Volume 44, Issue 6, Pages 1365-1378 (June 2016) Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock  Oliver H. Voss, Yousuke Murakami, Mirna Y. Pena, Ha-Na Lee, Linjie Tian, David H. Margulies, Jonathan M. Street, Peter S.T. Yuen, Chen-Feng Qi, Konrad Krzewski, John E. Coligan  Immunity  Volume 44, Issue 6, Pages 1365-1378 (June 2016) DOI: 10.1016/j.immuni.2016.05.005 Copyright © 2016 Terms and Conditions

Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 1 LPS Is a Ligand for CD300b (A–C) Sensorgrams of mCD300b-Fcγ (A), hCD300b-Fcγ (B), mCD300d-Fcγ, mCD300a-Fcγ, mCD300f-Fcγ, and NITR-Fcγ (C) protein binding to immobilized LPS (E. coli 0111:B4) over the indicated times. Binding was initiated at 60 s and the dissociation phase begun at 240 s and is expressed in resonance units (RU). (D) Streptavidin pull-down assays determining the binding of mCD300b-Fcγ (2, 5, and 10 μg [lanes 2–4]) or 10 μg of mCD300d-Fcγ, mCD300f-Fcγ, or NITR-Fcγ proteins to biotin-conjugated LPS (2 μg). Bound protein was determined by immunoblotting using an anti-human IgG Fcγ-specific Ab. hIgG indicates human Ig heavy chain. (E and F) mCD300b-, mCD300b-DAP12-, mCD300d-FcRγ-, mCD300f-, EV-expressing L929 cells were incubated with FITC-labeled LPS from E. coli or S. minnesota (10 μg/mL) for 1 hr at 37°C (E) or 4°C (F). Binding was analyzed by flow cytometry and expressed as mean fluorescence intensity (MFI). (G) mCD300b-DAP12-expressing L929 cells were incubated with FITC-labeled LPS from E. coli (10 μg/mL) for 1 hr and mixed with increasing concentrations of unlabeled LPS from E. coli, S. minnesota, or the TLR2 ligand Zymosan A (negative control). Samples analyzed by flow cytometry and MFI values from reactions without unlabeled E. coli LPS were considered as the maximum binding level (0% inhibition). Data in (A)–(D) are representative of three experiments. Error bars represent SEM (E–G); ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001. See also Figures S1 and S2. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 2 CD300b Augments the Pathogenesis of Lethal Endotoxemia and Septic Peritonitis (A) WT and Cd300lb−/− mice (n = 12) were injected i.p. with a toxic dose of LPS (37 mg/kg) or the diluent control PBS (n = 3). Survival was monitored every 6 hr for 7 days. (B) H&E staining of lung tissues from PBS- and LPS-treated WT and Cd300lb−/− mice. (C) Serum cytokine concentrations measured from PBS- and LPS-treated WT and Cd300lb−/− mice. (D) WT and Cd300lb−/− mice (n = 12) were subjected to cecal ligation puncture (CLP) or laparotomy without ligation and puncture (sham-control, n = 3). Survival was monitored every 6 hr for 7 days. (E) H&E staining of lung tissues from CLP-treated WT and Cd300lb−/− mice. (F) Serum cytokine concentrations measured from CLP-treated WT and Cd300lb−/− mice. Data in (B) and (E) are representative from five mice per group. Graphs in (C) and (F) show mean values ± SEM from five mice per group. NS, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. See also Figure S2. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 3 Neutralization of IL-10 Augments the Pathogenesis of Lethal Endotoxemia and Septic Peritonitis (A) Anti-IL-10- or control Ab-treated WT and Cd300lb−/− mice (n = 12) were i.p. injected with a toxic dose of LPS (37 mg/kg). Survival was monitored every 6 hr for 7 days. (B) H&E staining of lung tissues from anti-IL-10- or control Ab-treated WT and Cd300lb−/− mice after LPS treatment. Data are representative from five mice per group. (C) Serum cytokine concentrations measured from anti-IL-10- or control Ab-treated WT and Cd300lb−/− mice after LPS treatment. Graphs show mean values ± SEM from five mice per group. NS, not significant; ∗p ≤ 0.05 and ∗∗p ≤ 0.01. