Short-Term Western Diet Causes Rapid and Lasting Alterations of Bone Marrow Physiology

Dietary parameters have a strong influence on overall health in various ways. For instance, it affects the physiology of blood vessels. Consumption of western diet (WD) dysregulates the arterial homeostasis and promotes the development of atherosclerotic plaques in the intimal layer of arteries.[1] Interestingly, it was recently described that WD also alters the composition and function of blood vessels in the bone marrow,[2] structures which are considered to play a pivotal role in the microenvironment or niche that tightly controls hematopoiesis.[3] Endothelial cells as well as perivascular cells such as pericytes, mesenchymal stem cells, and CXCL12-abundant reticular cells that are found in bone marrow arteriolar and sinusoidal vessels regulate the maintenance of hematopoietic stem cells (HSCs).[4] [5] Thus, modifications of the architecture and/or the features of the bone marrow vascular network may have dramatic effects on hematopoiesis. Long-term and permanent WD was shown to modify the anatomy of the vascular bone marrow niche and HSC biology.[2] It remains unknown whether a short-term and acute WD regimen would be sufficient to initiate modifications of the bone marrow physiology and whether these alterations would be reversible.

We therefore investigated the effects of different short-term WD conditions ([Fig. 1A]) on the bone marrow vasculature and hematopoiesis in Apoe ^−/− hypercholesterolemic mice (see also [Supplementary Methods] [available in the online version]). First, microscopic three-dimensional visualization of optically cleared bones allowed us to assess the density of the bone marrow vessel network using laminin as a marker for vessel density and early remodeling ([Fig. 1B] and [Supplementary Fig. S1A], available in the online version), revealing that 4 weeks' regimen of WD (chronic WD group) promoted the remodeling of arterioles (laminin^high endoglin⁻)[6] but not sinusoids (laminin⁺ endoglin⁺; [Fig. 1B, C] and [Supplementary Fig. S1B, C], available in the online version). Moreover, 1 week of WD only (late WD group) was already sufficient to induce a similar effect with upregulated laminin expression ([Fig. 1B, C] and [Supplementary Fig. S1B, C], available in the online version), suggesting that remodeling of the bone marrow arterioles by WD is an acute and rapid process. Next, we tested whether these fast changes observed in the arterioles were reversible. To this end, WD was fed to a group of mice for 1 week only and thereafter was replaced by a chow diet for the following 3 weeks (early WD group). Surprisingly, the enhanced laminin expression in bone marrow arterioles was maintained after the interruption of WD for the remaining weeks. Of note, none of the different conditions (chronic, late, and early WD) altered the density of the bone marrow sinusoids ([Fig. 1B] and [Supplementary Fig. S1B, C], available in the online version). Altogether, these findings highlight that short-term WD not only induces a fast remodeling of the bone marrow arterioles, but also that these alterations remain present for a prolonged period of time.

Fig. 1 Short-term WD alters bone marrow physiology. (A) Schematic representation of dietary interventions. Apoe ^−/− mice were fed either a chow diet (CD) for 4 weeks (control), western diet (WD) for 4 weeks (chronic WD), 3 weeks of CD followed by 1 week of WD (late WD) or 1 week of WD followed by 3 weeks of CD (early WD). (B) Representative confocal microscopy 3D reconstruction of HSC (Lin⁻ CD48⁻ CD150⁺, white spheres), sinusoids (laminin⁺ endoglin⁺, red structure), and arterioles (laminin^high endoglin⁻, green structure) in optically cleared whole mount bone marrow from Apoe ^−/− fed CD (control) or WD (chronic, late and early groups). Scale bar = 50 µm. (C) Quantification of the density of bone marrow arterioles. n = 9 random field of views pooled from three mice per group. (D) Distance between HSCs and arterioles in bone marrow. Distribution quartiles were defined as Q1 ≤11.67 µm, Q2 ≤35.2 µm, Q3 ≤75.66 µm, and Q4 ≥75.67 µm. n = 601 HSCs (control group), n = 442 HSCs (chronic group), n = 405 HSCs (late group), and n = 455 HSCs (early group) from three mice per group. p < 0.001 (χ² test). (E) Numbers of HSCs (Lin⁻ Sca1⁺ c-Kit⁺ CD150⁺ CD48⁻) in the bone marrow of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups) assessed by flow cytometry. (F) Numbers of myeloid-biased MPP3s (Lin⁻ Sca1⁺ c-Kit⁺ CD150⁻ CD48⁺ Flt3⁻) in the bone marrow of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). (G) Numbers of polymorphonuclear neutrophils (CD45⁺ CD11b⁺ CD115⁻ Ly6G⁺) in the bone marrow of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). (H) Numbers of classical monocytes (CD45⁺ CD11b⁺ CD115⁺ Ly6C⁺) in the bone marrow of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). (I) Numbers of polymorphonuclear neutrophils (CD45⁺ CD11b⁺ CD115⁻ Ly6G⁺) in the blood of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). (J) Numbers of classical monocytes (CD45⁺ CD11b⁺ CD115⁺ Ly6C⁺) in the blood of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). (K) Percentage of Ki67⁺ HSCs Lin⁻ Sca1⁺ c-Kit⁺ CD150⁺ CD48⁻) in the bone marrow of Apoe ^−/− mice fed with CD (control) or WD (chronic, late, and early groups). All experiments presented in panels (C) and (E–J) were performed with n = 12 mice per group from three independent experiments, except the experiment depicted in (K) that was performed with n = 8 mice per group from two independent experiments. Mean ± SEM. One-way ANOVA test. *p < 0.05, **p < 0.01, and ***p < 0.001. 3D, three-dimensional; HSCs, hematopoietic stem cells; SEM, standard error of the mean.

