T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26 - PubMed (original) (raw)

doi: 10.1038/ni.3211. Epub 2015 Jul 13.

Jeremy Di Domizio 2, Kui S Voo 3, Heike C Friedrich 4, Georgios Chamilos 5, Dipyaman Ganguly 3, Curdin Conrad 2, Josh Gregorio 3, Didier Le Roy 6, Thierry Roger 6, John E Ladbury 7, Bernhard Homey 4, Stanley Watowich 8, Robert L Modlin 9, Dimitrios P Kontoyiannis 10, Yong-Jun Liu 11, Stefan T Arold 12, Michel Gilliet 13

Affiliations

T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

Stephan Meller et al. Nat Immunol. 2015 Sep.

Abstract

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

IL-26 is a cationic amphipathic protein that forms oligomers. (a) Protein ribbon of IL-26 obtained by homology modeling. Six predicted α-helices are indicated as αA–αF and are represented in different colors. (b) Color-coded electrostatic potentials mapped onto the surfaces of IL-26 (top row) and IL-22 (bottom row). Regions of positive charges are in blue, negative charges are in red and hydrophobic charges are in white. (c) Small-angle X-ray scattering analysis and rigid-body modeling (via Sasref) of IL-26 suggested the formation of elongated tetramers. The four IL-26 molecules (secondary-structure representation) are shown in different colors.

Figure 2

Figure 2

IL-26 has direct bactericidal properties. (a) Growth of Pseudomonas aeruginosa ATCC 27853 (P. aerug.), Escherichia coli O1:K1:H7 (E. coli), Staphylococcus aureus ATCC 6538 (S. aureus) and Klebsiella pneumoniae O1:K2 (K. pneumo.) in culture with increasing concentrations of rhIL-26. (b) Growth of P. aeruginosa in culture with increasing concentrations of rhIL-26, rhIL-17 and rhIL-22. (c) Growth kinetics of P. aeruginosa cultured with no IL-26, with 10 μM rhIL-26, and with rhIL-26 in the presence of either blocking anti–IL-26 or isotype control antibodies (ctl IgG). (d) Membrane potential of P. aeruginosa cultured for 24 h as in c and stained with the green fluorescent dye DiOC2(3), which forms red fluorescent aggregates in polarized membranes. A loss of red fluorescence indicates membrane disruption. The proton ionophore CCCP was used as a positive control. (e) Kinetics of membrane potential of P. aeruginosa cultured as in d. (f) P. aeruginosa cultured for 30 min with or without 10 μM rhIL-26 and visualized by scanning electron microscopy. Arrows in the top right image show the formation of membrane blebs; arrow in the lower right image shows extracellular leakage of bacterial content. (g) Capacity of IL-26 to bind LPS or LTA as assessed by enzyme-linked immunosorbent assay (ELISA). (h) Bacterial loads measured in mouse lungs, spleen and blood collected 3 d after intranasal infection with K. pneumoniae (100 CFU) followed by intranasal treatment with 20 μg rhIL-26 or 50 μg LL-37. As a control, K. pneumoniae was pretreated with rhIL-26 ex vivo. Each data point represents a separate mouse; ††† indicates a mouse that died from overwhelming infection within the 3-d period between initial infection and organ collection. Data are representative of three (a–c,e), two (g) or four (h) independent experiments and were obtained from eight mice per group; error bars represent ±s.d. of duplicate (a–c,e) or triplicate (g) wells. Data were statistically analyzed via unpaired non-parametric Mann-Whitney _U_-test. *P < 0.001. CFU, colony-forming units.

