Volume 27, Issue 2, Pages 561-571.e6 (April 2019) Sequential Sensing by TLR2 and Mincle Directs Immature Myeloid Cells to Protect against Invasive Group A Streptococcal Infection in Mice  Takayuki Matsumura, Tadayoshi Ikebe, Koji Arikawa, Masahito Hosokawa, Michio Aiko, Aoi Iguchi, Ikuko Togashi, Sayaka Kai, Sakiko Ohara, Naoya Ohara, Makoto Ohnishi, Haruo Watanabe, Kazuo Kobayashi, Haruko Takeyama, Sho Yamasaki, Yoshimasa Takahashi, Manabu Ato  Cell Reports  Volume 27, Issue 2, Pages 561-571.e6 (April 2019) DOI: 10.1016/j.celrep.2019.03.056 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 IL-6 Production in Late Phase Affords the Protection against Severe Invasive GAS Infection (A) C57BL/6 mice were intraperitoneally inoculated with 3.0 × 107 colony-forming units of non-invasive (K33) and severe invasive (NIH34) S. pyogenes clinical isolates, both of which are of the emm3 genotype. Plasma IL-6 was quantified using flow cytometry. Data are mean ± SD (n = 4 mice/group). Data are representative of two or four independent experiments. ∗p < 0.05 by Student’s t test versus K33-infected mice. (B) C57BL/6, Ifng−/−, and Il6−/− mice (n = 8 mice/group) were intraperitoneally inoculated with NIH34 (1.0 × 107 colony-forming units/mouse), and survival was monitored. Data are representative of two independent experiments. ∗∗p < 0.01 by log-rank test versus C57BL/6 mice. (C) C57BL/6 mice were intraperitoneally inoculated with NIH34 (2.0 × 107 colony-forming units/mouse). After 36 h post-infection, these mice were treated with IL-6 neutralizing monoclonal antibody (mAb) (clone MP5-20F3) (1 mg/mouse) or control rat IgG (1 mg/mouse) and survival was monitored (n = 6 mice/group). Data are representative of two independent experiments. ∗∗p < 0.01 by log-rank test versus control IgG-treated mice. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 γIMCs Are the Source of IL-6 at the Late Phase of Severe Invasive GAS Infection (A–E) C57BL/6 mice intraperitoneally non-infected (day 0) or infected for 18 h (day 1) or 42 h (day 2) with NIH34 (1.0 × 107 colony-forming units/mouse) were intravenously injected with monensin. Mice were sacrificed 6 h thereafter, and splenocytes were immediately stained for indicated markers and analyzed by intracellular cytokine synthesis assay. (A) Numbers indicate IL-6+ subsets as % of total splenocytes. (B) Data are expressed as mean ± SD (n = 4 mice/group) of the indicated marker positive and IL-6+ subsets as % of total splenocytes. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01 by Student’s t test. (C) Numbers indicate CD38+ IL-5Rα (T21)+ γIMC subsets as % of total splenocytes. Lower panels show cells gated on IL-6+. (D) Data are expressed as mean ± SD (n = 4 mice/group) of CD38+ IL-5Rα (T21)+ γIMC subsets as % of total splenocytes. ∗∗p < 0.01 by Student’s t test. (E) Data are expressed as mean ± SD (n = 4 mice/group) of CD38+ IL-5Rα (T21)+ γIMC subsets as % of IL-6+ subsets. ∗∗p < 0.01 by Student’s t test. Data are representative of two independent experiments. See also Figure S1. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 γIMC-Derived IL-6 Is Protective against Severe Invasive GAS Infection (A–C) C57BL/6 mice and Il6−/− mice intraperitoneally infected for 48 h with NIH34 (1.0 × 107 colony-forming units/mouse) were sacrificed. Splenocytes were immediately negative selected by magnetic separation, and CD11b+ CD11c− CD38+ IL-5Rα (T21)+ γIMC subsets were isolated by fluorescence-activated cell sorting. (A) The numbers (%) in the plots represent the population of CD38+ IL-5Rα (T21)+ γIMC subsets. (B and C) Il6−/− mice were then intravenously treated with 3.0 × 106 wild type γIMCs or with Il6−/− γIMCs combined with or without 500 ng IL-6. Finally, all mice were intraperitoneally inoculated with NIH34 (2.0 × 107 colony-forming units/mouse, n = 5 mice/group). (B) NIH34 colony-forming units in the peripheral blood 12 h post-infection. Bars indicate the average. ∗p < 0.05 by Mann-Whitney U test. (C) Survival post-infection. ∗p < 0.05 by log-rank test. Data are representative of two independent experiments. