Publications by authors named "K Mark Ansel"

84 Publications

Impaired antibacterial immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19.

medRxiv 2021 Mar 26. Epub 2021 Mar 26.

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections. Critically ill patients with coronavirus disease 2019 (COVID-19) face an elevated risk of VAP, although susceptibility varies widely. Because mechanisms underlying VAP predisposition remained unknown, we assessed lower respiratory tract host immune responses and microbiome dynamics in 36 patients, including 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill controls. We employed a combination of tracheal aspirate bulk and single cell RNA sequencing (scRNA-seq). Two days before VAP onset, a lower respiratory transcriptional signature of bacterial infection was observed, characterized by increased expression of neutrophil degranulation, toll-like receptor and cytokine signaling pathways. When assessed at an earlier time point following endotracheal intubation, more than two weeks prior to VAP onset, we observed a striking early impairment in antibacterial innate and adaptive immune signaling that markedly differed from COVID-19 patients who did not develop VAP. scRNA-seq further demonstrated suppressed immune signaling across monocytes/macrophages, neutrophils and T cells. While viral load did not differ at an early post-intubation timepoint, impaired SARS-CoV-2 clearance and persistent interferon signaling characterized the patients who later developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients who developed VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. Together, these findings demonstrate that COVID-19 patients who develop VAP have impaired antibacterial immune defense weeks before secondary infection onset.

One Sentence Summary: COVID-19 patients with secondary bacterial pneumonia have impaired immune signaling and lung microbiome changes weeks before onset.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.23.21253487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010763PMC
March 2021

Epithelial miR-141 regulates IL-13-induced airway mucus production.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

Department of Medicine, Division of Pulmonary and Critical Care Medicine.

IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.139019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021117PMC
March 2021

PICS2: Next-generation fine mapping via probabilistic identification of causal SNPs.

Bioinformatics 2021 Feb 24. Epub 2021 Feb 24.

Illumina, Inc.

 : The Probabilistic Identification of Causal SNPs (PICS) algorithm and web application was developed as a fine-mapping tool to determine the likelihood that each single nucleotide polymorphism (SNP) in LD with a reported index SNP is a true causal polymorphism. PICS is notable for its ability to identify candidate causal SNPs within a locus using only the index SNP, which are widely available from published GWAS, whereas other methods require full summary statistics or full genotype data. However, the original PICS web application operates on a single SNP at a time, with slow performance, severely limiting its usability. We have developed a next-generation PICS tool, PICS2, which enables performance of PICS analyses of large batches of index SNPs with much faster performance. Additional updates and extensions include use of LD reference data generated from 1000 Genomes phase 3; annotation of variant consequences; annotation of GTEx eQTL genes and downloadable PICS SNPs from GTEx eQTLs; the option of generating PICS probabilities from experimental summary statistics; and generation of PICS SNPs from all SNPs of the GWAS catalog, automatically updated weekly. These free and easy-to-use resources will enable efficient determination of candidate loci for biological studies to investigate the true causal variants underlying disease processes.

Availability: PICS2 is available at https://pics2.ucsf.edu.

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btab122DOI Listing
February 2021

Noncoding RNAs in B cell responses.

RNA Biol 2021 Feb 15:1-7. Epub 2021 Feb 15.

Department of Microbiology & Immunology, Sandler Asthma Basic Research Center, University of California San Francisco , San Francisco, CA, USA.

B cells constitute a main branch adaptive immune system. They mediate host defence through the production of high-affinity antibodies against an enormous diversity of foreign antigens. Remarkably, B cells undergo multiple types of somatic DNA mutation to achieve this effector function, including class switch recombination (CSR) and somatic hypermutation (SHM). These processes occur in response to antigen recognition and inflammatory signals, and require strict biological control at multiple levels. Transcription within the locus that encodes antibodies plays direct roles in CSR. Additional non-coding RNAs (ncRNAs), including both microRNAs (miRNAs) and long ncRNAs (lncRNAs), also play pivotal roles in B cell activation and terminal effector function through post-transcriptional gene regulation and chromatin remodelling, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15476286.2021.1885876DOI Listing
February 2021

COVID-19 ARDS is characterized by a dysregulated host response that differs from cytokine storm and is modified by dexamethasone.