Figure 3

Figure 3

TH17 cells exert direct antimicrobial activity via the production of IL-26. (a) Real-time PCR analysis of IL26, IL17A, IL17F, IL22, IL13 and IFNG mRNA expression by primary TH17, TH1, TH2 and TH0 cells generated in vitro from purified naive T cells and re-stimulated by plate-bound anti-CD3 and soluble anti-CD28. Cytokine mRNA expression was normalized to GAPDH mRNA expression. Each data point represents a separate donor (n = 10–11); horizontal bars denote mean values. (b) Growth of P. aeruginosa cultured with supernatants of primary TH0 and TH17 cells and measured by microbroth dilution assay. Assays were performed with and without blocking anti–IL-26 and control antibodies (Ctl IgG). (c) Growth of P. aeruginosa cultured with supernatants of three TH17 cell clones (1G3, 7H1 and 72G6) transfected with siRNA against IL-26 (si_IL26_, white bars) or a control siRNA (siCtl, black bars). n.s., not significant. (d) IL-26 production by re-stimulated primary TH0 and TH17 cells and transfected TH17 cell clones as measured by ELISA. Data are representative of at least three (b,c) or two (d) independent experiments. Error bars represent the s.d. of triplicate wells (b,c). Data were statistically analyzed via unpaired two-tailed Student’s _t_-test; *P < 0.05, **P < 0.01.

Figure 4

Figure 4

IL-26 promotes bacterial killing and concomitant sensing of DNA released by pDCs. (a) Confocal microscopy images of DNA–IL-26 complexes (yellow) in cultures of P. aeruginosa treated with rhIL-26 and stained with DAPI (green) and anti–IL-26 (red). Images shown are representative of three independent experiments. (b,c) Fluorimetric quantification of DNA staining by picogreen dye after mixing of bacterial DNA with increasing concentrations of IL-26 (b) or 1 μM IL-26, IL-17 or IL-22 (c). A.U., arbitrary units. (d) Number of particles formed after mixing of bacterial DNA with 1 μM IL-17, IL-22 or IL-26, counted as insoluble precipitates by microscopy. (e) Production of IL-1β, IL-6, TNF and IFN-α by PBMCs stimulated overnight with 1 μM IL-26 alone or in complex with bacterial DNA (bactDNA, 3 μg ml−1). Bars show mean ± s.d. of pooled data. (f) IFN-α produced by human PBMCs, monocytes, macrophages, NK cells, neutrophils, pDCs and pDC-depleted PBMCs stimulated overnight with 1 μM IL-26 alone or in complex with bactDNA. Bars show mean ± s.d. of pooled data. (g–i) IFN-α produced by pDCs stimulated overnight with increasing concentrations of live P. aeruginosa (titrated according to CFU) (g) or of P. aeruginosa lysate (titrated according to DNA content) (h) in the presence or not of IL-26 (10 or 1 μM, respectively). (i) IFN-α produced by pDCs stimulated overnight with P. aeruginosa lysate (1 μg ml−1 DNA) or purified bactDNA (1 μg ml−1) alone or in the presence of IL-26, with or without DNase pretreatment. Data are representative of four independent experiments (b–d,g–i) or four independent donors (e,f); error bars represent the s.d. of triplicate wells (b–d,g–i). Data were statistically analyzed via unpaired two-tailed Student’s _t_-test; *P < 0.05, **P < 0.01.

Figure 5

Figure 5

IL-26 promotes pDC sensing of human DNA released in the context of cell death. (a) Cell viability of monocytes, macrophages, NK cells, neutrophils, pDCs and HEK cells as determined by staining with 7-AAD after overnight culture in medium alone or in the presence of either 10 μM rhIL-26 or undiluted supernatants (SN) of primary TH17 cells. (b) IFN-α produced by pDCs stimulated overnight with live or UV-irradiated HEK293 cells in the presence or not of 10 μM rhIL-26. (c) Fluorimetric quantification of DNA staining by picogreen dye upon mixing of human DNA fragments (huDNA) with increasing concentrations of rhIL-26. (d) Visualization of DNA–IL-26 complexes (yellow) formed after mixing of human DNA with rhIL-26 and appearing as insoluble precipitates that stained with DAPI (green) and anti–IL-26 (red). (e) IFN-α produced by human PBMCs, monocytes, macrophages, NK cells, neutrophils, pDCs and pDC-depleted PBMCs stimulated overnight with IL-26 alone, human DNA alone or IL-26–human DNA complexes. Each data point represents an individual donor (n = 4 for each condition). Bars show mean ± s.d. of pooled data. Data were statistically analyzed via unpaired two-tailed Student’s _t_-test; *P < 0.05, **P < 0.0001. (f) IFN-α produced by pDCs stimulated with increasing concentrationsof human DNA either alone or in complex with 1 μM IL-26. In panels a–c and f, data are representative of three independent experiments, and error bars represent ±s.d. of triplicate wells.

