Fish Oil-New Study/"anti-inflammatory".....
"promotes hematopoiesis in the bone marrow and spleen"
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"In the 1970s, n-3 PUFAs-rich fish oil was initially realized as an underlying cause for the low rate of coronary heart disease (CHD) in Eskimos (55). Since then, many
health benefits of N-3 PUFAs have been reported, esp. in many inflammatory diseases, including rheumatoid arthritis (RA), diabetes, cardiovascular diseases and allergy (56-58). In 2004, FDA approved that fish oil can be used as a prescription drug in cardiovascular diseases. Consistently, our recent study showed that n-3 PUFAs-rich fish oil diet may induce myeloid-derived suppressor cell differentiation to suppress tissue inflammation (59). As this new study suggests that a chronic intake of fish oil rich diet may affect the hematopoietic system in mice, more studies are required in humans to investigate its long-term impact on hematopoiesis in healthy individuals as well as patients with bone marrow transplantation (BMT)."
"n-3 PUFAs are believed to have anti-inflammatory properties (18-20). Indeed, n-3 PUFAs are being used in the prevention and treatment of coronary artery disease (CAD), diabetes, hypertension, arthritis, cancer and other inflammatory and autoimmune disorders in humans (21, 22). However, its effect on stem cell proliferation and differentiation remains largely unknown."
"The self-renewal and differentiation of hematopoietic stem cells (HSCs) in bone marrow are essential to replenish all blood cell types, but how this process is influenced by diet remains largely unclear. Here we show that diet rich in fish oils promotes self-renewal of hematopoietic stem cells and extramedullary hematopoiesis. Chronic intake of fish-oil rich diet increases the abundance of HSCs, alters hematopoietic microenvironment and intriguingly, induces the expression of matrix metalloproteinase 12 (MMP12) in the bone marrow. Pointing to a direct effect of fish oil on MMP12 expression, omega-3 polyunsaturated fatty acids (n-3 PUFAs) induce the expression of MMP12 in a dose-dependent manner in bone marrow cells. Importantly, downregulation of MMP12 activity using an MMP12-specific inhibitor attenuates diet-induced myelopoiesis in both bone marrow and spleen. Thus, fish-oil rich diet promotes hematopoiesis in the bone marrow and spleen, in part, via the activity of MMP12. Taken together, these data provide new insights into diet-mediated regulation of hematopoiesis."
Fish Oil Rich Diet Promotes Hematopoiesis and Alters Hematopoietic Niche
Endocrinology June 10 2015
Sheng Xia1,2,3*, Xiao-ping Li2, Lu Cheng2, Mu-tian Han2, Miao-miao Zhang1,2,
Qi-xiang Shao1,2, Hua-xi Xu1,2, and Ling Qi1,3*
1Department of Immunology, 2Institute of Clinic Laboratory Diagnosis, School of Medicine, Jiangsu
University, Zhenjiang, Jiangsu 212013, China; 3Division of Nutritional Sciences, Cornell University, Ithaca,
NY 14853, USA
The self-renewal and differentiation of hematopoietic stem cells (HSCs) in bone marrow are essential to replenish all blood cell types, but how this process is influenced by diet remains largely unclear. Here we show that diet rich in fish oils promotes self-renewal of hematopoietic stem cells and extramedullary hematopoiesis. Chronic intake of fish-oil rich diet increases the abundance of HSCs, alters hematopoietic microenvironment and intriguingly, induces the expression of matrix metalloproteinase 12 (MMP12) in the bone marrow. Pointing to a direct effect of fish oil on MMP12 expression, omega-3 polyunsaturated fatty acids (n-3 PUFAs) induce the expression of MMP12 in a dose-dependent manner in bone marrow cells. Importantly, downregulation of MMP12 activity using an MMP12-specific inhibitor attenuates diet-induced myelopoiesis in both bone marrow and spleen. Thus, fish-oil rich diet promotes hematopoiesis in the bone marrow and spleen, in part, via the activity of MMP12. Taken together, these data provide new insights into diet-mediated regulation of hematopoiesis.