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 γIMC-Derived IL-6 Upregulates Mincle Expression (A–G) C57BL/6 mice, Il6−/− mice, and Mincle−/− mice intraperitoneally infected for 48 h with NIH34 (1.0 × 107 colony-forming units/mouse) were sacrificed. Splenocytes were immediately negatively selected by magnetic separation, and CD11b+ CD11c− CD38+ IL-5Rα (T21)+ γIMC subsets were isolated by fluorescence-activated cell sorting. (A and C) Il6−/− γIMCs were treated for 12 h with or without 100 ng/mL IL-6 (A) or with 1 μg/mL IL-6 neutralizing mAb (clone MP5-20F3) or control rat IgG in combination with 90% culture supernatant from wild type γIMCs stimulated with NIH34 for 24 h at a multiplicity of infection of 30 (C). Cells were analyzed for Mincle expression by flow cytometry using anti-Mincle mAb and an isotype control, represented by the solid and dashed lines, respectively. (B and D) Data are mean ± SD (B, n = 5 mice for each experimental cell group; D, n = 4 mice for each experimental cell group) of the geometric mean fluorescent intensity due to surface expression of Mincle. ∗∗p < 0.01 by Student’s t test. Data are representative of two independent experiments. (E) The numbers (%) in the plots represent the population of CD38+ IL-5Rα (T21)+ γIMC subsets. Cytospin preparations of each sorted cell subset were visualized with May-Grünwald-Giemsa staining. Scale bars, 20 μm. (F) Data are expressed as mean ± SD (n = 5 independent experiments) of CD38+ IL-5Rα (T21)+ γIMC subsets as % of total splenocytes. (G) RNA sequencing of wild type, Il6−/−, and Mincle−/− γIMCs (n = 3 independent experiments). Visualization of 18 cell types in two dimensions by principal component analysis of 20,596 protein-coding genes expressed in at least one sample. Left: first versus second principal component (PC1 versus PC2). Right: second versus third principal component (PC2 versus PC3). See also Figure S2 and Table S2. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 5 TLR2-Mediated GAS Recognition Induces IL-6 Production and Upregulates Mincle Signals Required for IFN-γ Production (A–E) Splenic CD11b+ CD11c− CD38+ IL-5Ra (T21)+ γIMCs were isolated by fluorescence-activated cell sorting from wild type, Il6−/−, or Mincle−/− mice infected for 48 h with NIH34 (1.0 × 107 colony-forming units/mouse). (A, C, and D) Cells were treated (A) or pretreated (C and D) for 12 h with or without 100 ng/mL IL-6. (B) Wild type γIMCs were pretreated for 1 h with 10 μg/mL TLR2 neutralizing mAb (clone T2.5) or control mouse IgG. (E) Il6−/− γIMCs were pretreated with 0, 1, or 100 ng/mL IL-6 for 12 h. (A) Cell lysates were analyzed by western blot for TLR2, TLR7, TLR9, TLR13, phosphorylated STAT3, STAT3, and β-actin. RAW264.7 cells were used as positive control for TLRs. (B, D, and E) Cells were treated with NIH34 at a multiplicity of infection of 30. Cell culture supernatants were harvested 24 h thereafter and assayed for IFN-γ (D and E) and IL-6 (B and D). Data are expressed as mean ± SD (n = 3 mice for each experimental cell group). ∗∗p < 0.01 by Student’s t test. (C) Cells were treated with NIH34 for the times indicated at a multiplicity of infection of 30, and cell lysates were analyzed by western blot for phosphorylated Syk, Syk, and β-actin. (F) C57BL/6 mice intraperitoneally non-infected (0 h) or infected for 30 h or 42 h with NIH34 (1.0 × 107 colony-forming units/mouse) were intravenously injected with monensin. Mice were sacrificed 6 h after that (36 h and 48 h), and splenocytes were immediately stained for IL-5Rα (T21) and indicated markers and analyzed by intracellular cytokine synthesis assay. Data are expressed as mean ± SD (n = 4 mice/group) of IL-6+, Mincle+, or IFN-γ+ subsets as % of IL-5Rα (T21)+ γIMCs. ∗∗p < 0.01 by Student’s t test. Data are representative of three (A–E) or two (F) independent experiments. See also Figure S3. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions

Figure 6 IL-6–Mincle Axis in γIMCs Confers Protection against Severe Invasive GAS Infection (A and B) Splenic CD11b+ CD11c− CD38+ IL-5Rα (T21)+ γIMCs were isolated by fluorescence-activated cell sorting from wild type, Il6−/−, or Mincle−/− mice infected for 48 h with NIH34 (1.0 × 107 colony-forming units/mouse). C57BL/6 mice were then treated intravenously with 3.0 × 106 wild type, Il6−/−, or Mincle−/− γIMCs, and intraperitoneally inoculated with NIH34 (5.0 × 107 colony-forming units/mouse, n = 6 mice/group). (A) NIH34 colony-forming units in peripheral blood 12 h post-infection. Bars indicate the average. ∗p < 0.05; ∗∗p < 0.01 by Mann-Whitney U test. (B) Survival post-infection. ∗∗p < 0.01 by log-rank test versus infected C57BL/6 mice. Data are representative of two independent experiments. (C) During severe invasive GAS infection, γIMCs are recruited, and upon constitutively expressed TLR2, instructed to produce IL-6 systemically. IL-6 serves as an alarm molecule, acting to upregulate Mincle expression in many γIMCs to induce effector IFN-γ production. Ultimately, Mincle-dependent IFN-γ may activate phagocytosis in macrophages and promote bacterial clearance and host defense. Cell Reports 2019 27, 561-571.e6DOI: (10.1016/j.celrep.2019.03.056) Copyright © 2019 The Author(s) Terms and Conditions