IL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells

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1 Immunity, Volume 40 Supplemental Information IL-17 Regulates Systemic Fungal Immunity by Controlling the Functional Competence of NK Cells Eva Bär, Paul G. Whitney, Kathrin Moor, Caetano Reis e Sousa, and Salomé LeibundGut- Landmann Inventory of Supplemental Information: 1. Supplemental Figures Figure S1, related to Figure 1 Figure S2, related to Figure 2 Figure S3, related to Figure 5 Figure S4, related to Figure 6 2. Supplemental Experimental Methods 3. Supplemental References 1

2 Figure S1 (Supplement to Figure 1): A) No impact of the genetic background on fungal control. Fungal burden in the kidneys of C. albicans infected WT C57Bl/6 (B6) and WT 129 mice. Each dot represents one mouse. Data are pooled from two independent experiments. B) Rorc-/- mice are highly susceptible to systemic candidiasis. Fungal burden in the kidneys of C. albicans infected WT and Rorc-/- mice. Each dot represents one mouse. Data are pooled from two independent experiments. C) F) IL-17A producing T cells are dispensable for fungal control. C) Kidney lymphocytes were isolated from naïve or infected WT and H2-Ab1-/- mice and were restimulated with recombinant IL-23 and IL-1 or left untreated. Intracellular IL-17A production by CD45 + TCRβ + cells was determined by flow cytometry. Representative FACS plots are shown. D) Summary graph of data shown in (A). The percentage IL-17A + cells within CD45 + TCRβ+ kidney lymphocytes is shown. Each symbol represents one mouse. Data are pooled from two independent experiments. E) Kidney lymphocytes isolated from naïve and infected WT, H2-Ab1-/-, Cd1d-/- and Rag1-/- mice were stimulated with platebound CD3ε and IL-17A cytokine secretion into the medium was quantified by ELISA. Data are mean + SD of duplicate wells and are representative of three independent experiments. F) Fungal burden in the kidneys of C. albicans infected WT, H2-Ab1-/-, Cd1d- /- and Tcrd-/- mice. Each dot represents data from one mouse. Data are pooled from two independent experiments. Differences are not statistically significant. 2

3 Figure S2 (Supplement to Figure 2): A) F) IL-17RA-/- NK cells do not display any major phenotypic alterations. A) Splenocytes from naïve WT and Il17ra-/- mice were analyzed for the expression of NKp46, CD49b and CD3 by flow cytometry. Data shown are pre-gated on CD3 -negative cells. Representative plots are shown. B - C) Frequencies and absolute numbers of CD49b + NKp46 + CD3 - NK cells (B) and CD3 + TCR + T cells (C) in the spleen of naïve WT and Il17ra-/- mice. Each symbol represents one mouse. Frequencies are % NK cells and % T cells, respectively, within total lymphocytes. Data shown are pooled from two independent experiments. D) Splenic NK cells from naïve WT and Il17ra-/- mice were analyzed for CD27 and CD11b expression. Numbers represent the % of CD49b + NKp46 + CD3 - splenocytes in each quadrant. E) Summary graph of the data shown in (D). Each symbol represents one 3

4 mouse. F) Expression levels of KLRG1, CD122, NKG2D, Ly49H, Ly49A and Ly49C/I on CD49b + NKp46 + CD3 - splenic NK cells in naïve WT and Il17ra-/- mice (filled histograms). Black lines represent the corresponding isotype control for each staining. All data shown are representative of two to six independent experiments. G-H) Functional impairment of Il17ra-/-, Il17rc-/-, Il17a-/- and Il17f-/- NK cells. WT C57Bl/6 (B6), WT 129, Il17ra-/- and Il17rc-/- mice (G) or WT (C57BL/6), Il17rc-/-, Il17a-/- and Il17f-/- mice (H) were primed with LPS and splenic CD49b + NKp46 + CD3 - NK cells were analyzed for CD69 expression and IFN- production. All data are representative of at least two to three independent experiments. I) IL-17 is produced in the bone marrow of naïve animals. IL-17A mrna expression in the bone marrow of naïve WT and Rorc-/- animals was analyzed by real-time RTPCR. Data were normalized to -actin. Each symbol represents one mouse (mean of duplicate measurements). 4

