Fig 2: B cell development is negatively affected whenTOP2B is mutated. a Spleen to body weight ratio for wild-type (n = 3), Top2b-/--mb1 cre (n = 3), and Top2b+/EE587E mice (n = 4) reveal smaller ratios in knockin and knockout mice compared to controls (shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. b Total serum IgG is reduced in Top2b-/- mb1 cre mice (n = 6) and Top2b+/EE587E (n = 4) mice compared to controls (n = 7, shown as mean ± SEM). *p < 0.05 by Kruskal–Wallis test. c, d Heterozygous mutant mice fail to produce specific IgG antibody in response to vaccination with ovalbumin (OVA, n = 4, (c) or pneumovax-23 (d) (n = 3 mice per group, shown as mean ± SEM, *p < 0.05 by the Kruskal–Wallis test). Bone marrow (e) and spleen (f) cells were isolated from Top2b+/EE587E mice and littermate controls. Phenotypic analysis by flow cytometry demonstrates that the bone marrow of Top2b+/EE587E mice (n = 3) have fewer PrePro B cells (Fr. A, B220+, CD43-, BP1-, CD24-), Pre B cells (Fr. D, B220+, CD43-, IgD-, IgM-), and immature B cells (Fr. E, CD19+, B220+, CD43-, IgM+, IgD-) compared to control mice (e). In the spleen (f), there are overall fewer B cells with significant reductions in transitional (CD19+, B220+, IgDlo, IgM+, CD21/35int, CD23 variable), follicular (CD19+, B220+, IgMlo, IgDhi, CD38+) and activated (CD19+, B220+, IgDhi, IgMhi, Flt3+) B cells compared to control mice (n = 4, shown as mean ± SEM, absolute numbers calculated from whole tissue numbers). *p < 0.05; **p < 0.01, two-tailed Student’s t test. Expression of mutant Top2b results does not affect E2A (g, n = 6)) or Rag1 (h, n = 8) expression in common lymphoid progenitor cells (compared to WT n = 6), whereas B cell-specific transcription factors Pax5 (i) and Foxo1 (j) show reduced expression of compared to wild-type mice. (n = 4 mice per group, shown as mean ± SEM, *p < 0.05, two-tailed Student’s t test)

Fig 3: Assessments of organ damage and mouse metabolites during HLSA or recovery from HLSA.A, upper left panel, MMP-1, IL-1ß, and CRP levels in plasma had no significant change between the control group, HLSA group, and R-HLSA group. Upper right panel, CK-MB as a marker of heart injury in the plasma had no significant change between the control group, HLSA group, and R-HLSA group. Lower left panel, creatinine and BUN as markers of kidney injury in the plasma slightly increased in the HLSA group and returned to normal level after recovery from HLSA. Lower right panel, AST and ALT as markers of liver injury in the plasma showed no change between the control group, HLSA group, and R-HLSA group. Data were presented as mean ± SD (N = 6; ANOVA: **p < 0.01). B, upper panel, food consumption and water consumption 3 days before and 3 days after a single ATP-induced HLSA depicted no change. Data were presented as mean ± SD (N = 6; ANOVA: p > 0.05). Lower panel, metabolomics analysis of scores plot from PCA analysis based on 1H NMR data from the plasma, brain, liver, and kidney of the control group, HLSA group, and R-HLSA group. The PCA score showcased clusters correspond to metabolic patterns in different groups, with each point representing one sample. Circles represent 95% confidence interval for each score in each group (see also Figs. S5–S8). C, draining lymph nodes of mice that underwent HLSA once a day for 10 consecutive days were analyzed with FACS. CD44 and CD62L were used to quantify the percentage of effector T cells. Upper left panel, flow cytometry quantification of CD4+, CD8+ T cells in draining lymph nodes. Upper right panel, representative flow cytometry analysis dot plots of effector CD4+ T cells (CD44+ CD62L- of total CD4+ T cells). Lower left panel, representative flow cytometry analysis dot plots of effector CD8+ T cells (CD44+ CD62L- of total CD8+ T cells) are shown. Lower right panel, the percentage of CD4+, CD8+ T cells, effector CD4+ T cells, effector CD8+ T cells. Data were presented as mean ± SD (N = 6; Student's t test: p > 0.05). D, the spleens of mice that underwent HLSA once a day for 10 consecutive days were analyzed with FACS. B220 and CD11b were used to quantify immune cell populations. Upper left panel, flow cytometry quantification of CD4+, CD8+ T cells in spleens. Upper right panel, representative flow cytometry analysis dot of B cell (B220+) populations in the spleens. Lower left panel, representative flow cytometry analysis dot of effector CD11b+ T cells was shown. Lower right panel, the percentage of CD4+, CD8+ T cells, B cells, and CD 11b+ cells. Data were presented as mean ± SD (N = 6; Student's t test: p > 0.05). Cre, creatinine; Ctrl, control group; HLSA group, hibernation-like suspended animation; R-HLSA group, mice recovered from hibernation-like suspended animation.

