Hematopoietic Progenitors Rapidly Respond to High Fat Diet Mediated Damage.

<p>(a) A representative gating scheme to isolate KLS and SP-KLS cells is shown. HSCs (SP-KLS) were assessed to determine the hematopoietic response to obesity driven leukocyte mis-allocation but no differences were found at any time point studied. (b) Hematopoietic progenitors rapidly respond to obesity mediated damage displaying an early response to HFD with HF animals having elevated KLS populations at 1 week. By 6 weeks this response was no longer evident in HF animals. *<i>P</i><0.05 vs. RD.</p

High Fat Diet Alters the Total Population Sizes of BM Leukocytes.

<p>(a) Total BM cells harvested for flow cytometry were not different between HF and RD animals at any time point, but a trend of increase was observed at 6W. (b) The total number of B-cells was not changed at 2D or 1W contrary to a decline in the phenotypic population at 1W. However, B-cells were reduced at 6W, despite a slight increase in total cellularity, demonstrating the degree of population reduction. (c,d) Total population sizes of both T-cells and Myeloid lineages tracked well with the phenotypic population proportions. *<i>P</i><0.05 vs. RD.</p

High Fat Diet Induces an Obese Phenotype.

<p>(a,b) At 6 weeks HF animals have expanded total abdominal adiposity with a prominent shift in the location of this adiposity from primarily subcutaneous to the visceral compartment. (c) The expansion and relocation of adiposity is illustrated in 3D reconstructions of abdominal μCT images which have been thresholded for adiposity (n = 9). In these images gray represents subcutaneous fat and pink represents visceral fat. *<i>P</i><0.05 vs. RD.</p

HFD Developmentally Decreases B Lymphopoiesis: RT-PCR was performed on whole BM extracted from the femur.

<p>(a,b) Il-7 and Ebf-1, early markers of lymphoid commitment showed strong trends of reduced expression after only 2D of HFD and were significantly reduced in HF animals from 1W onward. (c) Pax-5 expression, which represents an intermediate step in B lymphopoiesis did not appear reduced at 2D, but was significantly decreased subsequently. (d) Runx1 and Crebbp, which are critical regulators of hematopoiesis, were down regulated following 6 weeks of HFD demonstrating that obesity impacts the commitment and differentiation leukocytes as well as HSCs themselves. However, the expression of CXCL-12 and Angiopoietin-1 were not influenced by obesity (6W), demonstrating that the impact of obesity on IL-7 signaling is specific. *<i>P</i><0.05 vs. RD.</p

High Fat Diet Initiates an Aged Leukocyte Phenotype in the Bone Marrow.

<p>(a) A two-color flow cytometric method was used to isolate leukocyte populations of the lymphocyte (B and T-cells) and myeloid lineages, with the unstained control displayed in the left panel. (b–d) At two days a T-lymphopenia was present. At one week of HFD B-cell populations were diminished in the HF group, while T-cell populations were not significantly affected. After six weeks bone marrow lymphocyte populations were depressed and the myeloid lineage proliferated in response to HFD. The suppression of lymphocytes accompanied by myeloid expansion is a classic phenotype of hematopoietic aging. These changes may lead to defects in adaptive immunity. Each time point was drawn from distinct cohorts of animals, as such, comparisons can only be made within a time point due to variability in processing. *<i>P</i><0.05 vs. RD.</p

Six Weeks of HFD Rapidly Remodels the Bone Marrow Towards Adiposity.

<p>(a,b,d) The percentage of BM space taken up by adipose tissue, as assessed by histology, increased by almost 4 fold in HFD animals compared to control, an increase at least partially due to increasing adipocyte size (n = 6). (c) This leads to an increase in BM triglyceride storage (n = 10). The inclusion of adipocytes in the BM is significant because of the deleterious paracrine and inflammatory consequences adipocytes are thought to present to hematopoiesis. Additionally, BM adiposity in general represents a decline in BM quality and health, as is demonstrated by increased BM adiposity in osteoporosis and anorexia nervosa. *<i>P</i><0.05 vs. RD.</p

High Fat diet leads to a significant expansion of epidydimal fat after 1 and 6 weeks compared to RD controls, demonstrating the rapid accretion of adiposity in mice fed this diet.

<p>*<i>P</i><0.05 vs. RD.</p

Trabecular bone chemical composition using FTIRI.

<p>A) Level of mineralization as a proportion of the inorganic to organic matrix, in the trabecular bone of irradiated mice compared to control at 2d, 10d, and 8w following irradiation. *p<0.05 compared to age-matched control. B) A typical FTIRI heat map of the level of mineralization in a control (left) and an irradiated (right) trabecular strut at 2d. Increased intensity corresponds to increased degree of mineralization.</p

Mechanical properties of trabecular bone at 2d, 10d, and 8w following irradiation.

<p>As early as 2d following irradiation, alterations in the trabecular bone led to an increase in hardness and elastic modulus which was no longer present at 10d. *p<0.05 compared to control.</p

Light microscope image of mouse tibiae stained with Modified Wright Giemsa at 10d following irradiation.

<p>Image on the left is a control, and on the right is an irradiated mouse. The empty spaces in the marrow of the irradiated bone correspond to the rapid infiltration of fat cells, a consequence of which is that there is less space for immune cells to occupy the bone marrow space. Scale bar represents 1 mm.</p