G-CSF partially mediates effects of sleeve gastrectomy on the bone marrow niche

Abstract

Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.

Keywords: Adipose tissue; Bone Biology; Bone marrow; Endocrinology; Obesity.

Figure 1. Loss of bone and BMAT after VSG is independent of mechanical unloading and diet.

After consumption of HFD for 8 weeks, male C57BL/6J mice at 12 weeks of age received sham or VSG surgery. Each group was then fed NCD or HFD for a further 8 weeks. (A) Body weight and fat mass were measured before euthanasia (n = 6 for NCD+Sham; n = 7 for NCD+VSG; n = 6 for HFD+Sham, and n = 9 for HFD+VSG). (B) Tibial Tb. BV/TV, Tb. BMD, Tb. N, and Tb. Sp were measured with μCT. (C) Mid-tibial Ct. BA/TA and Ct. Th (n = 5 for NCD+Sham; n = 6 for NCD+VSG; n = 4 for HFD+Sham, and n = 5 for HFD+VSG). (D) Femoral stiffness, yield load, and maximum load in HFD-fed mice (n = 8 for Sham and n = 7 for VSG). (E) Representative sections from proximal and distal tibiae were stained with H&E and are shown at ×200 magnification. Scale bars, 100 μm. (F) Tibial BMAT was visualized and quantified relative to total bone volume within the indicated regions (n = 5 for NCD+Sham, n = 6 for NCD+VSG, n = 4 for HFD+Sham, and n = 5 for HFD+VSG). Growth plate (G/P) to tibia/fibula junction (T/F J). Distal tibia is T/F J to distal end. Box and whisker plots show a central median line, boxed 25th and 75th quartiles and the whiskers mark the range. Differences between treatments were evaluated by 1-way ANOVA with Tukey’s multiple comparisons test (A–D and F). P values in panels were adjusted across the experiment for multiple testing using limma package in R with FDR method. *Statistical difference at P < 0.05.

Figure 2. VSG causes rapid loss of bone and BMAT in lean mice.

Male mice on a NCD received sham or VSG surgery at 12 weeks of age and were euthanized 1, 2, or 4 weeks later. (A) Body weight and fat mass were determined at indicated times (n = 20 for Sham and n = 40 for VSG at 1 week; n = 12 for Sham and n = 30 for VSG at 2 weeks; n = 7 for Sham and n = 18 for VSG at 4 weeks). (B) Ct. BA/TA and Ct. Th were measured by μCT. (C) Tb. BV/TV, Tb. BMD, Tb. N, and Tb. Sp were also determined (n = 5 for Sham and n = 9 for VSG at 1 week; n = 5 for Sham and n = 12 for VSG at 2 weeks; n = 7 for Sham and n = 8 for VSG at 4 weeks). Representative sections from proximal (D) and distal (E) tibiae 1 or 2 weeks after surgery were stained with H&E and are shown at ×200 magnification. Scale bars, 100 μm. Decalcified tibiae were stained with osmium tetroxide and volume of BMAT determined for both locations relative to total bone volume (n = 10 for Sham; n = 9 for VSG at 1 week and n = 11 for VSG at 2 weeks). *Statistical difference at P < 0.05 by 2-way ANOVA with Sidak’s multiple comparisons test (A–C) and 1-way ANOVA with Tukey’s multiple comparisons test (D and E). P values across A to C were adjusted for multiple testing using limma package in R with FDR method.

Figure 3. Loss of bone after VSG is due to decreased bone formation and impaired mineralization.

Male mice on a NCD received either sham or VSG surgery at 12 weeks of age and were euthanized 2 weeks later. Mice were intraperitoneally injected with calcein (20 mg/kg) 9 and 2 days prior to euthanasia. (A) Tibial bone surface (BS) and osteoblast number (Ob. N) were determined on H&E-stained decalcified sections and calculated by Bioquant software. Tibial osteoclast surface (Oc. S) was determined from decalcified TRAP-stained sections and normalized by bone surface (n = 9 for each group). (B) Undecalcified femurs were embedded in plastic, sectioned, and stained with Masson Trichrome to calculate osteoid surface (S)/bone surface (BS) and osteoid width. Arrows indicate the borders of osteoid surface (n = 10 for Sham and n = 11 for VSG). (C) Calcein-labeled mineralized femurs were sectioned and fluorescence was visualized. Distance between calcein incorporation into mineralized matrix is shown by arrows. (D) Analyses of fluorescent double-labeled surface was used to calculate bone formation rate, mineral apposition rate, and osteoid maturation time using Bioquant software (n = 10 for Sham and n = 11 for VSG). (E) Expression of Sp7, Alpl, and Bglap mRNAs from whole femur tissue was analyzed by qPCR and normalized by the expression of the geometric mean of housekeeping genes Hprt, Rpl32A, and Tbp (n = 5 for Sham and n = 9 for VSG at 1 week; n = 5 for Sham and n = 12 for VSG at 2 weeks). *Statistical difference for indicated comparisons at P < 0.05 by 2-sample t test (A, B, and D) and 2-way ANOVA with Sidak’s multiple comparisons test (E). P values across A to D were adjusted for multiple testing using limma package in R with FDR method.

