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Activation of neutrophils by autocrine IL-17A–IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2

Nature Immunology volume 15, pages 143–151 (2014)Cite this article

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Abstract

Here we identified a population of bone marrow neutrophils that constitutively expressed the transcription factor RORγt and produced and responded to interleukin 17A (IL-17A (IL-17)). IL-6, IL-23 and RORγt, but not T cells or natural killer (NK) cells, were required for IL-17 production in neutrophils. IL-6 and IL-23 induced expression of the receptors IL-17RC and dectin-2 on neutrophils, and IL-17RC expression was augmented by activation of dectin-2. Autocrine activity of IL-17A and its receptor induced the production of reactive oxygen species (ROS), and increased fungal killing in vitro and in a model of Aspergillus-induced keratitis. Human neutrophils also expressed RORγt and induced the expression of IL-17A, IL-17RC and dectin-2 following stimulation with IL-6 and IL-23. Our findings identify a population of human and mouse neutrophils with autocrine IL-17 activity that probably contribute to the etiology of microbial and inflammatory diseases.

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Figure 1: Induction of IL-17A-producing neutrophils in vivo.
Figure 2: RORγt-dependent expression of IL-17 by neutrophils.
Figure 3: Translocation of RORγt to the nucleus of neutrophils stimulated with mouse IL-6 and IL-23.
Figure 4: Expression of RORγt and IL-17A in human peripheral blood neutrophils.
Figure 5: Expression of IL-17RA, IL-17RC and dectin-2 in mouse and human neutrophils.
Figure 6: The role of IL-17A and IL-17RC in the production of ROS by neutrophils and hyphal growth in vitro.
Figure 7: The role of IL-17, IL-17RC and NADPH oxidase from neutrophils in regulating hyphal growth of RFP-expressing A. fumigatus in vivo.

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  • 29 January 2014

    In the version of this article initially published, the far right labels in Figure 2d,e were truncated. The correct label for each is 'RorcGFP/GFP'. Also, a sentence was missing from the Flow Cytometry subsection of the Online Methods section. That subsection should begin as follows: "Neutrophils were incubated for 2 h at 37 °C and 5% CO2 with a brefeldin–monensin 'cocktail' (Protein Transport Inhibitor Cocktail; eBioscience) during in vitro stimulation. Total bone marrow cells...." The errors have been corrected for the HTML and PDF versions of this article.

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Acknowledgements

We thank W. Ouyang (Genentech) for Il17rc+/− and Il17rc −/− mice (under a material-transfer agreement with X. Li.); Y. Iwakura (University of Tokyo) for Il17a−/−, dectin-1-deficient (Clec7a−/−) and dectin-2 deficient (Clec4n−/−) mice (under a material-transfer agreement with E.P.); and M.-J. Chang and G. Yakubenko for technical assistance. Supported by the US National Institutes of Health (RO1 EY018612 and P30 EY011373 to E.P., F32 EY022278 to P.R.T., F31 EY019841 to S.M.L., R01GM082916 to B.A.C., and R01NS071996 to X.L.), the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.

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Author notes

  1. Sanhita Roy and Sixto M Leal: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, USA

    Patricia R Taylor, Sanhita Roy, Sixto M Leal Jr, Yan Sun, Scott J Howell & Eric Pearlman

  2. Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

    Brian A Cobb

  3. Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA

    Xiaoxia Li

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Contributions

P.R.T., experimental design, experiments, data analysis and manuscript preparation; S.R., S.M.L., Y.S. and S.J.H., experiments and data analysis; B.A.C., reagents; X.L., reagents, experimental design and manuscript preparation; and E.P., experimental design, data analysis and manuscript preparation.

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Integrated supplementary information

Supplementary Figure 1 Induction of murine IL-17A producing neutrophils.

