IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus
- National Heart, Lung, and Blood Institute, National Institutes of Health, United States
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
- National Heart, Lung and Blood Institute, National Institutes of Health, United States
- Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
Figures
Figure 1 with 2 supplements
IL-21 enhances killing of pulmonary MRSA in WT mice.
(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 …
Figure 1—figure supplement 1
IL-21 is expressed in the lung before and after MRSA infection.
(A–C) IL-21-mCherry transgenic reporter mice were either not infected or infected with MRSA for 24 hr. Lung cells were analyzed by flow cytometry for the percentage of Il21-mCherry+ CD4+ T cells (A, …
Figure 1—figure supplement 2
Effects of IL-21 on naïve lung.
(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 …
Figure 2 with 1 supplement
IL-21-mediated granzyme production and MRSA killing by lung neutrophils.
(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 …
Figure 2—figure supplement 1
Purity of neutrophil preparations.
(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 …
Figure 3 with 2 supplements
Human neutrophils express IL-21R, and IL-21 induces a program that leads to granzyme B-dependent neutrophil-mediated killing of MRSA.
(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 …
Figure 3—figure supplement 1
Purity of human neutrophils.
(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 …
Figure 3—figure supplement 2
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.
https://doi.org/10.7554/eLife.45501.009
Figure 4
Pulmonary MRSA infection is cleared more efficiently in Il21r KO than in WT mice and this requires neutrophils.
(A) WT and Il21r KO mice were infected intratracheally with MRSA (2 × 107) 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 …
Figure 5
RNA-Seq analysis revealed an enhanced IFN profile in Il21r KO lungs.
(A) Venn diagram showing the number of differentially expressed genes (1474) in WT vs Il21r KO lungs, with 664 genes downregulated and 810 genes upregulated in the Il21r KO. (B) Heat map showing a …
Figure 6 with 1 supplement
Blocking IL-21 signaling with IL-21R-Fc in WT mice phenocopies the enhanced MRSA killing seen in Il21r KO mice.
(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 …
Figure 6—figure supplement 1
Pathology and RNA-Seq analysis of lungs from mice treated with control Fc or IL-21R-Fc prior to i.t.
MRSA infection, as detailed in the legend to Figure 6A. (A, B) Pathology and neutrophil infiltration scores were assessed from H and E-stained lung sections shown in Figure 6B. (C) RNA-Seq was …
Figure 7
Neutrophils from patients with autosomal dominant hyper-IgE syndrome (AD-HIES) display reduced ex vivo IL-21-induced MRSA cytotoxic function.
(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 …
Figure 8
A model for the functional interplay of IL-21 with type I interferons in the response to MRSA.
(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 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. aureus) | FPR3757 | ATCC | ATCC BAA-1556 | |
| Strain, strain background (Streptococcus pyogenes) | NZ131 | ATCC | ATCC-BAA-1633 | |
| Genetic reagent (M. musculus) | Il21r-/- | Ozaki et al. Science 298:1630, 2002 | ||
| Genetic reagent (M. musculus) | mCherry Il21 | Wang et al. P.N.A.S. 108:9542, 2011 | ||
| Genetic reagent (M. musculus) | C57BL/6-Tg(Stat3*)9199Alau/J | Jackson Laboratory | Stock # 027952 | |
| Biological sample (S. aureus) | Staph enterotoxin B | List Biological Laboratories | #122 | |