Figure 4. Bone mass after VSG is inversely correlated with myeloid cell expansion.

Male mice at 4 weeks of age were fed a 60% HFD for 8 weeks and then received either sham or VSG surgery. Mice remained on a HFD until euthanasia 8 weeks after surgery. (A) Wright-Giemsa staining was performed on a femoral bone marrow touch preparation. Representative pictures at ×1000 magnification are shown. Scale bars, 20 μm. Yellow arrows show neutrophils and their myeloid precursors; red arrows show erythroid cells. (B) Blind counting of myeloid and erythroid cells was performed by a board-certified pathologist. Linear regression on the relationship between myeloid/erythroid cell ratio and Ct. BA/TA was performed (n = 15 for Sham and n = 10 for VSG). (C) Circulating proportion of neutrophils versus total white blood cells (WBCs) was calculated after performing a CBC. Linear correlations between neutrophil proportion and Tb. BV/TV or Ct. BA/TA are shown (n = 20 for Sham and n = 22 for VSG). (D) Hematocrit was calculated from the CBC. Linear regression demonstrates the positive correlation between hematocrit and Tb. BV/TV (n = 28 for Sham and n = 14 for VSG). *Statistical difference for indicated comparisons at P < 0.05 by 2-sample t test (B–D).

Figure 5. VSG in mice and humans rapidly increases circulating G-CSF, which in mice is sufficient to cause loss of bone and BMAT.

(A) Circulating G-CSF levels in male C57BL/6J mice were measured at 1, 2, 4, and 8 weeks (right panel) after sham or VSG surgery (Sham, n = 5 and VSG, n = 9 at 1 week; Sham, n = 5 and VSG, n = 11 at 2 weeks; Sham, n = 7 and VSG, n = 12 at 4 weeks; Sham, n = 10 and VSG, n = 16 at 8 weeks). (B) Female young patients underwent VSG surgery. Circulating G-CSF concentrations before (n = 22) and at 1 (n = 22), 3 (n = 17), 6 (n = 12), and 12 (n = 4) months after surgery. (C–G) C3H/HeJ mice at 12 weeks of age were implanted with osmotic minipumps containing saline (–) or recombinant murine G-CSF (+) (3 μg/mouse). Animals were sacrificed 4 weeks after implantation (n = 5 for each group). (C) Circulating G-CSF concentrations at 4 weeks. (D) Tibial trabecular characteristics including Tb. BV/TV, Tb. BMD, Tb. N, and Tb. Sp. The inverse correlation between Tb. BV/TV and circulating G-CSF concentrations is shown. (E) Ct. BA/TA and Ct. Th were measured. Linear regression reveals the inverse correlation between Ct. BA/TA and circulating G-CSF concentrations. (F) Proximal and distal tibial BMAT volume was determined by osmium staining and μCT. (G) Representative H&E-stained sections from proximal and distal tibiae with ×200 magnification are shown. Scale bars, 100 μm. *Statistical difference for indicated comparisons at P < 0.05 by 2-way ANOVA with Sidak’s multiple comparisons test (A), Wilcoxon matched-pair signed rank test (B), and 2-sample t test (C–F). P values across C to F were adjusted for multiple testing using limma package in R with FDR method.

Figure 6. G-CSF is not required for VSG-induced bone loss, but is necessary for complete effects on circulating neutrophils and BMAT.

Female Csf3^–/– mice and their littermates (WT) at 12 weeks of age received a sham (–) or VSG (+) surgery and were euthanized 4 weeks later. (A) Circulating G-CSF concentrations 1 week after surgery. (B) Ct. BA/TA and Ct. Th were measured by μCT. (C–D) Tb. BV/TV, Tb. BMD, Tb. N, Tb. Th, and Tb. Sp were determined (A–D: n = 9 for WT+Sham; n = 9 for WT+VSG; n = 14 for Csf3^–/–+Sham and n = 9 for Csf3^–/–+VSG). (E) Circulating neutrophil number at the indicated times after surgery (n = 10 for WT+Sham; n = 12 for WT+VSG; n = 15 for Csf3^–/–+ Sham and n = 15 for Csf3^–/–+VSG at 1 week; n = 9 for WT+Sham; n = 11 for WT+VSG; n = 15 for Csf3^–/–+Sham, and n = 13 for Csf3^–/–+VSG at 2 weeks; n = 6 for WT+Sham; n = 8 for WT+VSG; n = 10 for Csf3^–/–+Sham, and n = 7 for Csf3^–/–+VSG at 4 weeks). Representative H&E sections from (F) proximal and (G) distal tibiae at ×200 magnification are shown (n = 10 for WT+Sham; n = 8 for WT+VSG; n = 14 for Csf3^–/–+Sham, and n = 7 for Csf3^–/–+VSG). Scale bars, 100 μm. Decalcified tibiae were stained with osmium tetroxide and BMAT volume determined by μCT and normalized by bone marrow (BM) volume. *Statistical difference for indicated comparisons at P < 0.05 by 1-way ANOVA with Tukey’s multiple comparisons test (A–D and F–G) and 2-way ANOVA with Sidak’s multiple comparisons test (E). P values across the experiment were adjusted for multiple testing using limma package in R with FDR method.