Interferon- -producing immature myeloid cells confer protection. against severe invasive group A Streptococcus infections. Supplementary Information

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1 Supplementary Information Interferon- -producing immature myeloid cells confer protection against severe invasive group A Streptococcus infections Takayuki Matsumura, Manabu Ato, Tadayoshi Ikebe, Makoto Ohnishi, Haruo Watanabe, and Kazuo Kobayashi

2 Supplementary Figure S1: T cells and NK cells are the source of IFN- during late stage of severe invasive GAS infections. (a) C3H/HeN or C57BL/6 mice with or without i.p. infection of NIH34 ( CFU/mouse) for 1, 3 or 5 days (d) were i.v. injected with monensin. Six hours later, the mice were sacrificed and their splenocytes were immediately stained for the indicated markers, and analyzed by the ICS assay. The numbers (%) in the plots represent the proportion of IFN- + subsets to total splenocytes. (b) The CFU of NIH34 and each IFN- + cell number in spleen of C3H/HeN mice (C3H) and C57BL/6 mice (B6) were determined at the indicated days. These data are representative of 2 independent experiments.

3 Supplementary Figure S2: IFN- -producing myeloid cells are detected in various organs during early stage of severe invasive GAS infections. C3H/HeN or C57BL/6 mice with or without infection of NIH34 ( CFU/mouse i.p. or CFU/mouse s.c.) for 42 h were i.v. injected with monensin. (a) The CFU of NIH34 in the peritoneal cavity (P.C.), kidney, and spleen was determined at 48 h post-infection (n = 3, ±S.D). (b) Six hours later, the mice were sacrificed and their splenocytes, leukocytes in the P.C., kidney, or skin were immediately stained for IL-5R (T21) and IFN-, and analyzed by the ICS assay. The numbers (%) in the plots represent the proportion of IFN- + subsets to total splenocytes. These data are representative of 2 independent experiments.

4 Supplementary Figure S3: Gr-1 + CD49d + cells are the source of IFN- in severe invasive GAS infections. Non-infected C3H/HeN mice and C3H/HeN mice i.p. infected with severe invasive (STSS strain: NIH34 [emm3 genotype], NIH230 [emm49 genotype], NIH186 [emm1 genotype], or NIH202-2 [emm1 genotype]) GAS clinical isolates ( CFU/mouse) for 42 h were i.v. injected with monensin. Six hours later, the mice were sacrificed and their splenocytes were immediately stained for Gr-1, CD49d, and IFN-, and analyzed by the ICS assay. Lower panels show the cells gated on the IFN- + population. These data are representative of 3 independent experiments.

5 Supplementary Figure S4: Ly-6C low cells are the source of IFN- in severe invasive GAS infections. (a, b) C3H/HeN mice with or without i.p. infection of NIH34 ( CFU/mouse) for 42 h were i.v. injected with monensin. Six hours later, splenocytes and peripheral blood cells (a) or splenocytes and bone marrow cells (b) from non-infected mice or NIH34-infected mice were immediately stained for Ly-6C and IFN-, and analyzed by the ICS assay. The numbers (%) in the plots represent the proportion of IFN- + subsets to total cells. These data are representative of 3 independent experiments.

6 Supplementary Figure S5: IMCs differentiate into polymorphonuclear cells but not Eos in the presence of GM-CSF. (a) CD11b + CD11c - F4/80 low Ly-6G + IMCs in splenocytes from C3H/HeN mice infected with NIH34 ( CFU/mouse) for 48 h were isolated by FACS. Bone marrow-derived Eos were isolated and cultured. Cytospin preparations of GM-CSF [2 days]-cultured IMCs (GM2- IMCs) (left) and Eos (right) were visualized with May-Grünwald-Giemsa staining. Scale bars, 20 m. (b) IMCs and Eos were stained for IL-5R (H7), Siglec-F, and CCR3 or c-kit, (blue line: IMCs, red line: GM2- IMCs, orange line: CCR3 + H7 + Siglec-F + splenic Eos, green lines: c-kit low H7 + Siglec-F + bone marrow-derived Eos). These data are representative of 3 independent experiments.