(a) C57BL/6 and Rag2−/−Il2rg−/− mice were injected subcutaneously with heat-killed, swollen Aspergillus fumigatus conidia. Three days after injection (primed), splenocytes were incubated with anti-CD3, NK1.1, or NIMP-R14, permeabilized and incubated with anti-IL-17A, and examined by flow cytometry. As a positive control for IL-17 producing CD3+ cells, C57BL/6 mice were given a second injection of conidia 7 days later, and splenocytes were examined on day 10 (immunized). Scatter plots are representative of four mice per group and two repeat experiments. (b) Bone marrow neutrophils were isolated from naïve C57BL/6 mice by density centrifugation (Percoll) and incubated with: Ly6G antibodies NIMPR14 and 1A8, +/- CD11b, or with antibodies to CD4, CD3, B220, F4/80, γδ TCR, and NK1.1. Data are representative of neutrophils from four separate mice and two repeat experiments. (c) Bone marrow neutrophils were isolated from Il17a–GFP mice three days after subcutaneous injection of heat-killed, swollen A. fumigatus conidia (Primed). Cells were incubated with NIMP-R14, and co-expression with GFP (Merge) was detected by quantitative image analysis and flow cytometry. Representative cells from 4 naïve and 4 primed mice are shown. This experiment was repeated twice with similar results. (d, e) NIMP-R14+ cells were isolated from bone marrow (d) or spleen (e) of primed and naïve C57BL/6 mice by cell sorting using a FACSAriaIII (BD Biosciences), yielding > 98% pure population of NIMP-R14+ neutrophils. Il17a gene expression in purified neutrophil populations was examined by quantitative PCR, and q-PCR products were qualitatively examined on 1% agarose gel. (N: naïve). (a–e) representative data from 3 repeat experiments.

Supplementary Figure 2 Rorc, IL-6R, IL-23R, and IL-17A expression in bone marrow neutrophils.

(a) Total bone marrow cells from naïve Rorc+/GFP mice were incubated with PE conjugated NIMP-R14, and co-localization of GFP (Rorc) and PE was detected by quantitative image analysis and flow cytometry. Images are representative of three Rorc+/GFP mice and two repeat experiments. (b) Constitutive Rorc gene expression in bone marrow neutrophils isolated from naïve C57BL/6 mice and incubated 1h with recombinant murine IL-6 (20μg/ml) and IL-23 (2μg/ml) (IL-6+IL-23). PCR products were qualitatively analyzed on 1% agarose gel using Actb (β-actin) as a loading control. Cycling threshold scores are shown in Supplementary Table 1. (c) Co-expression of GFP and IL-6 and IL-23 receptors in bone marrow neutrophils from naïve Rorc+/GFP mice. (d) Constitutive IL-23R and IL-12Rβ gene expression in neutrophils isolated from naïve C57BL/6 mice. (e, f) Il17a gene expression in bone marrow neutrophils from C57BL/6 mice incubated 1h with recombinant mouse (rm)IL-6 and/or rmIL-23 at indicated concentrations per ml, and analyzed by quantitative PCR (e), or (f) incubated 15, 30 and 60 min with 20μg/ml of rmIL-6 and 2μg rmIL-23. (g) IL-17 protein in total cell lysates of bone marrow neutrophils from naïve C57BL/6 mice incubated 3h with rmIL-6, and/or rmIL-23 at indicated concentrations per ml. Data are mean+/- SD from 3 wells per group. All data shown are representative of two (a–d) or three (e-g) repeat experiments.

Supplementary Figure 3 Expression of IL-17A, receptors for IL-6 and IL-23, and expression of Rorc and RORγt in human neutrophils.