5 Figure S3 (Supplement to Figure 5): A) B) Il17ra-/- mice do not display a defect in neutrophil recruitment upon C. albicans infection. The frequency (A) and absolute numbers (B) of Ly6G + neutrophils were quantified in the spleen and kidney of WT and Il17ra-/- mice 24 hours post-infection. Frequencies are % Ly6G + cells of all CD45 + cells. 5

6 Figure S4 (Supplement to Figure 6): A) IFN- is not involved in the protective host response to C. albicans. Fungal burden in the kidneys of WT and Ifngr1-/- mice infected C. albicans. Data are representative of two independent experiments and each symbol represents one mouse. B) NK cells secrete GM-CSF in response to C. albicans infection. Splenic NK cells isolated from infected WT and Il17ra-/- mice were cultured for 24 hours and GM-CSF secretion into the supernatant was quantified by cytometric bead array. Each dot represents one mouse. Data are pooled from two independent experiments. 6

7 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Mice WT C57BL/6 (WT or WT B6) mice were purchased from Janvier Elevage. Il17ra-/- and Il17rc-/- mice were obtained from Amgen, and Rorc-/-, Il17a-/-, Il17f-/- and Csf2-/- mice were a gift from Burkhard Becher (University of Zürich, Switzerland). Rag1-/-, Rag2-/-Il2rg- /-, Cd1d-/-, H2-Ab1-/- TCR -/-, Ifngr1-/-, WT 129 and congenic Ly5.1 mice were bred at our animal facility Rodent Center HCI or at the Institute of Laboratory Animal Sciences (University of Zürich, Switzerland) under specific pathogen free conditions. All animals were on C57Bl/6 background. In Il17ra-/- mice, C57Bl/6 and 129 haplotypes still segregated at the NKC locus adjacent to the Il17ra locus on chromosome 6, and despite extensive backcrossing there was residual contribution of the 129 background. The presence of C57Bl/6 alleles at the NKC locus was confirmed by SNP analysis as well as Ly49H, Ly49A and Ly49C/I staining (Fig. S2F). Stratifying the mice of our Il17ra-/- colony on the basis of NK1.1 expression confirmed that the observed phenotype was not linked to a specific NKC haplotype but rather was due to a bona fide defect in IL-17 receptor signaling (data not shown). For the generation of mixed chimeras C57Bl/6 recipients were irradiated with 950 Rad and subsequently reconstituted with equal amounts of bone marrow cells from WT and Il17ra-/- mice. Six weeks after reconstitution, the mice were used for experiments. Fungal infections The C. albicans laboratory strain SC5314 was inoculated in YPD medium at a density of OD and grown for 18 hours at 30 C. Mice were infected with 10 5 cfu i.v.. The fungal burden was analyzed 72 hours post-infection by homogenization of the organs in sterile 0.05% NP40 in H 2 O and serial dilutions were plated on YPD agar containing 100 g/ml Ampicillin. Viral infections BAC-derived MCMV MW97.01 was previously described (Walton et al., 2008) and was propagated on C57BL/6 embryonic fibroblasts. Mice were infected with 5 x 10 6 pfu i.v. and viral titers were determined by standard virus plaque assay (Brune et al., 2001). Antibodies and staining reagents For NK cell depletion, mice were treated with anti-agm1 (Wako; 20 g i.p.) or anti-nk1.1 (clone PK136, BioXcell; 500 g i.p.) one day prior to infection and on the day of infection. In some experiments, anti-il-17a (clone 17F3, BioXCell, 200 g per mouse i.p.) and anti-il- 17F (Liang et al., 2007) (kindly provided by Pfizer Biotherapeutics, 200 g per mouse i.p.) was administered simultaneously with the infection and 1 day post infection. Anti-IL-17RA (clone M751, kindly provided by Amgen, 500 g per mouse i.p.) was administered on the day of infection. anti-gm-csf (clone MP1-22E9) was purchased from BioLegend. For in vitro blockade, antibodies were added at 10 g/ml. Plate-bound anti-cd3 (5 μg/ml in PBS, clone 145-2C11, BioLegend) was used for in vitro re-stimulation of lymphocytes. For flow cytometry, fluorophore-conjugated antibodies specific for the indicated mouse antigens were purchased from BioLegend, unless stated differently: CD4 (clone RM4-5), 7

