Journal: Cell systems

Article Title: Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor Alpha Bound Enhancers

doi: 10.1016/j.cels.2017.08.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DATA AND SOFTWARE AVAILABILITY Software: See Key Resources Table. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies H3K27ac rabbit polyclonal Active Motif Cat# 39133; RRID:AB_2561016 H3K9me3 rabbit polyclonal Abcam Cat# ab8898; RRID:AB_306848 FLAG mouse monoclonal Sigma-Aldrich Cat# F1804; RRID:AB_262044 RNA pol II 4H8 mouse monoclonal Abcam Cat# ab5408; RRID:AB_304868 ER alpha rabbit polyclonal Santa Cruz Cat#sc-544; RRID:AB_631469 Bacterial and Virus Strains Biological Samples Chemicals, Peptides, and Recombinant Proteins β-estradiol Sigma-Aldrich Cat# E2758 Trichostatin A Cayman Chemicals Cat# 89730; CAS 58880-19-6 Critical Commercial Assays ZR 96-well Quick-RNA Zymo Research Cat# R1053 KAPA Stranded mRNA-seq KAPA Biosystems Cat# KK8420 Power SYBR® Green RNA-to-CT™ 1-Step Applied Biosystems Cat# 4389986 Deposited Data Raw and analyzed ChIP-seq data This study {"type":"entrez-geo","attrs":{"text":"GSE99906","term_id":"99906"}} GSE99906 Raw and analyzed RNA-seq data This study {"type":"entrez-geo","attrs":{"text":"GSE99906","term_id":"99906"}} GSE99906 Experimental Models: Cell Lines Human: Ishikawa cells ECACC ECACC Cat# 99040201, RRID:CVCL_2529 Experimental Models: Organisms/Strains Ishikawa SID4x-dCas9-KRAB This study Oligonucleotides gBlocks, see Table S1 Primers for constructing and verifying dCas9 fusions and guide RNAs, see Table S2 Guide RNA targets, see Table S3 qPCR primers, see Table S4 Primers for sequencing ERBS deletions, see Table S5 Recombinant DNA dCas9-VP160 Cheng et al., 2013 Addgene 48227 pGL3-U6-PGK-Puro Shen et al., 2014 Addgene 51133 gRNA_cloningVector Mali et al., 2013 Addgene 41824 Software and Algorithms Bowtie Langmead et al., 2009 DOI: 10.1186/gb-2009-10-3-r25 MACS2 Zhang et al., 2008 DOI: 10.1186/gb-2008-9-9-r137 DESeq2 Love et al., 2014 DOI: 10.1186/s13059-014-0550-8 HISAT Kim et al., 2015 DOI: 10.1038/nmeth.3317 HTSeq Anders et al., 2015 DOI: 10.1093/bioinformatics/btu638 Bedtools Quinlan and Hall, 2010 DOI: 10.1093/bioinformatics/btq033 Prism 6 http://www.graphpad.com/scientific-software/prism/ Other Open in a separate window KEY RESOURCES TABLE Data produced in this study are available from GEO ( {"type":"entrez-geo","attrs":{"text":"GSE99906","term_id":"99906"}} GSE99906 ).