(a) A highly purified population of neutrophils was isolated from peripheral blood of healthy volunteers by gradient (Ficoll) centrifugation, and visualized by Wrights-Giemsa staining. (b, c) Neutrophils were stimulated 1h with recombinant human (rh)IL-6 and/or rhIL-23 at the indicated concentrations per ml, and Il17a gene expression was detected by q-PCR (b). IL-17A protein from neutrophil lysates was measured by ELISA after 3h incubation with 20μg/ml IL-6 and 2μg/ml IL-23 (c). (d, e) Peripheral blood neutrophils from a healthy donor were either left unstimulated (unstim) or were incubated 3h with rhIL-6 (20μg/ml) and rhIL-23 (2μg/ml) (IL-6+IL-23), and receptor expression was examined by flow cytometry (d) (numbers denote percent positive cells in IL-6+IL-23 – stimulated neutrophils). (e) IL-23R and IL-12Rβ1 expression in unstimulated neutrophils examined by quantitative PCR. (f) Rorc expression in either unstimulated neutrophils (N) or after incubation with rhIL-6 and IL-23 (IL-6+IL-23). (g) RORγt protein in total cell lysates and nuclear extracts of isolated neutrophils obtained after 1h incubation with supernatants from either unstimulated (unstim) total peripheral blood mononuclear cells or after 18h incubation with Aspergillus hyphal extract (AspHE). a,b, d-f: Representative data from a single donor; similar data were obtained from a second donor (data not shown). c: Mean +/- SD of three wells per group; data in (e-g) are from two donors and are representative of three repeat experiments.

Supplementary Figure 4 IL-6+IL-23–stimulated IL-17RC, dectin-1 and dectin-2 in human neutrophils, and IL-23R in RorcGFP/GFP mouse neutrophils.

(a) Cell surface IL-17RA and IL-17RC expression of peripheral blood neutrophils from a healthy volunteer (Donor 2) after 3h incubation with media alone (unstim), or media containing 20μg/ml rhIL-6, 2μg/ml rhIL-23, both cytokines, or both cytokines plus Aspergillus hyphal extract (AspHE). (b) Clec7a (dectin-1) and Clec6a (dectin-2) gene expression in recombinant human IL-6 and IL-23 (IL-6+IL-23) stimulated peripheral blood neutrophils from two healthy donors. (c) Neutrophils from naïve C57BL/6 or RorcGFP/GFP mice were isolated by gradient centrifugation, incubated with anti-IL-23R, and examined by flow cytometry. The histogram shows overlap of cell surface IL-23R in neutrophils from C57BL/6 and RorcGFP/GFP mice. Data in (a-c) are representative of two repeat experiments.

Supplementary Figure 5 Representative flow cytometry profiles showing ROS activity by bone marrow neutrophils.

Intracellular amine-reactive fluorescein diacetate (CFDA) was used as a ROS indicator and shown in representative populations of naïve C57BL/6 neutrophils, and recombinant mouse IL-6 and IL-23 (IL-6+ IL-23) – stimulated bone marrow neutrophils isolated from C57BL/6, Il17a-/- and Il17rc-/- mice after incubation with recombinant mouse IL-17A and anti-IL-17RC. Combined mean fluorescence intensity (MFI) data are representative of two repeat experiments with four mice per group.

Supplementary Figure 6 Aspergillus infected corneas of primed C57BL/6 mice and Cd18−/− mice.

(a) NIMP-R14+ neutrophils and intracellular IL-17A (IL-17 Ab) in A. fumigatus infected corneas of C57BL/6 mice that had either been primed by subcutaneous injection of heat-killed swollen conidia 72h prior, or were naïve prior to corneal infection. Mice were examined 24h after infection. (b) NIMP-R14+ cells from infected corneas 72h after priming by subcutaneous injection of heat killed swollen conidia. IL-17+ and IL-17- cells were examined for propidium Iodide (PI) uptake 24h post-infection. (c) Absence of infiltrating cells in corneas of A. fumigatus infected CD18-/– mice not given donor neutrophils. Flow cytometry of total cells pooled from four corneas 24h after infection. Cells were incubated with anti- CD11b, 1A8, NIMP-R14, F4/80, CD4, CD3, γδ TCR, NK1.1 antibodies or anti-IL-17A (intracellular).

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Taylor, P., Roy, S., Leal, S. et al. Activation of neutrophils by autocrine IL-17A–IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2. Nat Immunol 15, 143–151 (2014). https://doi.org/10.1038/ni.2797

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