Fig 2: Neutrophil depletion rescues the enhanced bacterial burden in the lungs of CD200R-/- mice. WT and CD200R-/- C57BL/6 mice were treated IP with 50 µg anti-Ly6G or Rat IgG2a at day -1, 1, 3 and 5 of F. tularensis infection. The challenge dose of ˜1000 CFU F. tularensis LVS was administered IN. a Representative dot plots confirming neutrophil depletion in the lung, with neutrophil gated as single, live, CD45+, Gr1HiLy6CINT. b Quantification of lung neutrophils at day 7 p.i. c Bacterial burden (CFU/g) of the lung was enumerated at day 7 p.i. Data represent three independent experiments and data are shown as mean ± SD (n = 15). Statistical analysis was performed using two-way ANOVA (*p < 0.01, ****p < 0.0001)

Fig 3: Lack of CD200R in mice causes exacerbated pulmonary F. tularensis infection via neutrophil-dependent mechanisms. Pulmonary challenge with F. tularensis LVS causes an excessive neutrophil influx in the lung of CD200R-/- mice compared with WT. Due to a reduced ROS output from CD200R-/- neutrophil, they provide a replicative niche for F. tularensis LVS, leading to an increased burden in CD200R-/- neutrophils. This ultimately leads to an increased overall F. tularensis LVS burden in the lung of CD200R-/- mice compared with WT

Fig 4: CD200R-/- neutrophils show enhanced expression of proteins associated with reactive oxygen species (ROS) clearance and reduced ROS output. Neutrophils were isolated by negative selection from WT and CD200R-/- C57BL/6 mouse bone marrow. a Heatmap of selected genes identified in RNA-seq analysis of primary neutrophils isolated from 3 WT and 3 CD200R-/- mice. Signal intensity represents gene expression change following mean centring scaling. b, c WT and CD200R-/- neutrophils were incubated for 15 min with Dihydrorhodamine 123 (DHR123) (25 ng/ml), followed by 1 h stimulation with Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml). b Representative histograms of FITC DHR123 expression in unstimulated or PMA-treated WT and CD200R-/- neutrophils. c Oxidative index was quantified using the following equation: mean fluorescence intensity (MFI) Stimulated/MFI Unstimulated. Alternatively, neutrophils were infected with F. tularensis MOI 100 and incubated with DHR123 for 24 h. d Representative histograms of FITC DHR123 expression in unstimulated and F. tularensis-infected neutrophils. e Oxidative index was quantified using the following equation: MFI F. tularensis-infected/MFI unstimulated. Data represents b, c four or d, e two independent experiments and is shown as mean ± SD (n = 12 and 6, respectively). Statistical analysis was performed using unpaired t-test (*p < 0.01)

Fig 5: TGFß in obese mouse livers limits NK cell degranulation and alters their metabolic profile. (A) Tgfb1 mRNA in the livers of lean and obese mice, normalized so that mean Tgfb1 transcript expression in the livers of lean mice = 1. (B,C) TGFß1 in lean and obese liver conditioned medium (B) and lean and obese mouse plasma (C) measured by ELISA. n = 8 mice per group (A,C) and six conditioned media per group (B); significance was determined using Mann Whitney U-Tests; medians and IQRs are shown. (D) Splenic NK cells were cultured for 24 h in medium alone, lean or obese liver conditioned medium, or 10 ng/ml TGFß1. For the last 4 h of culture, CD107a antibody and Brefeldin A were added to media. n = 6 culture conditions per group; significance was determined using Mann Whitney U-Tests with Holm's correction for multiple comparisons; medians and IQRs are shown. (E) Splenic NK cells were cultured as described in (D), with the addition of 10 µg/ml anti-TGFß antibody or isotype control. n = 6 culture conditions per group; significance was determined using a Wilcoxon signed ranks test, where each pair is obese liver conditioned medium from the same mouse, with anti-TGFß antibody or isotype control. Degranulation in NK cells cultured in lean liver conditioned medium with isotype control are shown for comparison, but were not statistically tested. (F) Splenic NK cells were cultured for 24 h in medium alone, lean or obese liver conditioned medium, or 10 ng/ml TGFß1, before staining for pS6. n = 6 culture conditions per group; significance was determined using Mann Whitney U-Tests with Holm's correction for multiple comparisons; medians and IQRs are shown. (G,H) Splenic NK cells were cultured for 5 d in 10 ng/ml TGFß1. CD49a (G) and CD200R (H) staining before and after culture are shown. For this small dataset, no statistical testing was carried out.