8 CD3 (clone 145-2C11), TCRβ (clone H57-597), NKp46 (clone 29A1.4), CD49b (clone DX5), CD11b (clone M1/70), CD122 (clone TM-β1), Ly49H (clone 3D10), Ly49A (clone YE1/ ), Ly49C/I (clone 5E6, BD Biosciences) and CD27 (clone L128), CD69 (clone H1.2F3), NKG2D (clone A10), CD94 (clone 18D3), Ly6G (clone 1A8, BD Biosciences), CD18 (clone M18/2), IFN- (clone XMG1.2), GM-CSF (clone MP1-22E9, BD Biosciences), IL-17RA (clone M751, kindly provided by Amgen) and perk1/2 (clone 20A). For the analysis of cell viability, Annexin V (BioLegend) and 7-AAD (BD Biosciences) were used according the manufacturer's instructions. Recombinant proteins Recombinant mouse GM-CSF was purified from the supernatant of the X63-GMCSFproducing cell line (Stockinger et al., 1996). For in vivo experiments, 5 g per mouse were injected i.p. simultaneously with the infection and again 4 and 24 hours post-infection. For in vitro experiments, recombinant mouse GM-CSF was added at 0.25 g/ml. Heterodimeric IL- 17AF (BioLegend) was used at 1 g/ml. IL-23 and IL-1 (both BD Biosciences) were added at 100 ng/ml. Flow cytometry Cells were stained for cell surface markers in ice-cold PBS supplemented with 2mM EDTA and 10% FCS. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and then resuspended in Perm/Wash buffer (BD Biosciences) containing antibodies specific for intracellular antigens. For perk1/2 staining, cells were fixed and lysed in Phosflow Lyse/Fix buffer and Phosflow Perm Buffer III (both BD Biosciences) according to manufacturer`s instructions. FACS data were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tristar). NK cell isolation and adoptive transfer Single cell suspensions of spleens were obtained by enzymatic digestion with Collagenase I (4.8 mg/ml, Invitrogen) and DNase (2.4 mg/ml, Roche) and NK cells were then enriched with anti-dx5 microbeads (Miltenyi Biotech) according to the manufacturer s instructions. NK cell purity was routinely >92%. For adoptive transfer, purified NK cells were injected i.v. one hour before infection (8 x 10 6 cells pooled from 4 donor mice per one recipient mouse). Ex vivo analysis of NK cell function Cytokine production by splenic NK cells was determined by intracellular cytokine staining 5 h after i.v. injection of LPS or 24 h after C. albicans infection. Alternatively, NK cells were purified from the spleen at 24 h post-infection as described above, cultivated in medium for 24 hours at 37 C, 5% CO2, and GM-CSF secretion into the supernatant was measured by cytometric bead array (BD Biosciences) according to the manufacturer s instructions. To determine NK cell cytotoxic activity, splenic NK cells were purified 18 to 24 hours after LPS injection as described above and co-cultured with 10 5 FITC-labeled YAC-1 cells at different ratios (1:1; 1:5; 1:10) for 6 hours. Viability of YAC-1 cells was assessed by 7-AAD staining combined with flow cytometry and killing efficiency was calculated as % 7-AADpositive YAC-1 cells. 8

