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

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Microglial Upregulation of Progranulin as a Marker of Motor Neuron Degeneration

doi: 10.1097/nen.0b013e3181fc9aea

Figure Lengend Snippet: FIGURE 1. Progranulin (PGRN) mRNA and protein levels increase with motor neuron degeneration in mutant SOD1G93A mice. (A) Spinal cord PGRN mRNA levels measured by quantitative PCR and normalized to presymptomatic stage and A-actin levels in nontransgenic (NTG) and mutant SOD1G93A mice (analysis of variance, F value = 6.5; overall, p = 0.002). (B) Spinal cord PGRN protein levels measured by ELISA normalized to protein content (analysis of variance, F value = 4.9; overall, p = 0.002).

Article Snippet: Cryosections (10 Km) were stained with a goat polyclonal human PGRN antibody (R&D Systems) and a mouse monoclonal CD68 antibody (KP1; Biogenex, San Ramon, CA).

Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Microglial Upregulation of Progranulin as a Marker of Motor Neuron Degeneration

doi: 10.1097/nen.0b013e3181fc9aea

Figure Lengend Snippet: FIGURE 2. Proganulin (PGRN) expression in the mouse spinal cord. (AYC) PGRN+ motor neurons in the ventral horn of the spinal cord. Progranulin staining (A), SMI32 staining (B), and overlay (C) of ventral part of spinal cord from nontransgenic mouse; inset shows detail of motor neuron. (DYL) Proganulin staining of the ventral part of the spinal cord in presymptomatic, early, and late symptomatic mutant SOD1G93A mice (in red, D, G, J), CD11b staining (in green, E, H, K), and overlay (F, I, L); 2 motor neurons are marked by white arrows in D. (DYL) There is a gradual increase in the number of PGRN+ microglia in the spinal cord of mutant SOD1G93A mice. (MYO) At the end stage, surviving cells with motor neuron morphology (CD11b-negative, white arrows) remained PGRN+ in mutant SOD1G93A mice. Scale bar = 50 Km.

Article Snippet: Cryosections (10 Km) were stained with a goat polyclonal human PGRN antibody (R&D Systems) and a mouse monoclonal CD68 antibody (KP1; Biogenex, San Ramon, CA).

Techniques: Expressing, Staining, Mutagenesis

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Microglial Upregulation of Progranulin as a Marker of Motor Neuron Degeneration

doi: 10.1097/nen.0b013e3181fc9aea

Figure Lengend Snippet: FIGURE 4. Progranulin (PGRN) mRNA and protein levels increase with motor neuron degeneration in VEGFC/C and mutant tauP301L

Article Snippet: Cryosections (10 Km) were stained with a goat polyclonal human PGRN antibody (R&D Systems) and a mouse monoclonal CD68 antibody (KP1; Biogenex, San Ramon, CA).

Techniques: Mutagenesis

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Microglial Upregulation of Progranulin as a Marker of Motor Neuron Degeneration

doi: 10.1097/nen.0b013e3181fc9aea

Figure Lengend Snippet: FIGURE 5. Motor neurons and reactive microglia express progranulin (PGRN) in the human spinal cord. (AYI) Progranulin staining (red, A, D, I) and CD68 staining (green, B, E, I) of control (AYC) and amyotrophic lateral sclerosis (ALS) (DYF, I) spinal cords. Surviving cells with motor neuron morphology at a higher magnification in the ventral horn of ALS spinal cord on H&E staining remained positive for PGRN (G and H). Fluorescent staining of a CD68-negative PGRN+ cell with motor neuron morphology, surrounded by 2 microglial cells (white arrows) is shown in panel I. Scale bar = 50 Km.

Article Snippet: Cryosections (10 Km) were stained with a goat polyclonal human PGRN antibody (R&D Systems) and a mouse monoclonal CD68 antibody (KP1; Biogenex, San Ramon, CA).

Techniques: Staining, Control

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Microglial Upregulation of Progranulin as a Marker of Motor Neuron Degeneration

doi: 10.1097/nen.0b013e3181fc9aea

Figure Lengend Snippet: FIGURE 6. Increased progranulin (PGRN) in CSF with disease progression in mutant SOD1G93A mice. Cerebrospinal fluid PGRN levels measured by ELISA in nontransgenic and mutant SOD1G93A mice (analysis of variance, F value = 15.8; overall, p = 0.000001; ***, p G 0.0001 compared with other groups).

Article Snippet: Cryosections (10 Km) were stained with a goat polyclonal human PGRN antibody (R&D Systems) and a mouse monoclonal CD68 antibody (KP1; Biogenex, San Ramon, CA).

Techniques: Biomarker Discovery, Mutagenesis, Enzyme-linked Immunosorbent Assay