Page 54 - Haematologica-April 2018

M.J. Kraakman et al.
obesity to be associated with a small but significant increase in the risk of developing AML.11,12 While the exact biological mechanisms underpinning the increased leukemia risk in obesity are likely to be complex and mul- tifactorial, the presence of chronic low-grade inflamma- tion induced by obesity is thought to contribute to the increased cancer risk.13 Chronic low-grade inflammation is broadly characterized by alterations in circulating immuno-modulatory cytokines and leukocytes within the adipose tissue as well as the activation of stress path- ways in metabolically important tissues.14 We and others have recently reported that obesity causes prominent monocytosis due to enhanced myelopoiesis and altered hematopoiesis driven by interleukin-1β (IL-1β).15,16 Interestingly, IL-1β, along with other myeloid promoting cytokines (i.e., IL-3, granulocyte-macrophage colony- stimulating factor [GM-CSF]) has also been shown to pro- mote the progression of AML.17 Thus, we sought to inves- tigate the influence of obesity on the transition of MDS to AML and survival. We hypothesized that obesity- induced inflammation would promote the progression of MDS to AML through heightened myelopoiesis and hematopoietic stress.
Methods
Detailed methods are available in Online Supplementary Methods
10-week-old male Ob/Ob mice, along with littermate lean controls, purchased from the Jackson Laboratory (USA), under- went a bone marrow transplant (BMT), receiving marrow from 6/8-week-old male wild-type (WT) C57bl/6 mice (Jackson Laboratory) or male NHD13 mice sourced from colonies main- tained within the Alfred Medical Research Education Precinct (AMREP) Animal Centre. All animal experiments were approved by the AMREP Animal Ethics Committee and conducted in accordance with the National Health and Medical Research Council of Australia Guidelines for Animal Experimentation (Ethics E/1444/2014/B).
Results
Obese mice exhibit a hematopoietic phenotype after seven months of myelodysplastic syndrome
To determine the impact of obesity on the develop- ment of MDS, we performed bone marrow (BM) trans- plantation studies using NHD13 transgenic donor mice or WT littermate BM as a control into lean (Ob/+) and obese (Ob/Ob) recipient mice (Figure 1A). We considered the diet-induced obesity (DIO) model, but explicitly chose to conduct this experiment in Ob/Ob mice for the following reasons: i) Ob/Ob mice are guaranteed to maintain and gain weight following a BM transplant, which is in con- trast to our prior experience whereby WT mice had lim- ited weight gain post-BMT on a high fat diet, ii) the loss of leptin means these mice will feed consistently, and removes a variable of changes in feed patterns as the myelodysplasia progresses, iii) the diets are matched, rul- ing out effects of altered nutritional composition of stan- dard chow and high fat diets, and iv) we have previously shown that Ob/Ob and DIO mice display enhanced myelopoiesis through the same mechanism (i.e., increased IL-1β production emanating from the adipose
tissue).15 We acknowledge that leptin plays a role in hematopoiesis, but this is mainly restricted to the lym- phoid system,18 which is suppressed in MDS and not like- ly to be a confounding factor in our experiment. Moreover, we have shown that supplementing Ob/Ob mice with leptin, while causing weight reduction, had no impact on myelopoiesis.15
The MDS model we chose to employ was the NHD13 transgenic mouse, which overexpresses a NUX98- HOXD13 fusion protein that has been associated with human MDS.19 These mice have an MDS phenotype that can develop into AML with a penetrance of ∼30% within 14 months.19 Accordingly, we initially assessed the impact of obesity on hematopoiesis, and specifically myelopoiesis, seven months post-transplantation with BM from NHD13 transgenic donor mice, a time point at which all mice remained alive (i.e., the predicted half- way survival point of the model).
Total blood cell counts revealed the expected MDS fea- tures of decreased white blood cells (WBC) and red blood cells (RBC), effects that were observed in both lean and obese MDS mice (Figure 1B). As expected, the decrease in WBC counts was primarily due to lymphope- nia (Figure 1C). Platelets were also significantly decreased in both lean and obese MDS mice. These changes occurred despite obese mice presenting with increased platelet numbers in the healthy state (Figure 1D). Both lean and obese MDS mice failed to compen- sate this thrombocytopenia with enhanced platelet pro- duction, as suggested by the increased percentage of newly formed reticulated platelets (Figure 1D). Next, we analyzed numbers of circulating myeloid cells to deter- mine whether obesity influenced myelopoiesis. Consistent with our previous findings, obesity induced a significant increase in the proportion of monocytes when compared with lean controls, and this effect was strongly amplified in the presence of MDS (Figure 1E). Both Ly6- Chi and Ly6-Clo subsets contributed to this increase in the proportion of monocytes (Figure 1E). Interestingly, while the proportion of neutrophils was elevated in the lean NHD13/WT mice, this effect was absent in obese MDS mice (Figure 1E). Consistent with the known phenotype of MDS mice, defective hematopoiesis in BM progenitor cells was evident, with a decrease in the abundance and proliferation of all of the hematopoietic stem and pro- genitor cells occurring in both lean and obese MDS mice (Figure 1F,G). The obesity-induced increase in circulating monocytes could be explained by extramedullary myelopoiesis in the MDS mice, made apparent by increased hematopoietic stem and progenitor cells (HSPCs, also referred to as LSKs), common myeloid pro- genitors (CMPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs) (Figure 1H). Consequently, a significant accumu- lation of CD11b+ myeloid cells and F4/80+ macrophages was also observed in the spleen (Figure 1I). Of note, the MDS pathology did not appear to significantly affect the metabolic function of lean or obese mice relative to their littermate controls (Online Supplementary Figure S1). Taken together, these data, at the half-way point in the pathogenesis of this model, demonstrated an enhanced monocyte response in obese MDS mice compared with lean MDS mice. Further, failing BM hematopoiesis appeared to promote a shift of this process to the spleen in the mice suffering from MDS.
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haematologica | 2018; 103(4)