The hematopoietic system provides the body with a constant supply of all blood lineages. In order to maintain hematopoietic homeostasis, hematopoietic stem cells (HSCs) have the clonal capacity to provide life-long regeneration of all blood lineages. Bone marrow provides hematopoietic microenvironment or niche to support and regulate the self-renewal, migration and differentiation of HSCs (1). Many cellular components including osteoclasts, osteoblasts, mesenchymal progenitor cells and adipocytes as well as components of the extracellular matrix (ECM) play regulatory roles in the homeostasis of HSCs (2-5). The interplay between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) also serves an important role in hematopoiesis through remodeling the ECM in the hematopoietic niche of the bone marrow (6, 7). Injuries to these components brought by inflammation and/or metabolic conditions influence the size and fate of HSCs. Indeed, systemic inflammation alters the regulation of HSCs and increases more granulocytes and monocytes in peripheral tissues (8, 9).
Moreover, a number of intracellular regulatory molecules including FoxOs, mTORC1, Fbw7, Egr1, Pbx1, pRb, c- Cbl, Myc, and Bmi1 mediate the processes of dormancy, cycling, self-renewal, differentiation, and survival of HSCs (10-15).
The LKB1/AMPK signaling pathway, a metabolic sensing pathway, plays a key role in the HSCs self-renewal and differentiation (16, 17). These studies have provided strong link between HSCs homeostasis and metabolism. However, how intermediary metabolites influence stem cell behavior remains largely unclear, especially in regards to the effect of dietary fatty acids.
Of particular interest are two biologically active fatty acids (FA), omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs). Both n-3 and n-6 FAs are long chain PUFAs of 18-22 carbons in length with first double bond positioned at the third or sixth carbon atom, respectively, from the methyl end of the FAs. N-3 PUFAs are present in vegetable oil, but the richest source is fish oil. Unlike n-6 PUFAs which promote inflammation and insulin resistance, n-3 PUFAs are believed to have anti-inflammatory properties (18-20). Indeed, n-3 PUFAs are being used in the prevention and treatment of coronary artery disease (CAD), diabetes, hypertension, arthritis, cancer and other inflammatory and autoimmune disorders in humans (21, 22). However, its effect on stem cell proliferation and differentiation remains largely unknown.
We speculate that diet rich in n-3 PUFAs is likely to play a critical role in the homeostasis of HSCs by influencing the niche and/or energy metabolism of stem cells and thereby their proliferative potential. Indeed, here we show that diet rich in fish oils promotes hematopoiesis in the bone marrow and spleen in mice. The increased abundance of HSCs and hematopoietic progenitors in the spleen is associated with morphological changes in hematopoietic microenvironment of the bone and the up regulation of MMP12, also known as macrophage elastase, in bone marrow. The effect of dietary PUFAs on hematopoietic microenvironment is likely mediated in part by MMP12 as downregulation of MMP12 activity attenuates dietary PUFAs-induced hematopoiesis.
Mice and diets
6-week-old male CD45.2 or CD45.1 C57BL/6 wild type mice (from The Jackson laboratory) were fed with low-fat diet (LFD, 2.9 kcal/g,13%kcal from fat) (Teklad 2914), high-fat diet (HFD, 5.24 kcal/g, 60% kcal from fat) (Bioserv F3282), or high fish oil diet (5.24 kcal/g, 60% kcal from fat) (Bioserv F5424) for 4 weeks. Fatty acid compositions of each diet provided by the manufacturers are shown in Table 1. Of note, LFD vs. high fat diets may not only vary in fat content, but also in the source of protein (grain vs. casein) as well as sucrose and cornstarch. In some experiments, mice on high fish oil diet were orally gavaged on alternate days with an MMP12 inhibitor MMP408 (Calbiochem, Darmstadt, Germany) at 10 mg/kg body weight as previously described (23) for a total 4 weeks. All animal protocols have been approved by the Cornell Institutional Animal Care and Use Committee, and by the Scientific Investigation Board of Jiangsu University.