Res Sq 2021 Jan 14. Epub 2021 Jan 14.

We performed comparative lower respiratory tract transcriptional profiling of 52 critically ill patients with the acute respiratory distress syndrome (ARDS) from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a cytokine storm, we observed reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS was characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity that were predicted to be modulated by dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 was characterized by impaired interferon-stimulated gene expression (ISG). We found that the relationship between SARS-CoV-2 viral load and expression of ISGs was decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients with COVID-19 ARDS did not demonstrate cytokine storm but instead revealed a unique and dysregulated host response predicted to be modified by dexamethasone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21203/rs.3.rs-141578/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814832PMC
January 2021

miR-29 Sustains B Cell Survival and Controls Terminal Differentiation via Regulation of PI3K Signaling.

Cell Rep 2020 Dec;33(9):108436

Department of Pathology, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

The phosphatidylinositol 3-kinase (PI3K) signaling cascade downstream of the B cell receptor (BCR) signalosome is essential for B cell maturation. Proper signaling strength is maintained through the PI3K negative regulator phosphatase and tensin homolog (PTEN). Although a role for microRNA (miRNA)-dependent control of the PTEN-PI3K axis has been described, the contribution of individual miRNAs to the regulation of this crucial signaling modality in mature B lymphocytes remains to be elucidated. Our analyses reveal that ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and dampening of the PI3K pathway in mature B cells. This dysregulation has a profound impact on the survival of B lymphocytes and results in increased class switch recombination and decreased plasma cell differentiation. Furthermore, we demonstrate that ablation of one copy of Pten is sufficient to ameliorate the phenotypes associated with miR-29 loss. Our data suggest a critical role for the miR-29-PTEN-PI3K regulatory axis in mature B lymphocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730937PMC
December 2020

Author Correction: Profiling immunoglobulin repertoires across multiple human tissues using RNA sequencing.

Nat Commun 2020 09 4;11(1):4499. Epub 2020 Sep 4.

Department of Computer Science, University of California, Los Angeles, 404 Westwood Plaza, Los Angeles, CA, 90095, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-18509-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474053PMC
September 2020

Profiling immunoglobulin repertoires across multiple human tissues using RNA sequencing.

Nat Commun 2020 06 19;11(1):3126. Epub 2020 Jun 19.

Department of Computer Science, University of California, Los Angeles, 404 Westwood Plaza, Los Angeles, CA, 90095, USA.

Profiling immunoglobulin (Ig) receptor repertoires with specialized assays can be cost-ineffective and time-consuming. Here we report ImReP, a computational method for rapid and accurate profiling of the Ig repertoire, including the complementary-determining region 3 (CDR3), using regular RNA sequencing data such as those from 8,555 samples across 53 tissues types from 544 individuals in the Genotype-Tissue Expression (GTEx v6) project. Using ImReP and GTEx v6 data, we generate a collection of 3.6 million Ig sequences, termed the atlas of immunoglobulin repertoires (TAIR), across a broad range of tissue types that often do not have reported Ig repertoires information. Moreover, the flow of Ig clonotypes and inter-tissue repertoire similarities across immune-related tissues are also evaluated. In summary, TAIR is one of the largest collections of CDR3 sequences and tissue types, and should serve as an important resource for studying immunological diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16857-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305308PMC
June 2020

An anti-siglec-8 antibody depletes sputum eosinophils from asthmatic subjects and inhibits lung mast cells.

Clin Exp Allergy 2020 08 8;50(8):904-914. Epub 2020 Jul 8.

Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, CA, USA.

Background: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on mast cells and eosinophils, but information about Siglec-8 expression and function in the lung is limited. A humanized antibody, AK002, targeting Siglec-8 is undergoing development for treatment of diseases associated with mast cell and eosinophil-driven inflammation.

Objective: To characterize Siglec-8 expression in the airway in asthma and determine whether antibodies that target Siglec-8 (S8mAbs) can decrease airway eosinophils in asthma or inhibit lung mast cell activation.

Methods: Gene expression profiling and flow cytometry were used to characterize Siglec-8 expression in sputum cells from stable asthma. An antibody-dependent cellular cytotoxicity (ADCC) assay was used to determine whether an S8mAb can decrease eosinophils in sputum from asthma patients ex vivo. A mast cell activation assay was used to determine whether an S8mAb can inhibit mast cell activation in human lung tissue ex vivo.

