As treatment of the early, inflammatory phase of sepsis improves, post-sepsis immunosuppression and secondary infection have increased in importance. How early inflammation drives immunosuppression ...remains unclear. Although IFN-gamma typically helps microbial clearance, we found that increased plasma IFN-gamma in early clinical sepsis was associated with the later development of secondary Candida infection. Consistent with this observation, we found that exogenous IFN-gamma suppressed macrophage phagocytosis of zymosan in vivo, and antibody blockade of IFN-gamma after endotoxemia improved survival of secondary candidemia. Transcriptomic analysis of innate lymphocytes during endotoxemia suggested that NKT cells drove IFN-gamma production by NK cells via mTORC1. Activation of invariant NKT (iNKT) cells with glycolipid antigen drove immunosuppression. Deletion of iNKT cells in Cd1d.sup.-/- mice or inhibition of mTOR by rapamycin reduced immunosuppression and susceptibility to secondary Candida infection. Thus, although rapamycin is typically an immunosuppressive medication, in the context of sepsis, rapamycin has the opposite effect. These results implicated an NKT cell/mTOR/IFN-gamma axis in immunosuppression following endotoxemia or sepsis. In summary, in vivo iNKT cells activated mTORC1 in NK cells to produce IFN-gamma, which worsened macrophage phagocytosis, clearance of secondary Candida infection, and mortality.
Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA ...sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.
Hematopoietic stem cells (HSCs) are rare cells that generate all the various types of blood and immune cells. High-quality transcriptome data have enabled the identification of significant genes for ...HSCs. However, most genes are expressed in various forms by alternative splicing (AS), extending transcriptome complexity. Here, we delineate AS to determine which isoforms are expressed in mouse HSCs. Our analysis of microarray and RNA-sequencing data includes differential expression of splicing factors that may regulate AS, and a complete map of splicing isoforms. Multiple types of isoforms for known HSC genes and unannotated splicing that may alter gene function are presented. Transcriptome-wide identification of genes and their respective isoforms in mouse HSCs will open another dimension for adult stem cells.
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•This study presents the splicing landscape in mouse hematopoietic stem cells•Many HSC genes express multiple isoforms, some with alternative isoforms•High-resolution analysis allows the discovery of unannotated splicing•HSC-expressed isoforms can have similar or divergent functions
Gazit and colleagues mapped all splicing within mouse hematopoietic stem cells by analysis of microarrays and meta-analysis of RNA-sequencing datasets. They validated co-expression of minor isoforms, variant isoform predominance, multiple isoforms from one gene, and discovery of unannotated splicing. Functionally, variant isoforms may alter the activity of HSC genes, or not. Whole-transcriptome splicing data at nucleotide resolution is providing a useful tool for the community.
Alternative splicing (AS) is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Different types of AS have been observed; these ...include exon skipping, alternative donor or acceptor site and intron retention. In humans, exon skipping is the most common type while intron retention is rare. In contrast, in Arabidopsis, intron retention is the most prevalent AS type (approximately 40%). Here we show that direct transcript expression analysis using high-density oligonucleotide-based whole-genome microarrays (WGAs) is particularly amenable for assessing global intron retention in Arabidopsis. By applying a novel algorithm retained introns are detected in 8% of the transcripts examined. A sampling of 14 transcripts showed that 86% can be confirmed by RT-PCR. This rate of detection predicts an overall total AS rate of 20% for Arabidopsis compared with 10-22% based on EST/cDNA-based analysis. These findings will facilitate monitoring constitutive and dynamic whole-genome splicing on the next generation WGA slides.
Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less AS than animals. An algorithm, based on expressed sequence tag (EST) pairs gapped alignment, ...was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for AS. EST pairs gapped alignment was first evaluated in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum) for which annotated genome sequence is available and was shown to accurately predict splicing events. The method was then applied to 11 plant species that include 17 cultivars for which enough ESTs are available. The results show a large, 3.7-fold difference in AS rates between plant species with Arabidopsis and rice in the lower range and lettuce (Lactuca sativa) and sorghum (Sorghum bicolor) in the upper range. Hence, compared to higher animals, plants show a much greater degree of variety in their AS rates and in some plant species the rates of animal and plant AS are comparable although the distribution of AS types may differ. In eudicots but not monocots, a correlation between genome size and AS rates was detected, implying that in eudicots the mechanisms that lead to larger genomes are a driving force for the evolution of AS.
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