| Biological sample (S. aureus) | Heat-killed S. aureus | In Vivo Gen | tlrl-hksa | |
| Antibody | Mouse monoclonal anti-granzyme B | Biolegend | GB11 RRID:AB_2562195 | flow cytometry 1:100 |
| Antibody | Mouse monoclonal anti-granzyme B | Biolegend | clone M3304B06 RRID:AB_2565266 | WB (1:500) |
| Antibody | Mouse monoclonal anti-Ly6G (1A8) | BioX Cell | RRID: AB_1107721 | Blocking Ab |
| Antibody | Mouse monoclonal anti-IFNAR (MAR1-5A3) | BioX Cell | RRID: AB_2687723 | Blocking Ab |
| Antibody | Mouse monoclonal MOPC-21 | BioX Cell | RRID: AB_1107784 | Blocking Ab |
| Antibody | Rat monoclonal 2A3 | BioX Cell | RRID: AB_1107769 | Blocking Ab |
| Antibody | Mouse monoclonal anti-human IL21 | BD Biosciences | clone 3A3-N2.1 RRID:AB_2738933 | flow cytometry 1:100 |
| Antibody | Rat monoclonal anti-mouse IL-21R | BD Biosciences | clone 4A9 | flow cytometry 1:100 |
| Antibody | Mouse monoclonal anti-human IL-21R | Biolegend | clone 2G1-K12 RRID:AB_2123988 | flow cytometry 1:100 |
| Antibody | Mouse monoclonal anti-human CD66b | Biolegend | clone G10F5 RRID:AB_2563294 | flow cytometry 1:100 |
| Antibody | Mouse monoclonal anti-CD11b | Biolegend | clone M1/70 RRID:AB_312789 | flow cytometry 1:100 |
| Peptide, recombinant protein | Recombinant mouse IL-21 | R and D Systems | 594 ML-100/CF | |
| Peptide, recombinant protein | Recombinant human IL-21 | R and D Systems | 8879-IL-050/CF | |
| Peptide, recombinant protein | Recombinant human GM-CSF | R and D Systems | 215 GM | |
| Peptide, recombinant protein | Recombinant human IL-4 | R and D Systems | 204-IL | |
| Peptide, recombinant protein | Z-AAD-CMK | Enzo Life Sciences | BML-P165 | |
| Commercial assay or kit | IL-21 ELISA | R and D Systems | DY594 | |
| Commercial assay or kit | Granzyme B ELISA | Ebioscience | BMS2027 RRID:AB_2575322 | |
| Commercial assay or kit | IFNalpha ELISA | Ebioscience | 50-246-672 | |
| Commercial assay or kit | Cell Rox Deep Red staining kit | InVitrogen/Molecular Probes | C10491 | |
| Commercial assay or kit | Mouse neutrophil isolation kit | Miltenyi | 130-097-658 | |
| Commercial assay or kit | Human neutrophil isolation kit | Stem Cell Technologies | 19666 | |
| Commercial assay or kit | KAPA stranded mRNA-Seq library kit | Kapa Biosystems | KK8580 | |
| Chemical compound, drug | Peptidoglycan from S. aureus | Sigma | #77140 |
Additional files
-
Supplementary file 1
RNA-Seq analysis was performed on lung mRNA isolated 7 or 24 hr after treatment of WT mice with PBS or IL-21.
- https://doi.org/10.7554/eLife.45501.016
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Supplementary file 2
RNA-Seq was performed on highly purified human neutrophils incubated for 4 or 24 hr with PBS or IL-21 in the absence or presence of heat-killed S. aureus.
- https://doi.org/10.7554/eLife.45501.017
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Supplementary file 3
RNA-Seq analysis from WT and Il21r KO mouse lungs 4 and 24 hr after MRSA infection.
- https://doi.org/10.7554/eLife.45501.018
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Supplementary file 4
RNA-Seq was performed on total lung mRNA either before MRSA infection or 4 hr after infection of mice that were pre-treated with either control Fc protein or IL-21R-Fc fusion protein.
- https://doi.org/10.7554/eLife.45501.019
-
Supplementary file 5
RNA-Seq analysis of neutrophils from normal human donors and AD-HIES patients; cells were either not treated or treated for 4 hr with IL-21, IFNβ, or IL-21 +IFNβ.
- https://doi.org/10.7554/eLife.45501.020
-
Transparent reporting form
- https://doi.org/10.7554/eLife.45501.021