9 In vitro analysis of NK cell function In vitro priming of NK cells was determined by co-culturing 2.5x10 5 purified splenic NK cells (see above) from naïve mice with 10 5 bone marrow-derived DCs in the presence or absence of LPS (25 ng/ml) for 18 hours. In some instances, anti-il-17a and anti-il-17f or anti-il-17ra antibodies were added to the cultures. Brefeldin A was added for the last 15 hours of stimulation. IFN- production by NK cells was accessed by intracellular cytokine staining as described above. To evaluate NK cell responsiveness to IL-17, total bone marrow cells were stimulated with heterodimeric IL-17AF (1 g/ml) or PMA (20 ng/ml, Sigma) and Ionomycin (1 g/ml, Sigma) for 30 min. NK cell subsets were then analyzed by flow cytometry as described above. Neutrophil isolation and functional assays Blood was drawn by cardiac puncture and diluted with PBS containing 2 mm EDTA, l/ml Heparin and 1% FCS. Neutrophils were isolated over a density gradient of Histopaque 1119 and Histopaque 1077 (both Sigma). Neutrophil purity was routinely >80% Ly6G +. To test neutrophil killing activity, 10 4 C. albicans cells were exposed to 10 4 neutrophils in protein low binding tubes (Sarstedt) for two hours. Then, neutrophils were lysed with water and the number of surviving yeast cells was assessed by plating on YPD agar and colony counting. Killing activity is expressed as % of yeast cells surviving in presence of neutrophils compared to yeast cells surviving in absence of neutrophils. To analyze the crosstalk of NK cells and neutrophils in vitro, 10 6 blood neutrophils isolated from naïve mice were co-cultured with 10 6 purified NK cells from naïve or infected mice (see above) for 18 hours and then analyzed by flow cytometry. ROS production was quantified by measuring de-esterification of 2,7 -Dichlorofluorescin diacetate (Sigma). Neutrophil viability was determined by Annexin V staining. In some experiments NK cells were added into a transwell (0.4 m pore size), or NK-neutrophil co-cultures were supplemented with recombinant mouse GMCSF or anti-gmcsf antibodies. Isolation and analysis of kidney cells Mice were anaesthetized with a sublethal dose of Ketamin, Xylazin and Acepromazin, and perfused by injection of PBS into the right heart ventricle to remove circulating blood cells from the kidneys. Kindeys were then removed, cut into small pieces and digested with DNAse I (2.4 mg/ml, Roche) and Colagenase I (4.8 mg/ml, Invitrogen) in PBS for 30 min at 37 C. Lymphocytes were purified by a discontinuous gradient of 70% and 40% of Percoll (Sigma) that was overlaid with the cell suspension. Lymphocytes were collected from the 70%-40% interphase. For analysis of cytokine production, kidney lymphocytes were restimulated in vitro for 18 hours with recombinant IL-23 and IL-1 or left untreated. Brefeldin A was added for the last 15 hours before staining and analysis by flow cytometry. Alternatively, kidney lymphocytes were re-stimulated on plate-bound anti-cd3e for 24 hours and cytokine production was quantified by sandwich ELISA. RNA isolation and real-time RT-PCR. 9

10 Isolation of total RNA from bone marrow and generation of cdna was carried out according to standard protocols. Quantitative PCR was performed using SYBR Green (Roche) and a Rotor-Gene 3000 (Corbett Research). The primers were IL-17A fwd, 5'- GCTCCAGAAGGCCCTCAGA-3'; IL-17A rev, 5'-AGCTTTCCCTCCGCATTGA-3'. Data were normalized to -actin ( -actin fwd, 5'-CCCTGAAGTACCCCATTGAAC-3'; -actin rev, 5'- CTTTTCACGGTTGGCCTTAG-3'). 10

11 SUPPLEMENTAL REFERENCES Brune, W., Hengel, H., and Koszinowski, U.H. (2001). A mouse model for cytomegalovirus infection. Current protocols in immunology / edited by John E. Coligan... [et al.] Chapter 19, Unit Liang, S.C., Long, A.J., Bennett, F., Whitters, M.J., Karim, R., Collins, M., Goldman, S.J., Dunussi-Joannopoulos, K., Williams, C.M., Wright, J.F., and Fouser, L.A. (2007). An IL- 17F/A heterodimer protein is produced by mouse Th17 cells and induces airway neutrophil recruitment. J Immunol 179, Stockinger, B., Zal, T., Zal, A., and Gray, D. (1996). B cells solicit their own help from T cells. The Journal of experimental medicine 183,