Fish oil rich diet promotes myelopoiesis in the
To explore the effect of high fish oil diet on hematopoiesis, we separated wildtype littermates into three dietary groups, LFD, high fish oil diet and HFD.
diets do not contain any -3 PUFAs, high fish oil diet contains high levels of n-3 PUFAs, including eicosapentaenoic acid (C20:5n3), docosapentaenoic acid (C22:5n3) and DHA (C22:6n3). By contrast, HFD contains higher content of saturated stearic (C18:0) and monounsaturated oleic acid (C18:1n9) relative to high fish oil diet. The content of n-6 PUFAs, mainly in the form of linoleum acid (C18:2n6), was about the same between HFD and high fish oil diet (Table 1).
Following 4 weeks of feeding, the HFD cohort gained a significant amount of body weight, while the fish oil cohort exhibited a moderate increase of body weight (Figure 1A). Spleens of the fish oil cohort were enlarged and weighed significantly more than the other two cohorts (Figure 1A). Although total number of circulating white blood cells was comparable among the cohorts (Figure 1B), FSClowSSChi granulocytes in the spleen were significantly increased by 4-fold in the fish oil cohort when compared to other two cohorts (Figure 1C). Pointing to the impact of n-3 PUFAs (rather than the impact of high fat), HFD had no obvious effect on the granulocyte population in the spleen compared to that of the LFD cohort (Figure 1C). While both high fish oil diet and HFD increased the abundance of FSChiSSClow monocytes in the spleen, and the effect of high fish oil diet was much more pronounced (Figure 1C). This was further confirmed by flow cytometric analysis of CD11b myeloid cells in the spleen (Figure 1D). Indeed, while HFD increases the abundance of myeloid cells in the spleen as we previously shown (26), fish oil diet exerted more profound impact on this population (Figure 1D-F). Moreover, histological assessment of the spleen revealed the alteration of splenic compartmentalization in the fish oil cohort (Figure 1G) with a large number of CD11b monocytes present in the marginal zone (Figure 1H-I). Thus, 4-week intake of high fish oil diet promotes myelopoiesis in the spleen.
High fish oil diet induces extra medullary hematopoiesis in the spleen.
To understand how high fish oil intake induced splenic myelopoiesis, we next examined the status of extra medullary hematopoiesis, ie, hematopoiesis occurring in organs other than bone marrow (28). We measured the levels of hematopoietic progenitors (Lin- c-KitSca-1-) and HSCs (Lin- c-Kit Sca-1) in the spleen. As shown in Figure 2A, high fish oil diet increased the percent of Linc-KitSca-1- hematopoietic progenitors and Lin- c-Kit Sca-1 HSCs by 4- and 10-fold, respectively, when compared to the HFD cohort. As a control, fish oil diet did not affect the percent of nonprogenitor Lin- c-Kit- Sca-1 populations in the spleen.
We next measured the impact of high fish oil diet on proliferative capacity of splenic HSCs and progenitors using the colony forming unit assays (CFU) both in vitro and in vivo. Figure 2B shows representative images ofGEMM, GM and BFU-E colonies after in vitro culture of splenocytes in methylcellulose complete culture medium. Numbers of GEMM, GM and BFU-E colonies were all significantly increased in mice fed with the high fish oil diet while HFD had a negligible to moderate effect (Figure 2B). Similarly, percent of Ter-119 erythroid precursors was significantly upregulated by nearly 6-fold in the spleens of the high fish oil cohort (Figure 2C). These changes were associated with elevated expression of stem cell factor (SCF) and granulocyte- macrophage colony stimulating factor (GM-CSF), two factors involved in the hematopoietic
cell proliferation (29), in the spleens of the fish oil cohort (Figure 2D). To further demonstrate cell-autonomouseffect of the diet on hematopoiesis,we performed the spleen colony forming unit (CFU-S) assay in vivo where splenocytes from different cohorts were transferred into sublethally irradiated mice on LFD. Indeed, donor splenocytes from the high fish oil cohort formed significantly more colonies in the spleens of recipients when compared to other dietary cohorts (Figure 2E). Thus, high fish oil diet enhances extramedullary hematopoiesis and increases progenitor and primitive stem cell populations in the spleen.