Results: Gene expression for Siglec-8 is increased in sputum cells in asthma and correlates with gene expression for eosinophils and mast cells. Gene expression for Siglec-8 is inversely and significantly correlated with measures of airflow obstruction in asthma patients. Siglec-8 is prominently expressed on the surface of eosinophils and mast cells in sputum. S8mAbs decrease eosinophils in sputum from patients with asthma and inhibit FcεR1-activated mast cells in lung tissues.

Conclusions And Clinical Relevance: Siglec-8 is highly expressed on eosinophils and mast cells in asthmatic sputum and targeting Siglec-8 with an antibody is a plausible strategy to decrease sputum eosinophils and inhibit lung mast cells in asthma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cea.13681DOI Listing
August 2020

Distinct associations of sputum and oral microbiota with atopic, immunologic, and clinical features in mild asthma.

J Allergy Clin Immunol 2020 11 13;146(5):1016-1026. Epub 2020 Apr 13.

Department of Internal Medicine, Division of Pulmonary/Critical Care Medicine, University of Michigan, Ann Arbor, Mich. Electronic address:

Background: Whether microbiome characteristics of induced sputum or oral samples demonstrate unique relationships to features of atopy or mild asthma in adults is unknown.

Objective: We sought to determine sputum and oral microbiota relationships to clinical or immunologic features in mild atopic asthma and the impact on the microbiota of inhaled corticosteroid (ICS) treatment administered to ICS-naive subjects with asthma.

Methods: Bacterial microbiota profiles were analyzed in induced sputum and oral wash samples from 32 subjects with mild atopic asthma before and after inhaled fluticasone treatment, 18 atopic subjects without asthma, and 16 nonatopic healthy subjects in a multicenter study (NCT01537133). Associations with clinical and immunologic features were examined, including markers of atopy, type 2 inflammation, immune cell populations, and cytokines.

Results: Sputum bacterial burden inversely associated with bronchial expression of type 2 (T2)-related genes. Differences in specific sputum microbiota also associated with T2-low asthma phenotype, a subgroup of whom displayed elevations in lung inflammatory mediators and reduced sputum bacterial diversity. Differences in specific oral microbiota were more reflective of atopic status. After ICS treatment of patients with asthma, the compositional structure of sputum microbiota showed greater deviation from baseline in ICS nonresponders than in ICS responders.

Conclusions: Novel associations of sputum and oral microbiota to immunologic features were observed in this cohort of subjects with or without ICS-naive mild asthma. These findings confirm and extend our previous report of reduced bronchial bacterial burden and compositional complexity in subjects with T2-high asthma, with additional identification of a T2-low subgroup with a distinct microbiota-immunologic relationship.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2020.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554083PMC
November 2020

MicroRNA regulation of CD8 T cell responses.

Noncoding RNA Investig 2019 Aug 26;3. Epub 2019 Aug 26.

Sandler Asthma Basic Research Center, Department of Microbiology & Immunology, University of California San Francisco, San Francisco, CA, USA.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that play critical roles in the regulation of a broad range of biological processes. Like transcription factors, miRNAs exert their effects by modulating the expression of networks of genes that operate in common or convergent pathways. CD8 T cells are critical agents of the adaptive immune system that provide protection from infection and cancer. Here, we review the important roles of miRNAs in the regulation of CD8 T cell biology and provide perspectives on the broader emerging principles of miRNA function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/ncri.2019.07.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913524PMC
August 2019

Universal Principled Review: A Community-Driven Method to Improve Peer Review.

Cell 2019 Dec;179(7):1441-1445

Department of Immunology, University of Washington, Seattle, WA 98109, USA.

Despite being a staple of our science, the process of pre-publication peer review has few agreed-upon standards defining its goals or ideal execution. As a community of reviewers and authors, we assembled an evaluation format and associated specific standards for the process as we think it should be practiced. We propose that we apply, debate, and ultimately extend these to improve the transparency of our criticism and the speed with which quality data and ideas become public.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.11.029DOI Listing
December 2019

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

Cell Rep 2019 09;28(11):2795-2806.e3

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752737PMC
September 2019

miR-15/16 Restrain Memory T Cell Differentiation, Cell Cycle, and Survival.

