Figure 2. Ketogenic diet (KD) feeding alters HSC populations and bone marrow-derived macrophages (BMDMs) from KD-fed mice show a hyper-inflammatory response to lipopolysaccharide (LPS) ex vivo.

Bone marrow was extracted from the femurs and tibias of age-matched (6–8 weeks) female BALB/c mice fed standard chow (SC), Western diet (WD), or KD for 2 weeks. (A) Fluorescence-Activated Cell Sorting (FACS) plots of total HSCs (CD201⁺CD27⁺) and (B) LT-HSCs, ST-HSCs, and multipotent progenitors (MPPs) from mice fed SC, WD, or KD for 2 weeks. Quantification of (C) the total numbers of LT- and ST-HSCs, and MPPs in bone marrow from mice fed SC, WD, or KD for 2 weeks. Next, BMDMs were plated at 5×10^⁶ cells/mL and differentiated for 7 days in media supplemented with macrophage colony-stimulating factor. Cells were split and plated in 24-well plates to adhere for 12 hr and treated with media (Ctrl) or LPS (24 hr; 10 ng/mL). Supernatants were assessed via ELISA for (D) TNF and (E) IL-6 secretion at 24 hr post-LPS treatment. IL-6 Ctrl supernatants were below the limit of detection; ND = no data. For (A-E), all experiments were run three times, and data are representative of one experiment, n=5 per diet group. (C) A Mann Whitney test was used for pairwise comparisons. (D, E) For all plates, all treatments were performed in triplicate, and a student’s t-test was used for statistical significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Error bars show the mean ± SD.

Figure 2—figure supplement 1. Ketogenic diet (KD) does not alter multipotent progenitor (MPP) differentiation or basal inflammation in bone marrow-derived macrophages (BMDMs), and monocytes and splenocytes show a hyper-inflammatory response to lipopolysaccharide (LPS) ex vivo.

Age-matched (6–8 weeks) conventional, wild-type, female BALB/c mice were fed standard chow (SC), Western diet (WD), or KD for 2 weeks. Bone marrow was extracted from the femurs and tibias of mice, HSCs were isolated via FACS, and (A) MPPs were quantified. BMDMs were plated at 5×10⁶ cells/mL and differentiated for 7 days in media supplemented with macrophage colony-stimulating factor. Cells were split and plated in 24-well plates to adhere for 12 hr and treated with media (Ctrl) for 24 hr. Supernatants were assessed via ELISA for (B) TNF and IL-6 secretion. Monocytes were isolated from the femurs and tibias of mice and plated at 2×10⁶ cells/mL. RNA was extracted from (C) untreated monocytes (0 hr) or (D) monocytes with LPS (10 ng/mL) for 2 hr. Expression of Tnf and Il6 was analyzed via qRT-PCR. Splenocytes were isolated and plated at 1×10⁶ cells/mL. RNA was isolated from (E) untreated splenocytes (0 hr) or (F) splenocytes treated with LPS (10 ng/mL) for 2 hr. Expression of Tnf and Il6 was analyzed via qRT-PCR. For (A, B), all experiments were run three times, and data are representative of one experiment, n=5 per diet group. For (C-F), all experiments were run twice, and data are representative of one experiment, n=3 per diet group.(A) A Mann Whitney test was used for pairwise comparisons. (B-F) For all plates, all treatments were performed in triplicate, and a student’s t-test was used for statistical significance. For all panels, * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Error bars show mean ± SD.

Figure 2—figure supplement 2. Gating strategy for HSCs, related to Figure 2.

Cells were gated in FSC-A against SSC-A. Doublets were excluded using FSC-A against FSC-H and subsequently SSC-A against SSC-H. Viable cells were gated, and lineage-committed cells were excluded. Within the lineage-negative cells, the CD201⁺CD27⁺ population was gated. In a CD150 against CD48 plot, the CD201⁺CD27⁺ cells were divided into LT-HSC, ST-HSC, multipotent progenitor (MPP), and the remaining CD150⁻CD48⁻ population. MPPs were characterized as MPP3 and MPP4 by their surface expression of CD34 and Flt3.