Figure 6

Figure 6

IL-26–DNA complexes are endocytosed via heparan-sulfate proteoglycans and activate TLR9 in pDCs. (a) Flow cytometry of CD123+ pDCs stimulated for 4 h with Alexa Fluor 488–conjugated human DNA (huDNAA488) alone or complexed with increasing concentrations of IL-26. (b) Confocal microscopy images of pDCs stimulated with huDNAA488 alone or with IL-26–huDNAA488 complexes. We used DAPI to stain the nucleus, and we used phycoerythrin-conjugated anti-CD123 to visualize the contour of pDCs (left and middle panels) or biotin-conjugated anti-CD71 plus phycoerythrin-conjugated streptavidin to visualize early endocytic compartments (right panel). (c) Flow cytometry of human DNA–positive CD123+ pDCs stimulated for 4 h with huDNAA488 complexed with IL-26 after pretreatment with 1 μg ml−1 cytochalasin D (Cyto D) to block endocytosis, 0.5 μg ml−1 trypsin to cleave membrane proteins and 100 mU Heparinase III to cleave surface heparan-sulfate proteoglycans. Cells were also cultured at 4 °C to inhibit active internalization processes such as endocytosis. (d) Quantification of human DNA–positive CD123+ pDCs stimulated as in c. *P < 0.01, **P < 0.001, ***P < 0.0001. (e) IFN-α produced by pDCs stimulated overnight with IL-26–human DNA complexes in the presence of increasing concentrations of chloroquine to inhibit endosomal TLR activation. (f) Fluorimetric quantification of embryonic alkaline phosphatase secreted by HEK293 cells transfected with either TLR9 or TLR4 along with an NF-κB–inducible secreted embryonic alkaline phosphatase reporter gene and stimulated under the indicated conditions. CpG, synthetic phosphothioate TLR9 agonist CpG-2006. *P < 0.0001. Data are representative of three independent experiments. Error bars in d–f represent the s.d. of triplicate wells. Data were statistically analyzed via unpaired two-tailed Student’s _t_-test.

Figure 7

Figure 7

TH17 cells activate pDCs via IL-26 production. (a) Confocal microscopy images of insoluble DNA–IL-26 particles in supernatants of re-stimulated TH17 cells stained with DAPI and unlabeled anti–IL-26 plus Alexa Fluor 546–conjugated mouse anti-IgG. Images shown are representative of three independent experiments. (b) DNA–IL-26 particle count in supernatants of re-stimulated TH17 and TH0 cells. Each data point represents an independent donor (n = 4–6). Horizontal bars represent means. Data were statistically analyzed via unpaired two-tailed Student’s t_-test; *P < 0.05. (c–g) IFN-α produced by pDCs stimulated overnight with (c) various dilutions of TH17 cell–derived supernatants in culture medium; (d) supernatants of re-stimulated TH17 or TH0 cells derived from multiple donors (n = 9); (e) supernatant derived from TH17 cells re-stimulated in the presence of anti–IL-26, anti–IL-17 or DNase I treatment or untreated; (f) TH17 cell supernatants derived from multiple donors (n = 3–7), either alone or supplemented by exogenous human DNA (3 μg ml−1), with or without blocking anti–IL-26; and (g) supernatants of TH17 cell clones (n = 3) transfected with siRNA against IL-26 (si_IL26) or a control siRNA (siCtl) supplemented or not by human DNA. In a–g, data are representative of at least three independent experiments and were statistically analyzed via unpaired two-tailed Student’s _t_-test; *P < 0.05, **P < 0.01. Horizontal bars in d, f and g represent means. (h) IL-26 concentrations in healthy skin and psoriatic skin lesions measured by ELISA of total skin extracts derived from healthy donors (n = 15) or psoriasis patients (n = 15). Each data point represents an individual donor. Data were statistically analyzed via unpaired non-parametric Mann-Whitney _U_-test; *P < 0.001.

Comment in

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