearance and localization of cells observed here was consistent with previous observations in other marsupial species. CD3+ cells were first detected at one day postpartum (dpp) in the thymus, followed by ModoUG+ cells at 5-7 dpp in the thymus and lymph nodes. CD79b+ cells were first detected at 12-14 dpp in bone marrow, spleen, and lymph nodes. IgM+ cells were first detected at 12-14 dpp in thymus, bone marrow, spleen, and lymph nodes. MHC class II+ cells were first detected at 12-14 dpp in thymus, bone marrow, and lymph nodes. CD4+ cells were detected in adult thymus and spleen only. The presence of the mature immune cell populations and their localization to characteristic T and B cell zones in mature lymphoid tissues with normal histological structure indicates that red-tailed phascogales develop immunocompetence by the end of pouch life. Anat Rec, 302:1985-2002, 2019. © 2019 American Association for Anatomy.

Molecular identification and gene expression profiles of the T cell receptors and co-receptors in developing red-tailed phascogale (Phascogale calura) pouch young.

Expressed coding sequences were identified for the T cell receptors TCRα, TCRß, TCRγ, TCRδ, TCRµ, and co-receptors CD3ε, CD4, and CD8α in the red-tailed phascogale (Phascogale calura). Expression of these genes was examined in the developing thymus and spleen of pouch young and juvenile individuals to detect any changes in expressionrelative to developmental stages. Transcripts of CD3ε, CD4, TCRδ and TCRµ were first detected in the thorax at 1 day postpartum (dpp), and transcripts of all target genes were detected in the thymus from 3 dpp, and the spleen from 17 dpp. The relative expression of all target genes changed significantly over the course of pouch life and was associated with histological milestones of both the thymus and the spleen. Peak gene expression was observed early in pouch life in the thymus, with a decrease in expression associated with timing of thymic involution, while expression peaked later in the spleen and was associated with the initial differentiation of the red and white pulp and a population of T cells forming the periarterial lymphatic sheath (PALS). Active expression of these target genes within the thymus and spleen indicates that the young marsupials are actively developing immunocompetence during pouch life.