Cell Rep 2019 08;28(8):2169-2181.e4

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Sandler Asthma Basic Research Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters, and their generation is the goal of many vaccination strategies. microRNAs (miRNAs) are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immunoprecipitation of AGO2 combined with gene expression analysis in miR-15/16-deficient T cells indicates that these effects are mediated through the direct inhibition of an extensive network of target genes within pathways critical to cell cycle, survival, and memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.07.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715152PMC
August 2019

Antigen Complexed with a TLR9 Agonist Bolsters c-Myc and mTORC1 Activity in Germinal Center B Lymphocytes.

Immunohorizons 2019 08 19;3(8):389-401. Epub 2019 Aug 19.

Sandler Asthma Basic Research Center, University of California, San Francisco, San Francisco, CA 94143; and

The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88 mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88 mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88 mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88 mice, indicating a B cell-extrinsic effect on c-Myc protein expression combined with a B cell-intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, "TLR help," thus lowering the threshold for selection and increasing the magnitude of the GC response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/immunohorizons.1900030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738343PMC
August 2019

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

Genome Res 2019 06 31;29(6):896-906. Epub 2019 May 31.

Department of Microbiology and Immunology and Sandler Asthma Basic Research Center, University of California San Francisco, San Francisco, California 94143, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.242552.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581050PMC
June 2019

The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

Cell 2019 04;177(2):231-242

Department of Obstetrics, Gynecology, and Reproductive Sciences and Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601620PMC
April 2019

Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses.

Cell Rep 2019 01;26(4):933-944.e4

Sandler Asthma Basic Research Center, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365014PMC
January 2019

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

Cell Rep 2018 12;25(12):3356-3370.e4

Sandler Asthma Basic Research Center and Department of Microbiology & Immunology, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.11.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392044PMC
December 2018

Identification of Functionally Relevant microRNAs in the Regulation of Allergic Inflammation.

Methods Mol Biol 2018 ;1799:341-351

Sandler Asthma Basic Research Center, Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.

Transgenic methods to manipulate CD4 T lymphocytes in vivo via forced expression of TCR transgenes and targeted "knockout" of individual genes by Cre-lox technology are fundamental to modern immunology. However, efforts to scale up functional analysis by modifying expression of larger numbers of genes in T cells ex vivo have proven surprisingly difficult. Early RNA interference experiments achieved successful small RNA transfection by using very high concentrations of short-interfering RNA (siRNA) [1], but primary T cells are generally resistant to standard electroporation, cationic liposome-, and calcium phosphate-mediated transfection methods. Moreover, although viral vectors can successfully introduce DNA fragments of varying length, expression of these constructs in primary T cells is low efficiency and the subcloning process laborious. In this context, the relatively recent discovery of dozens of highly expressed microRNAs (miRNAs) in the immune system provides both an opportunity and a new challenge [2, 3]. How can we query the miRNAome of a cell to assign particular roles to individual miRNAs? Here, we describe an optimized technique for efficient and reproducible transfection of primary mouse CD4 T cells in vitro with synthetic miRNA mimics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7896-0_24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193755PMC
February 2019

Author Correction: Discovery of stimulation-responsive immune enhancers with CRISPR activation.

Nature 2018 07;559(7715):E13

Department of Microbiology and Immunology, University of California, San Francisco, California, 94143, USA.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0227-7DOI Listing
July 2018

Bacterial biogeography of adult airways in atopic asthma.

Microbiome 2018 06 9;6(1):104. Epub 2018 Jun 9.

Department of Medicine, Division of Pulmonary/Critical Care Medicine, University of California San Francisco, San Francisco, CA, USA.

Background: Perturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma.

Results: Bacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p < 0.001), compositional similarity was greatest for BB-IS, particularly in AAs and ANAs. The abundance of genera detected in BB correlated with those detected in IS and OW (r median [IQR] 0.869 [0.748-0.942] and 0.822 [0.687-0.909] respectively), but not with those in NB (r = 0.004 [- 0.003-0.011]). The number of taxa shared between IS-BB and NB-BB was greater in AAs than in HCs (p < 0.05) and included taxa previously associated with asthma. Of the genera abundant in NB, only Moraxella correlated positively with abundance in BB; specific members of this genus were shared between the two compartments only in AAs. Relative abundance of Moraxella in NB of AAs correlated negatively with that of Corynebacterium but positively with markers of eosinophilic inflammation in the blood and BAL fluid. The genus, Corynebacterium, trended to dominate all NB samples of HCs but only half of AAs (p = 0.07), in whom abundance of this genus was negatively associated with markers of eosinophilic inflammation.

