Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell

IL-21 and IL-6 Are Critical for Different Aspects of B Cell Immunity and Redundantly Induce Optimal Follicular Helper CD4 T Cell (Tfh) Differentiation

Cytokines are important modulators of lymphocytes, and both interleukin-21 (IL-21) and IL-6 have proposed roles in T follicular helper (Tfh) differentiation, and directly act on B cells. Here we investigated the absence of IL-6 alone, IL-21 alone, or the combined lack of IL-6 and IL-21 on Tfh differentiation and the development of B cell immunity in vivo. C57BL/6 or IL-21−/− mice were treated with a neutralizing monoclonal antibody against IL-6 throughout the course of an acute viral infection (lymphocytic choriomeningitis virus, LCMV). The combined absence of IL-6 and IL-21 resulted in reduced Tfh differentiation and reduced Bcl6 protein expression. In addition, we observed that these cytokines had a large impact on antigen-specific B cell responses. IL-6 and IL-21 collaborate in the acute T-dependent antiviral antibody response (90% loss of circulating antiviral IgG in the absence of both cytokines). In contrast, we observed reduced germinal center formation only in the absence of IL-21. Absence of IL-6 had no impact on germinal centers, and combined absence of both IL-21 and IL-6 revealed no synergistic effect on germinal center B cell development. Studying CD4 T cells in vitro, we found that high IL-21 production was not associated with high Bcl6 or CXCR5 expression. TCR stimulation of purified naïve CD4 T cells in the presence of IL-6 also did not result in Tfh differentiation, as determined by Bcl6 or CXCR5 protein expression. Cumulatively, our data indicates that optimal Tfh formation requires IL-21 and IL-6, and that cytokines alone are insufficient to drive Tfh differentiation

MicroRNA target site identification in helper T cell differentiation

MicroRNAs are important mediators in the control of helper T cell differentiation, where small perturbations in responses to extracellular signals leads to early polarization and specialization. Identifying gene targets of highly expressed helper T cell miRNAs can lead to identification of novel players in these networks. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a powerful tool for identifying these gene targets by pulling down the miRNA binding protein argonaute (Ago) and sequencing the co-immunoprecipitated miRNA and targeted cognate mRNA fragment. We performed HITS-CLIP on mouse CD4+ helper T cells and identified miRNA binding sites. While a majority of these sites were in well annotated 3’ untranslated regions (UTRs), we also identified a number of miRNA sites in coding regions, downstream of annotated 3’UTRs and in difficult to annotate regions of the genome. We identified hundreds of binding sites that were dependent of the presence of miR-29a, a miRNA highly expressed in CD4 T cells and implicated for its roles in T cell differentiation. We also observed a role for miR-29 in IL-17 production, which led us to identify a number of miR-29 targeted genes with roles in Th17 differentiation, one of which, ICOS, was a new target that we found to be directly regulated by miR-29. Our study identifies Ago dependent miRNA binding sites important in Th17 biology and identifies a role for miR-29 in IL17 production. Additionally, our lab has generated a tool that aims to create an easy to use interface for labs working with CLIP-Seq data to create high quality graphics that are easily edited in a vector based graphics program. ClipPlot is a webapp that can be run on a local server and used by all members of a lab simultaneously, requiring only the technical expertise of a single user. While this is not a replacement for sophisticated visualization tools like IGV, ClipPlot provides an easy to use platform for researchers working with CLIP-Seq or RNA-Seq data to quickly create presentable and easily manipulated graphics, visualizing the shape of sequencing data to defined regions

Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation

Summary: The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5′ end and 3′-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation. : Chiou et al. show that T cells release extracellular vesicles that carry RNA cargo enriched in tRNA fragments. Immune activating signals enhance multivesicular body formation and the secretion of vesicles containing specific tRNA fragments. Within cells, these tRNA fragments inhibit T cell activation and cytokine production. Keywords: tRNA fragment, tsRNA, extracellular vesicle, exosome, T lymphocyt

GCLiPP Genome Biology scripts

<p>Code used for data analysis and production of figure for Zhu, Litterman, et al, Genome Biology</p&gt

A massively parallel 3' UTR reporter assay reveals relationships between nucleotide content, sequence conservation, and mRNA destabilization.

Compared to coding sequences, untranslated regions of the transcriptome are not well conserved, and functional annotation of these sequences is challenging. Global relationships between nucleotide composition of 3' UTR sequences and their sequence conservation have been appreciated since mammalian genomes were first sequenced, but the functional relevance of these patterns remain unknown. We systematically measured the effect on gene expression of the sequences of more than 25,000 RNA-binding protein (RBP) binding sites in primary mouse T cells using a massively parallel reporter assay. GC-rich sequences were destabilizing of reporter mRNAs and come from more rapidly evolving regions of the genome. These sequences were more likely to be folded in vivo and contain a number of structural motifs that reduced accumulation of a heterologous reporter protein. Comparison of full-length 3' UTR sequences across vertebrate phylogeny revealed that strictly conserved 3' UTRs were GC-poor and enriched in genes associated with organismal development. In contrast, rapidly evolving 3' UTRs tended to be GC-rich and derived from genes involved in metabolism and immune responses. Cell-essential genes had lower GC content in their 3' UTRs, suggesting a connection between unstructured mRNA noncoding sequences and optimal protein production. By reducing gene expression, GC-rich RBP-occupied sequences act as a rapidly evolving substrate for gene regulatory interactions

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells

CD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17∼92 is induced by T cell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17∼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17∼92 target genes that include several negative regulators of T cell activation. CD28-deficient T cells exhibit derepressed miR-17∼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17∼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ T cells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17∼92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent T cell activation

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells.

Funder: Horizon 2020 Framework ProgrammeFunder: National Institutes of HealthCD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17∼92 is induced by T cell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17∼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17∼92 target genes that include several negative regulators of T cell activation. CD28-deficient T cells exhibit derepressed miR-17∼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17∼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ T cells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17∼92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent T cell activation