High fish oil diet increases HSCs self-renewal in the bone
To address mechanistically how high fish oil diet induced extramedullary hematopoiesis in the spleen, we next examined the status of hematopoiesis in the bone marrow. While total numbers of bone marrow cells were comparable among the cohorts after 4 weeks of feeding (Figure 3A), Lin- c-Kit Sca-1 HSCs, but not Lin- c-Kit Sca-1- progenitors, were significantly increased by 50%-60% in the fish oil cohort compared to the other two cohorts (Figure 3B). In line with the observation that fish oil diet had no effect on the number of progenitors in the bone marrow, CFU assays in vitro revealed no significant differences in theGEMM,GMand BFU colony formation among the three cohorts (Figure 3C). We next compared repopulating capability of HSCs using an in vivo competitive repopulation assay where a mixed bone marrow cells from the fish oil (CD45.1) and LFD (CD45.2) cohorts at the ratio of 1:1 were transferred into lethally irradiated recipients. Three months later, the ratio of CD45.1 to
CD45.2lymphocytes changed to near 1.5:1 in peripheral blood, pointing to a higher proliferative capacity and self renew of HSCs of the fish oil cohort (Figure 3D-E). Collectively, these data suggest that high fish oil diets promote self-renewal of HSCs in the bone marrow.
Fish-oil rich diet alters hematopoietic niche and induces
MMP12 expression in the bone marrow.
We next addressed how fish oil diet affected hematopoietic stem cell (HSC) self-renewal in the bone marrow. As the homeostasis of HSCs largely depends on the niche, we assessed the change of hematopoietic microenvironment in the bone marrow. Strikingly, fish-oil rich diet reduced the amount of medulla ossiumrubra or the red marrow in distal tibia of the bone (Figure 4A), pointing to a possible altered hematopoietic microenvironment. Given the important role ofMMP-9and 12 in hematopoiesis and tissue remodeling (30, 31), we next determined whether MMPs were responsive to fish oil diet or n-3 PUFAs. Unlike Mmp9, Mmp12 mRNA level in the bone marrow was
highly responsive to fish-oil rich diet, but not HFD (Figure 4B). This upregulation was further confirmed at the protein level using Western blot (Figure 4C) and immunohistochemical staining of MMP12 in the bone marrow (Figure 4D).
We next analyzed the effect of fish oil diet on MMP12 expression in different cellular components made up of the hematopoietic niche including stromal cells and macrophages. Fish oil-rich diet dramatically enhanced Mmp12 expression in stromal cells, while having a moderate effect on CD11b macrophages or CD11b- bone marrow cells (Figure 4E). Moreover, as extracellular signal-regulated kinase (ERK)-mediated signaling pathway is known to regulate the expression ofMmp12(32), we next measured ERK phosphorylation in bone marrow cells. Indeed, fish oil rich diet promoted ERK phosphorylation of bone marrow cells when compared to that of LFD cohort (Figure 4F).
To directly assess the effect of n-3 PUFAs on Mmp12 expression in bone marrow cells, we treat with bone marrow cells with DHA (n-3 PUFA). DHA increased the expression of Mmp12 in a dose-depend manner, but had no obvious effect on Mmp9 (Figure 4G). Thus, MMP12 expression in the bone marrow is responsive to n-3 PUFAs.
MMP12 activity contributes to fish oil diet-induced
Lastly, we asked whether there was a causal relationship between MMP12 expression and diet-induced myelopoiesis. To this end, mice on fish oil rich diet were orally gavaged with MMP12-specific inhibitor MMP408 (23) on alternate days for a total of 4 weeks (Figure 5A). MMP408 treatment reduced CD11b myeloid cells in the spleen by nearly 50% (Figure 5B-C). In both spleen and bone marrow, percent of Lin- c-Kit Sca-1 HSCs was decreased while Lin- c-Kit Sca-1- progenitors were not affected (Figure 5D-G). These data were further confirmed by using the CFU-S assay where MMP408 reduced the number of colonies formed by the donor splenocytes from mice on high fish oil diet (Figure 5H). Thus, chronic intake of fish oil rich diet promotes HSCs self-renewal and proliferation, at least in part, via MMP12.