Conclusions: Induced sputum is superior to nasal brush or oral wash for assessing bronchial microbiota composition in asthmatic adults. Although compositionally similar to the bronchial microbiota, the microbiota in induced sputum are distinct, reflecting enrichment of oral bacteria. Specific bacterial genera are shared between the nasal and the bronchial mucosa which are associated with markers of systemic and bronchial inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40168-018-0487-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994066PMC
June 2018

MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation.

J Exp Med 2017 Dec 9;214(12):3627-3643. Epub 2017 Nov 9.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA

MicroRNAs (miRNAs) exert powerful effects on immunity through coordinate regulation of multiple target genes in a wide variety of cells. Type 2 innate lymphoid cells (ILC2s) are tissue sentinel mediators of allergic inflammation. We established the physiological requirements for miRNAs in ILC2 homeostasis and immune function and compared the global miRNA repertoire of resting and activated ILC2s and T helper type 2 (T2) cells. After exposure to the natural allergen papain, mice selectively lacking the miR-17∼92 cluster in ILC2s displayed reduced lung inflammation. Moreover, miR-17∼92-deficient ILC2s exhibited defective growth and cytokine expression in response to IL-33 and thymic stromal lymphopoietin in vitro. The miR-17∼92 cluster member miR-19a promoted IL-13 and IL-5 production and inhibited expression of several targets, including SOCS1 and A20, signaling inhibitors that limit IL-13 and IL-5 production. These findings establish miRNAs as important regulators of ILC2 biology, reveal overlapping but nonidentical miRNA-regulated gene expression networks in ILC2s and T2 cells, and reinforce the therapeutic potential of targeting miR-19 to alleviate pathogenic allergic responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20170545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716040PMC
December 2017

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

Nature 2017 09 30;549(7670):111-115. Epub 2017 Aug 30.

Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature23875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675716PMC
September 2017

A Distinct Inhibitory Function for miR-18a in Th17 Cell Differentiation.

J Immunol 2017 07 12;199(2):559-569. Epub 2017 Jun 12.

Institute for Immunology, Biomedical Center Munich, Ludwig Maximilians University, 82152 Planegg-Martinsried, Germany;

Th17 cell responses orchestrate immunity against extracellular pathogens but also underlie autoimmune disease pathogenesis. In this study, we uncovered a distinct and critical role for miR-18a in limiting Th17 cell differentiation. miR-18a was the most dynamically upregulated microRNA of the miR-17-92 cluster in activated T cells. miR-18a deficiency enhanced CCR6 RAR-related orphan receptor (ROR)γt Th17 cell differentiation in vitro and increased the number of tissue Th17 cells expressing CCR6, RORγt, and IL-17A in airway inflammation models in vivo. Sequence-specific miR-18 inhibitors increased CCR6 and RORγt expression in mouse and human CD4 T cells, revealing functional conservation. miR-18a directly targeted , , and , all key transcription factors in the Th17 cell gene-expression program. These findings indicate that activating signals influence the outcome of Th cell differentiation via differential regulation of mature microRNAs within a common cluster.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1700170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508756PMC
July 2017

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation.

J Vis Exp 2017 04 13(122). Epub 2017 Apr 13.

Department of Microbiology & Immunology, Sandler Asthma Basic Research Center, University of California, San Francisco;

CD4 T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4 T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/55546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564694PMC
April 2017

Alternative splicing of interleukin-33 and type 2 inflammation in asthma.

Proc Natl Acad Sci U S A 2016 08 18;113(31):8765-70. Epub 2016 Jul 18.

Department of Pulmonary and Critical Care Medicine, University of California, San Francisco, CA 94143; Cardiovascular Research Institute, University of California, San Francisco, CA 94143; Sandler Asthma Basic Research Center, University of California, San Francisco, CA 94143;

Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an "alarmin" during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1601914113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978244PMC
August 2016

Quantitative systematic review of the effects of non-pharmacological interventions on reducing apathy in persons with dementia.