Techniques: Recombinant, SYBR Green Assay, Sequencing, Software

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Intra-tracheal infection with the USA 300 strain of MRSA induced a significant increase in pulmonary Il21 mRNA ( A ) and IL-21 protein ( B ) 24 hr after infection. ( C ) PBS or 2 μg of IL-21 was administered i.t. to WT mice one day prior to MRSA infection, and lung MRSA CFU were quantitated 7 and 24 hr later. ( D, E ) Lung immunopathology was assessed in H and E-stained sections of lung tissue from naïve uninfected mice and mice pre-treated with PBS or IL-21 and then infected for 7 or 24 hr with MRSA (in upper left panel, the bar = 500 μm and inset bar = 10 μm) ( D ), and pathology ( E ) scores were assessed. ( F–K ) animals were infected with MRSA as above. ( F ) Total lung neutrophil cellularity was quantitated by flow cytometry after staining with Ly6G and CD11b. ( G ) RNA-Seq analysis was performed on total lung tissue mRNA (pools of 5 animals) isolated 7 or 24 hr after treatment of WT mice with PBS or IL-21. Boxed regions include genes mentioned in the text. ( H–K ) RT-PCR was used to assess expression of Gzma ( H ) and Gzmb ( I ) mRNA in lungs treated with IL-21 for 7 hr, and ELISA was used to assess the induction of granzyme B ( J ) and IFNγ protein ( K ) in corresponding bronchoalveolar lavage fluid at 7 hr. Data are representative of either three ( A, B, C, F, H–K ) or two ( D, E, G ) independent experiments and validation of RNA-Seq was performed by RT-PCR of mRNA from additional mice.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, RNA Sequencing, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A–B ) Total cellularity of lung was assessed in BAL fluid ( A ) and lung ( B ) 24 hr after IL-21 intratracheal instillation. ( C ) Percentage of neutrophils was assessed by flow cytometry at 24 hr after IL-21 treatment. ( D–E ) Levels of chemokines were measured in BAL fluid at 24 hr after IL-21 treatment.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Bone-marrow neutrophils were purified by negative selection using a neutrophil isolation kit (Miltenyi) and either not stimulated or stimulated in vitro with IL-21 (100 ng/ml) for 24 hr in the absence (control) or presence of peptidoglycan (PG) (2 or 5 μg/ml); IL-21R expression on gated Ly6G + CD11b + neutrophils was detected by flow cytometry (blue = isotype control; red = anti-IL-21R). ( B, C ) IL-21 lowers the MRSA CFU in a neutrophil-dependent fashion. Mice were treated i.p. with an isotype control mAb or neutrophil-depleted with anti-Ly6G (1A8 mAb), and the efficacy of neutrophil depletion is shown in BAL and lung ( B ). Mice were then treated with PBS or IL-21 i.t., infected i.t. with MRSA, and CFU quantitated at 24 hr ( C ). CFU quantitation was performed on the left single lobe, whereas immune populations and RNA were determined using the right lobes. ( D, E ) Levels of Gzmb and Gzma mRNAs in lung tissue of untreated or neutrophil-depleted mice (treated as in panel C ) were quantitated by RT-PCR. ( F ) Levels of Gzma and Gzmb mRNAs in purified cell-sorted lung neutrophils (>98% pure Ly6G + CD11b + ) from either untreated or IL-21-treated WT or Il21r KO mice were measured by RT-PCR and normalized to Rpl7 expression. ( G, H ) Lung neutrophils were elicited by i.t treatment with heat-killed S. aureus 24 hr prior to isolation, purified, stimulated in vitro for 4 hr with either PBS or IL-21 (100 ng/ml), and Gzma ( G ) and Gzmb ( H ) mRNAs assayed by RT-PCR and normalized to Rpl7 expression. ( I ) Purified HKSA-elicited lung neutrophils were incubated in vitro with MRSA for 3 hr in the presence of PBS or IL-21 either without or with the granzyme B inhibitor, Z-AAD-CMK. MRSA CFU was quantitated by plating serial dilutions on blood agar plates. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Isolation, In Vitro, Control, Expressing, Flow Cytometry, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Quantitation of IL-21R MFI in neutrophils treated with or without IL-21 in the presence or absence of peptidoglycan (PG). ( B ) Lung neutrophils from HKSA-stimulated mice were purified via a magnetic neutrophil purification kit (Miltenyi). Shown is CD11b versus Ly6G staining.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Quantitation Assay, Purification, Staining