7 Supplementary Figure S6: BMPCs differentiate into BM- IMCs and PMNs in the presence of GM-CSF and IFN-. (a) CD11b + CD11c - F4/80 - Ly-6G high immature bone marrow PMNs in non-infected C3H/HeN mice and CD11b + CD11c - F4/80 low Ly-6G + BM- IMCs or CD11b + CD11c low F4/80 + Ly-6G low BMPCs in NIH34-infected mice were isolated by FACS. The numbers (%) in the panels represent the proportion of PMNs, BM- IMCs, and BMPCs to total bone marrow cells. (b) Sorted BM- IMCs and BMPCs were cultured with GM-CSF in the presence or absence of control rat IgG or an IFN- neutralizing mab (clone R4-6A2). On days 2 or 4, the cells were harvested for May-Grünwald-Giemsa staining. Scale bars, 20 m. These data are representative of 3 independent experiments.

8 Supplementary Figure S7: No IMCs were detected in Ifng -/- mice infected with severe invasive GAS isolates. (a) C57BL/6 mice and Ifng -/- mice were i.p. infected with NIH34 ( CFU/mouse) for 48 h. Those mice were sacrificed and their splenocytes were immediately stained for F4/80 and Ly-6G, and analyzed by FACS. The numbers (%) in the plots represent the proportion of IMC to total splenocytes. (b) Bone marrow cells from non-infected C57BL/6 mice and Ifng -/- mice were stained for F4/80 and Ly-6G, and analyzed by FACS. The numbers (%) in the plots represent the proportion of PMNs to total bone marrow cells. These data are representative of 3 independent experiments.

9 Supplementary Table S1. Phenotypes of IFN- + cells in GAS infections, monocytes, and MDSCs. IFN- + cells in GAS infections Inflammatory monocytes Resident monocytes Monocytic MDSCs Granulocytic MDSCs Monocyte markers Mac-1 (CD11b) F4/80 +/ or +/- Ly-6C +/- ++ +/- ++ +/- Granulocyte marker Ly-6G Chemokine receptors CCR CCR3 - ND ND ND ND CX3CR Other receptors and lineage markers CD4 - ND ND - ND CD8 - ND ND - ND CD11c CD27 - ND ND ND ND PECAM-1 (CD31) CD34 - ND ND - - CD38 + ND ND ND ND CD44 ++ ND ND B220 (CD45R) ND DX5 (CD49b) ND CD49d + ND ND ++ + L-selectin (CD62L) CD69 + ND ND ND ND c-kit (CD117) - ND ND - - IL-5R (CD125) H7 - ND ND ND ND IL-5R (CD125) T21 ++ ND ND ND ND IL-7R (CD127) - ND ND ND ND MHC-II - +/- or + +/- or Siglec-F - ND ND ND ND Siglec-H - ND ND ND ND TCR ND ND TCR- / - ND ND ND ND Surface expression levels have been arbitrarily designated as undetectable (-), low (+/-), positive (+), and high (++) on the basis of flow cytometry analysis 9, 11, 36 ; ND, not determined.

10 Supplementary Table S2. List of antibodies used in this study. Antigen Clone Final Supplier concentration ( g/100 L) CD4 GK ebioscience CD ebioscience CD11b M1/ ebioscience CD11c N ebioscience CD27 LG.3A BD CD ebioscience CD34 RAM ebioscience CD BioLegend CD44 IM BD CD45R (B220) RA3-6B ebioscience CD49b (DX5) DX5 0.2 ebioscience CD49d R ebioscience CD62L MEL ebioscience CD69 H1.2F3 0.1 ebioscience CD117 (c-kit) 2B ebioscience CD125 (IL-5R ) H7 1 Dr. K. Takatsu (University of Toyama) CD125 (IL-5R ) T BD CD127 (IL-7R ) A7R ebioscience CD161 (NK1.1) PK ebioscience CCR2 E68 1 Lifespan Biosciences CCR R&D Systems CX3CR1 rabbit polyclonal 1 ebioscience F4/80 CI:A3-1 1 BioLegend Gr-1 (Ly-6C/G) RB6-8C5 0.1 ebioscience Ly-6C ER-MP AbD Serotec Ly-6G 1A8 0.1 BD MHC-II (I-A/E) M5/ ebioscience Siglec-F E BD Siglec-H ebio440c 0.1 ebioscience TCR- H ebioscience TCR- / ebiogl3 0.1 ebioscience