J Adv Nurs 2016 Nov 23;72(11):2612-2628. Epub 2016 Jun 23.

Wayne State University College of Nursing, Detroit, Michigan, USA.

Aim: To review the quantitative evidence concerning the effects of non-pharmacological interventions on reducing apathy in persons with dementia.

Background: Apathy, a prevalent behavioural symptom among persons with Alzheimer Disease, is defined as a disorder of motivation with deficits in behavioural, emotional and cognitive domains and is associated with serious social and physical obstacles. Non-pharmacological interventions show promise as symptom control modalities among persons with dementia.

Design: Quantitative systematic review.

Data Sources: CINAHL, PubMed, PSYCHinfo and Cochrane Trials databases were searched for published English language research inclusive through December 2014, with no early year limiters set.

Review Methods: Comprehensive searches yielded 16 international randomized controlled trials or quasi-experimental studies based on inclusion criteria and a rigorous quality appraisal process.

Results: A narrative summary analysis revealed that non-pharmacological interventions for apathy varied substantially and lacked specificity, conceptual clarity and were methodologically heterogeneous. Select interventions demonstrated effectiveness, but lacked systematic long-term follow-up. Limitations include publication bias and lack of a meta-analytic approach due to the methodological heterogeneity of included studies.

Conclusion: Study results demonstrate promise for the use of non-pharmacological interventions, particularly music-based interventions, in reducing apathy levels in individuals with dementia. Intervening to reduce apathy may have a positive clinical impact and healthcare providers should be encouraged to incorporate positive sources of interest and intellectual stimulation into care. However, future research is needed to examine the aetiologic mechanism and predictors of apathy, to improve evidence-based interventions and specificity and to optimize dosage and timing of non-pharmacological interventions across the disease trajectory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jan.13026DOI Listing
November 2016

MicroRNAs 24 and 27 Suppress Allergic Inflammation and Target a Network of Regulators of T Helper 2 Cell-Associated Cytokine Production.

Immunity 2016 Apr 2;44(4):821-32. Epub 2016 Feb 2.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Sandler Asthma Basic Research Center, University of California, San Francisco, San Francisco CA, USA. Electronic address:

MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2016.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838571PMC
April 2016

IFN-γ-Producing T-Helper 17.1 Cells Are Increased in Sarcoidosis and Are More Prevalent than T-Helper Type 1 Cells.

Am J Respir Crit Care Med 2016 06;193(11):1281-91

1 Division of Pulmonary and Critical Care, Department of Medicine.

Rationale: Pulmonary sarcoidosis is classically defined by T-helper (Th) cell type 1 inflammation (e.g., IFN-γ production by CD4(+) effector T cells). Recently, IL-17A-secreting cells have been found in lung lavage, invoking Th17 immunity in sarcoidosis. Studies also identified IL-17A-secreting cells that expressed IFN-γ, but their abundance as a percentage of total CD4(+) cells was either low or undetermined.

Objectives: Based on evidence that Th17 cells can be polarized to Th17.1 cells to produce only IFN-γ, our goal was to determine whether Th17.1 cells are a prominent source of IFN-γ in sarcoidosis.

Methods: We developed a single-cell approach to define and isolate major Th-cell subsets using combinations of chemokine receptors and fluorescence-activated cell sorting. We subsequently confirmed the accuracy of subset enrichment by measuring cytokine production.

Measurements And Main Results: Discrimination between Th17 and Th17.1 cells revealed very high percentages of Th17.1 cells in lung lavage in sarcoidosis compared with controls in two separate cohorts. No differences in Th17 or Th1 lavage cells were found compared with controls. Lung lavage Th17.1-cell percentages were also higher than Th1-cell percentages, and approximately 60% of Th17.1-enriched cells produced only IFN-γ.

Conclusions: Combined use of surface markers and functional assays to study CD4(+) T cells in sarcoidosis revealed a marked expansion of Th17.1 cells that only produce IFN-γ. These results suggest that Th17.1 cells could be misclassified as Th1 cells and may be the predominant producer of IFN-γ in pulmonary sarcoidosis, challenging the Th1 paradigm of pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1164/rccm.201507-1499OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910899PMC
June 2016