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 4 CD300b-Expressing Macrophages Are Responsible for Augmenting the Pathogenesis of LPS-Induced Lethal Endotoxemia (A) Liposome-encapsulated PBS- or dichloromethylene biphosphate (Cl2MBP)-treated WT and Cd300lb−/− mice (n = 12) were i.p. injected with a toxic dose of LPS (37 mg/kg). Survival was monitored every 6 hr for 7 days. (B) H&E staining of lung tissues from PBS- and Cl2MBP-liposome-injected WT and Cd300lb−/− mice after LPS treatment. Data are representative from five mice per group. (C) Serum cytokine concentrations measured from PBS- and Cl2MBP-liposome-injected WT and Cd300lb−/− mice after LPS treatment. Graphs show mean values ± SEM from five mice per group. NS, not significant; ∗p ≤ 0.05 and ∗∗p ≤ 0.01. See also Figure S3. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 5 CD300b-Expressing BMMs Increase the Severity of Lethal Endotoxemia by Altering the Levels of Cytokines Produced (A) BMMs from WT or Cd300lb−/− mice were stimulated with 2 μg/mL of LPS for various lengths of time. Cytokine levels were assessed by flow cytometry. No differences in cytokine levels between diluent control-treated WT and Cd300lb−/− BMMs was observed (data not shown). (B) BMMs from WT, Cd300lb−/−, or Cd300lb−/−Il10−/− mice were intravenously transferred into WT or Cd300lb−/− animals (n = 15) 24 hr prior to i.p. injection with a toxic dose of LPS (37 mg/kg). Survival was monitored every 6 hr for 7 days. (C) Serum cytokine concentrations measured from WT, Cd300lb−/−, or Cd300lb−/−Il10−/− BMM-injected WT or Cd300lb−/− animals 2 hr after LPS treatment. The graphs show mean values + SEM from three independent experiments (A) or ± SEM from five mice per group (C). NS, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001. See also Figure S4. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions

Figure 6 LPS Binding by CD300b Promotes the Association with TLR4-MyD88-TIRAP Complex and Dampens the Production of IL-10 via DAP12-Syk-PI3K Recruitment (A) BMMs from WT, Cd300lb−/−, Cd14−/−, or Tlr4−/− mice were lysed and analyzed by immunoblotting with the indicated Abs. GAPDH was used as a loading control. (B) Purified mCD300b, mTLR4, and mCD14 proteins were incubated in the presence or absence of LPS (2 μg/mL), followed by the addition of dithiobissuccinimidyl propionate (DSP) where indicated. Samples were immunoprecipitated with anti-CD300b or anti-IgG isotype control Ab and analyzed by immunoblotting with the indicated Abs. (C and D) BMMs from WT or Cd300lb−/− mice were stimulated with LPS (2 μg/mL) for various lengths of time. Reactions were immunoprecipitated with anti-CD300b (C), anti-TLR4 (D), or anti-IgG isotype control Ab (C and D), and then analyzed by immunoblotting with the indicated Abs. (E) BMMs from WT mice were pretreated for 12 hr with anti-IgG isotype control Ab, anti-CD300b Ab, anti-CD14 Ab, or both anti-CD300b and anti-CD14 Abs before the addition of LPS (2 μg/mL). Cell lysates were immunoprecipitated with anti-TLR4 or anti-IgG isotype control Ab and samples were analyzed by immunoblotting with the indicated Abs. (F and G) BMMs from WT, Cd300lb−/−, or Cd300lf−/− mice were stimulated with LPS (2 μg/mL) for various lengths of time and cell lysates were analyzed for the levels of phosphorylated (F) or total protein expression (G) by immunoblotting with the indicated Ab. (H) BMMs from WT or Cd300lb−/− mice were treated with p38 inhibitor SB203580 (10 μM) and/or the ERK1/2 inhibitor PD98059 (25 μM) for 1 hr prior to stimulation with LPS (2 μg/ml) for additional 2 hr. The levels of p38 and ERK1/2 phosphorylation were analyzed by immunoblotting with the indicated Ab. (I) IL-10 cytokine levels in the culture medium from WT or Cd300lb−/− BMMs treated with SB203580 or PD98059 as determined by flow cytometry. Data in (A)–(H) are representative of three experiments. Error bars represent SEM from three experiments (I). ∗p ≤ 0.05 and ∗∗p ≤ 0.01. See also Figures S4 and S5. Immunity 2016 44, 1365-1378DOI: (10.1016/j.immuni.2016.05.005) Copyright © 2016 Terms and Conditions