Fig 4: Mouse bone marrow contains abundant non-proliferative adipocyte precursors.(A) Representative fluorescent images of 1-month-old Adipoq:Td:Col1a1-GFP (a–d) or Adipoq:Td (f–g) femur reveal many bone marrow Td+ cells. (a) A low magnification image of a distal femur. (b–g) At a high magnification, Td does not label chondrocytes in articular cartilage (b) and growth plate (c), osteoblasts and osteocytes (arrows, d) but labels pericytes (arrows, e), Perilipin+ adipocytes (f), and CD45- stromal cells (g). (B) According to BODIPY lipid staining, those Td+ stromal cells in a reticular shape (arrow) have no lipid accumulation. (C) In vivo EdU injection reveals that Td+ cells in Adipoq:Td mice do not proliferate. (D) CFU-F assay of bone marrow cells from Adipoq:Td mice shows that all CFU-F colonies are made of Td- cells (a). (b) Some Td+ cells do attach to the dish but have minimum proliferation ability. n = 5 mice. (E) Endosteal bone marrow Td+ cells from 1-month-old Col2:Td mice (5 out of 5 transplants), but not Adipoq:Td mice (0 out of 3 transplants) form bone-like structure after transplanted under the kidney capsule. Representative fluorescent images of transplants were shown here. Osterix: osteoblasts; CD45: hematopoietic cells; Calcein: new bone surface. (F) In vitro adipogenic differentiation assay demonstrates that non-lipid-laden adipocytes exist as an intermediate state between mesenchymal progenitors and lipid-laden adipocytes. Mesenchymal progenitors, which are Td-, were obtained from culturing endosteal bone marrow cells from 1-month-old Adipoq:Td mice. Upon confluency, cells were cultured in adipogenic differentiation medium. The same area was imaged daily by inverted fluorescence microcopy. (G) CFU-F assay of bone marrow cells from P23 AdipoqER:Td mice (Tamoxifen injections at P14-16) shows that all CFU-F colonies are made of Td- cells (a). (b) Some Td+ cells do attach to the dish but have minimum proliferation ability. n = 3 mice. (H) Immunofluorescence staining shows that Perilipin+bone marrow adipocytes are derived from non-lipid-laden adipocytes in 1-month-old AdipoqER:Td mice (Tamoxifen injections at P6, 7). (I) Representative images of co-localization of Td and Lepr in stromal (white arrows) and pericytes (yellow arrows) in bone marrow of 1-month-old Adipoq:Td mice.

Fig 5: miR-155 is required for ILC2 expansion in response to IL-25 and IL-33. WT and miR155-/- mice were treated with intra-peritoneal (i.p.) IL-33 (A–C) or IL-2/anti-IL-2/IL-25 complex (D), for 3 days. (A) Representative staining of ILC2 (CD45+Lin-ICOS+KLRG1+) isolated from mesenteric lymph node (MLN). (B) Quantification of ILC2 in MLN. (C) Percentage ST2+ WT and miR155-/- ILC2. (D) Quantification of ILC2 following IL-2/anti-IL-2/IL-25 (IL-25/IL-2C) complex treatment. Data (A,B,D) are representative of three independent experiments; (C) a single experiment. *p = 0.05 with Student's t-test.