Both HFD and fish-oil rich diets are known to alter myeloid cell differentiation (26, 33-35), but underlying molecular mechanisms were not well understood. Here, our data show that fish-oil rich diet, not HFD, promotes extra medullary hematopoiesis in the spleen with elevated HSCs and progenitors. In the bone marrow, fish-oil rich diet alters hematopoietic microenvironment and increases the abundance of HSCs.MMP12is likely involved in fish oil diet-induced hematopoiesis as inhibition of MMP12 partially attenuates myelopoiesis in both the spleen and bone marrow. This is consistent with early studies showing an important role ofMMP12in hematopoiesis (30, 36, 37). Thus, these data suggest that dietary fish oil intake may alter hematopoietic niche in the bone marrow and thereby affecting self-renewal of HSCs.
The size of HSCs pool largely depends on their intrinsic properties and hematopoietic microenvironment of the bone (1, 38). Many cellular elements, including stromal cells, adipocytes, osteoblasts and multiple components of the extracellular matrix constitute the hematopoietic niche to regulate self-renewal and differentiation of HSCs (39, 40). Moreover, as the renewal of HSCs, progenitors and even stromal cells is highly active, the metabolism of these cells may be influenced by the changes in dietary composition. Indeed, previous studies have shown that different PUFAs and their metabolites have distinct osteoclastogenic effect (41, 42), suggesting their potential roles in hematopoiesis. A recent study showed that adipocytes are present in the bone marrow of HFD mice, which may modify hematopoietic microenvironment and alter myelopoiesis and lymphopoiesis (43). Our data show that fish-oil rich diet alters hematopoietic microenvironment and induces the expression of MMP12 in stromal cells and hence hematopoiesis. However, whether other cell types, such as adipocytes, play a role in fish oil diet induced bone marrow remodeling remain to be determined.
Both MMP9 and MMP12 belong to the family of zinc dependent proteases and degrade the components of extracellular matrix in hematopoietic microenvironment, thereby playing a critical role in hematopoiesis under both physiological and pathological conditions. MMP9 is involved in the recruitment of HSCs from the bone marrow (44-46) and in the absence of MMP9, HSCs mobilization is impaired (44). MMP12 secreted by macrophages and osteoclasts is involved in inflammation and tissue remodeling through the degradation of ECM (47). MMP12-deficient mice are defective in macrophage recruitment and tissue inflammation under many pathological settings in-cluding emphysema (48, 49). While it remains unclear how hematopoiesis is altered in MMP12-deficient mice, over expression of MMP12 promotes myelopoiesis and increases the frequencies and numbers of myeloid progenitors (30, 36, 37). Moreover, our observation that n-3 PUFAs regulate Mmp12 expression in stromal cells of the bone marrow is interesting. More studies are required to identify key transcription factors and signaling pathways linking n-3 PUFAs to Mmp12 expression.
Additionally, the effect of n-3 PUFA-rich diet may be mediated through the alteration in membrane lipid composition. Lipid microdomains, highly enriched in glycosphingolipids and cholesterol, are important signaling platforms. It has been reported that n-3PUFAs may alter cell signaling and functions by disrupting lipid raft formation in various cell types (50, 51). In addition, n-3 PUFA-rich diet may affect the activation and differentiation of T, B cells and antigen presenting cells through disrupting membrane domain organization and thereby altering signaling networks (52-54). Thus, dietary n-3 PUFAs may regulate hematopoiesis and hematopoietic niche through the modification of lipids rafts on HSCs and hematopoietic stromal cells.
In the 1970s, n-3 PUFAs-rich fish oil was initially realized as an underlying cause for the low rate of coronary heart disease (CHD) in Eskimos (55). Since then, many health benefits of N-3 PUFAs have been reported, esp. in many inflammatory diseases, including rheumatoid arthritis (RA), diabetes, cardiovascular diseases and allergy (56-58). In 2004, FDA approved that fish oil can be used as a prescription drug in cardiovascular diseases. Consistently, our recent study showed that n-3 PUFAs-rich fish oil diet may induce myeloid-derived suppressor cell differentiation to suppress tissue inflammation (59). As this new study suggests that a chronic intake of fish oil rich diet may affect the hematopoietic system in mice, more studies are required in humans to investigate its long-term impact on hematopoiesis in healthy individuals as well as patients with bone marrow transplantation (BMT).