An Early Islet Transcriptional Signature Is Associated With Local Inflammation in Autoimmune Diabetes.

Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. In this article, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the prediabetic period. Single-cell RNA-sequencing and differential expression analyses of islets from prediabetic rats reveal subsets of ß- and α-cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ ß- and α-cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed ß- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

The Impact of Integrated Behavioral Healthcare on Glycemic Control.

Integrated behavioral healthcare (IBH) is the "standard of care" to address psychosocial factors impacting diabetes outcomes; it is not standard in practice. This longitudinal, retrospective, chart-review examines IBH impact on glycemic control in an adult diabetes clinic. Adults (n = 374) with ≥ 1 behavioral health encounter, ≥ 2 hemoglobin A1c (HbA1c) values, and HbA1c value > 8% at initial IBH visit were included. Mixed effects linear piecewise models examined differences in slope trajectories for 365 days pre- and post-IBH intervention. Pre-intervention slope was not significant (z = - 1.09, p = 0.28). The post-intervention slope was significant (z = - 6.44, p < 0.001), indicating a significant linear decrease in HbA1c values. Results demonstrated that prior to engaging with behavioral health, there was no change in HbA1c. After initial IBH visit, there was a predicted reduction of > 1% in HbA1c over the following year. These results suggest that IBH significantly improves patients' metabolic status. Next steps for IBH research are offered.

Type I IFN-Driven Immune Cell Dysregulation in Rat Autoimmune Diabetes.

Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing ß cells of pancreatic islets. Essential components of the innate immune antiviral response, including type I IFN and IFN receptor (IFNAR)-mediated signaling pathways, likely contribute to human type 1 diabetes susceptibility. We previously showed that LEW.1WR1 Ifnar1 -/- rats have a significant reduction in diabetes frequency following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR loss on immune cell populations in KRV-induced diabetes, we performed flow cytometric analysis in spleens from LEW.1WR1 wild-type (WT) and Ifnar1 -/- rats after viral infection but before the onset of insulitis and diabetes. We found a relative decrease in CD8+ T cells and NK cells in KRV-infected LEW.1WR1 Ifnar1 -/- rats compared with KRV-infected WT rats; splenic regulatory T cells were diminished in WT but not Ifnar1 -/- rats. In contrast, splenic neutrophils were increased in KRV-infected Ifnar1 -/- rats compared with KRV-infected WT rats. Transcriptional analysis of splenic cells from KRV-infected rats confirmed a reduction in IFN-stimulated genes in Ifnar1 -/- compared with WT rats and revealed an increase in transcripts related to neutrophil chemotaxis and MHC class II. Single-cell RNA sequencing confirmed that MHC class II transcripts are increased in monocytes and macrophages and that numerous types of splenic cells harbor KRV. Collectively, these findings identify dynamic shifts in innate and adaptive immune cells following IFNAR disruption in a rat model of autoimmune diabetes, providing insights toward the role of type I IFNs in autoimmunity.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Restore Thymic Architecture and T Cell Function Disrupted by Neonatal Hyperoxia.

Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3+ regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrated preferential impact of MEx treatment on the thymic medullary antigen presentation axis, as evidenced by enrichment of antigen presentation and antioxidative-stress related genes in dendritic cells (DCs) and medullary epithelial cells (mTECs). Our study demonstrates that MEx treatment represents a promising restorative therapeutic approach for oxygen-induced thymic injury, thus promoting normal development of both central tolerance and adaptive immunity.

HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells.

Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet ß-Cells From Donors With Type 1 Diabetes.

Type 1 diabetes studies consistently generate data showing islet ß-cell dysfunction and T cell-mediated anti-ß-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the ß-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single ß-cells. Consistent with immunohistological studies, ß-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These ß-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- ß-cells, specifically found in islets with lymphocytic infiltrates. ß-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ ß-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic ß-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. ß-Cell expression of Class II molecules suggests that ß-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data.

RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3'-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct ß-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

DNA-binding-domain fusions enhance the targeting range and precision of Cas9.

The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9's inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

A genome-wide map of diversity in Plasmodium falciparum.

Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.

Genomic patterns of pathogen evolution revealed by comparison of Burkholderia pseudomallei, the causative agent of melioidosis, to avirulent Burkholderia thailandensis.

BACKGROUND: The Gram-negative bacterium Burkholderia pseudomallei (Bp) is the causative agent of the human disease melioidosis. To understand the evolutionary mechanisms contributing to Bp virulence, we performed a comparative genomic analysis of Bp K96243 and B. thailandensis (Bt) E264, a closely related but avirulent relative. RESULTS: We found the Bp and Bt genomes to be broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands, which we experimentally validated to be differentially present in multiple Bt isolates. By examining species-specific genomic regions, we derived molecular explanations for previously-known metabolic differences, discovered potentially new ones, and found that the acquisition of a capsular polysaccharide gene cluster in Bp, a key virulence component, is likely to have occurred non-randomly via replacement of an ancestral polysaccharide cluster. Virulence related genes, in particular members of the Type III secretion needle complex, were collectively more divergent between Bp and Bt compared to the rest of the genome, possibly contributing towards the ability of Bp to infect mammalian hosts. An analysis of pseudogenes between the two species revealed that protein inactivation events were significantly biased towards membrane-associated proteins in Bt and transcription factors in Bp. CONCLUSION: Our results suggest that a limited number of horizontal-acquisition events, coupled with the fine-scale functional modulation of existing proteins, are likely to be the major drivers underlying Bp virulence. The extensive genomic similarity between Bp and Bt suggests that, in some cases, Bt could be used as a possible model system for studying certain aspects of Bp behavior.