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Human peripheral blood neutrophils were purified by a neutrophil negative selection kit (StemCell Technologies). Shown is CD56 versus CD66b and CD8 versus CD66b staining. ( B ) Western blotting of granzyme B from neutrophils, PBMC, or A549 cells containing 0, 1, or 5% PBMC extracts, confirming that the low level of contamination of purified neutrophils cannot account for the granzyme B expression observed in this population. Also shown is western blotting for actin as a loading control.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Staining, Western Blot, Expressing, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Purified peripheral blood neutrophils were stimulated with PBS (control) or IL-21 (100 ng/ml) for 4 hr, and IL21R mRNA was measured by real-time PCR and normalized to RPL7 expression ( A ), and IL-21R protein levels were measured by flow cytometry ( B ); left panel shows isotype control shaded and anti-IL21R black line, with a summary in the right panel). ( C ) RNA-Seq was performed on neutrophils after 4 or 24 hr incubation with PBS or IL-21 in the absence or presence of heat-killed S. aureus (10 6 /ml). We used HKSA rather than live bacteria in order to allow analysis at 24 hr, as live bacteria would have overgrown the system by then. Genes differentially expressed (fold-change >2.0) are shown. Shown is a representative RNA-Seq analysis. ( D – G ) Human peripheral blood neutrophils were stimulated for 4 hr in vitro in the presence or absence of IL-21 and GZMA ( D ), GZMB ( E ), GNLY ( F ), and IFNG ( G ) mRNA levels were quantitated by RT-PCR and normalized to RPL7 expression. ( H ) Purified human neutrophils were stimulated with IL-21 for 4 hr, fixed, permealized, and stained for intracellular granzyme B protein (gated on CD66b + cells); MFIs are summarized in right panel. ( I ) Granzyme B protein was measured by ELISA in supernatants from human peripheral blood neutrophils cultured for 24 hr in either the absence or presence of IL-21 (100 ng/ml). ( J, K ) Human neutrophils were incubated in vitro with either MRSA ( J ) or S. pyogenes ( K ) for 3 hr with PBS or IL-21 (100 ng/ml). MRSA and S. pyogenes CFU were quantitated by plating serial dilutions on blood agar plates. Results shown are representative of 3 independent experiments, except panel C shows one of two similar independent RNA-Seq experiments, each from a different donor.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Control, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, RNA Sequencing, Incubation, Bacteria, In Vitro, Reverse Transcription Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: Reactive oxygen species were measured by flow cytometry in CellRox Red loaded peripheral blood neutrophils that were stimulated at 37°C for 30 min with PBS, IL-21, HKSA, or HKSA +IL-21.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT and Il21r KO mice were infected intratracheally with MRSA (2 × 10 7 ) and CFU in the lung were quantitated at 4 and 24 hr post-infection. ( B ) WT and Il21r KO mice were infected in parallel with WT mice that had been treated with PBS or 2 μg IL-21, and CFU in the lung were quantitated at 7 and 24 hr post infection. ( C–E ) Lung immunopathology was assessed in H and E-stained lung sections (bar in upper left panel = 500 μm; bar in inset = 10 μm) ( C ); lung neutrophil cellularity as assessed by pathology score ( D ) and flow cytometry ( E ) were assessed at 4 and 24 hr post-infection. ( F ) WT or Il21r KO mice were pre-treated with an isotype control antibody or anti-Ly6G to deplete neutrophils, infected with MRSA, and CFU quantitated at 24 hr. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT mice were treated intratracheally with 50 μg of control Fc (from IgG1) or IL-21R-Fc protein for 2 days prior to infection with MRSA and lung CFU were quantitated at 4 and 24 hr post-infection with MRSA. ( B ) Histology of lungs in control Fc or IL-21R-Fc pre-treated mice at 4 and 24 hr post-infection (bar in upper left panel = 500 μm; bar in inset = 10 μm). ( C ) IFNα levels in BAL fluid were measured by ELISA 4 hr after infection in mice pretreated with control Fc or IL-21R-Fc. ( D ) Gzmb mRNA was measured in lungs of WT or Il21r KO mice pre-treated with isotype control or anti-IFNAR1 antibodies prior to MRSA infection, and normalized to Rpl7 mRNA expression. ( E ) Gzmb mRNA was measured in lungs of mice treated with either control Fc or IL-21R-Fc prior to MRSA infection and normalized to Rpl7 expression. ( F ) Like IL-21, IFNβ also induced GZMB mRNA in human peripheral blood neutrophils and normalized to RPL7 expression. ( G ) IFNβ induces increased in vitro killing of MRSA by human peripheral blood neutrophils and this was prevented by a granzyme B inhibitor, Z-AAD-CMK. ( H–J ) Co-cultures of CD4 + T cells and dendritic cells were stimulated with SEB either in the presence of control Fc or IL-21R-Fc, and mRNA was quantitated by RT-PCR after 24 hr and normalized to Rpl7 expression. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: MRSA infection, as detailed in the legend to . ( A, B ) Pathology and neutrophil infiltration scores were assessed from H and E-stained lung sections shown in . ( C ) RNA-Seq was performed on total lung mRNA either before infection (Ctrl) or at 4 hr after MRSA infection of mice pretreated with either Fc protein or an IL-21R-Fc fusion protein. Representative of 2 independent RNA-Seq experiments.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, RNA Sequencing

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Peripheral blood neutrophils from normal donors (NDs) or AD-HIES patients (PTs) were assayed using an in vitro MRSA killing assay in the absence or presence of IL-21 (100 ng/ml). ( B ) RT-PCR was used to quantitate GZMB mRNA in neutrophils 4 hr after in vitro stimulation without or with IL-21. Expression was normalized to RPL7 expression ( C ) RNA-Seq was performed on normal donor and AD-HIES patient neutrophils stimulated for 4 hr with PBS, IL-21, IFNβ, or IL-21 +IFNβ. Genes differentially expressed (fold-change >1.5) in two independent RNA-Seq analyses are shown. ( D ) RT-PCR normalized to RPL7 expression was used to validate the expression pattern of GBP1 and GBP2 in neutrophils from additional normal donors and AD-HIES patients. ( E–G ) Mutant Stat3 transgenic mice were treated i.t. with PBS or 2 μg IL-21, infected i.t. 24 hr later with MRSA, and at 7 hr post-infection lung MRSA CFU quantitated ( E ), and IFNα levels were measured in the serum ( F ) and BAL fluid ( G ).

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Mutagenesis, Transgenic Assay, Infection

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) In wild-type (WT) mice, MRSA produces SEB, which bridges between T cells and dendritic cells by interacting with TCR and MHCII, leading to the production of IL-21 by CD4 + T cells. IL-21 stimulates neutrophils to release granzymes, with enhanced killing of MRSA. Type I IFN (e.g., produced by dendritic cells, as shown in the cartoon) can also induce granzyme production to promote killing of MRSA, but IL-21 inhibits dendritic cell production of type I IFN. ( B ) In the absence of either IL-21R or a functional STAT3 signaling response, the IL-21-induced neutrophil granzyme production does not occur. However, IL-21-mediated repression of type I IFN production by dendritic cells also no longer occurs, which leads to enhanced production of type I IFN and hence increased killing of MRSA through this pathway. The functional cross-talk of and relative potency of the IL-21- and type 1 IFN-mediated pathways and levels of each cytokine influence the outcome.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Produced, Functional Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet:

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vivo, Flow Cytometry, Blocking Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Isolation