The bone marrow vascular network was suggested to serve as a niche and under steady state, HSCs reside in close vicinity of arterioles or sinusoids.[4] Since arteriolar laminin expression was already increased by short-term WD, we investigated the relationship of HSCs with these vessels.[7] Three-dimensional computation of HSC–vessel spatial relationships at a single-cell level revealed that permanent WD strongly increased the proximity between HSCs (Lin⁻, CD150⁺, CD48⁻)[7] [8] and arterioles ([Fig. 1D]). Although less pronounced, we could detect a similar effect after 1 week of WD at late or early time point ([Fig. 1D]). Given the importance of bone marrow niche on HSC homeostasis, we subsequently evaluated the contribution of the short-term WD on HSC counts. Flow cytometry analysis revealed that, in chronic, late, and early conditions of WD, numbers of HSCs were decreased ([Fig. 1E]). We hypothesized that this reduction was the consequence of a deterioration in the maintenance of HSCs. However, we did not observe any changes in apoptosis ([Supplementary Fig. S1D], available in the online version). We then reasoned that short-term WD induced the differentiation of HSCs into a downstream population, namely multipotent progenitors (MPPs).[9] Indeed, the numbers of myeloid-biased MPP (a.k.a. MPP3)[9] were greater in all groups of mice that were fed a short-term WD compared to control mice ([Fig. 1F]), whereas proportions of lymphoid-primed MPP (a.k.a. MPP4)[9] remained unchanged ([Supplementary Fig. S1E], available in the online version). We then investigated whether the expansion of the myeloid-biased MPP population correlated with an increase in myeloid cells. The different WD conditions led to elevated numbers of neutrophils and monocytes in the bone marrow ([Fig. 1G, H]). Subsequently, we examined whether this expansion had an impact on blood cell counts. Circulating monocytes showed an increase in all WD conditions ([Fig. 1J]), while higher neutrophil counts were only detected in the late WD group ([Fig. 1I]). To understand the mechanisms involved in neutrophil and monocyte mobilization, we assessed the plasma levels of the chemokines CXCL1[10] and CCL2.[11] We observed higher levels of CXCL1 in the plasma of the late WD group ([Supplementary Fig. S1F], available in the online version), providing an explanation for the increased presence of neutrophils in circulation within this group. However, CCL2 levels remained unaffected in all WD groups ([Supplementary Fig. S1G], available in the online version), suggesting the involvement of another mechanism in their mobilization. Together, these findings indicate that short-term WD after 1 week (late WD group) induces an accelerated differentiation of HSC to MPP3 and led to an increase of myeloid cell production. Interestingly, our data further imply that these effects are long lasting since the upregulation of myelopoiesis could still be observed even after suspension of WD (early WD group).

HSCs are by definition key in maintaining proper hematopoiesis, and a loss of HSCs can have significant consequences on the production of hematopoietic cells. In our study, we observed a significant decrease in HSC numbers across all short-term WD groups ([Fig. 1E]). To further investigate their maintenance, we examined whether the proliferation of the remaining HSCs was affected. Utilizing Ki67 staining, we found that 62% of HSCs in Apoe^−/− mice on a chronic WD were positive, whereas only 24% of HSCs in chow-fed Apoe^−/− mice showed positivity ([Fig. 1K]). Notably, we also observed an increased percentage of Ki67-positive HSCs in the late WD group (55% of HSCs) and the early WD group (43% of HSCs). Collectively, these findings suggest that, after differentiating into MPPs, the remaining HSCs undergo proliferation, likely to maintain their pool. Furthermore, this proliferation of HSCs occurs rapidly and continues over an extended period, as evidenced by increased HSC proliferation in both the late and early WD groups.

Clonal hematopoiesis (CH) is characterized by the proliferation of a specific clone of HSCs, with or without a genetic mutation, and is commonly associated with the aging process.[12] Certain somatic mutations in CH have been found to contribute to atherosclerosis by affecting the behavior of myeloid cells.[13] More recently, it has been shown that increased proliferation of HSCs by long-term WD in atherosclerosis condition accelerates the emergence of CH.[14] Our data suggest that short-term WD can trigger the initiation of CH, primarily through neutral drift and more specifically via a bottleneck effect, and potentially provides an explanation for the subsequent appearance of CH described with prolonged exposure to WD. Indeed, the initial drastic reduction of HSC pool size following short-term WD, especially observed after 1 week of WD (in the late group in [Fig. 1E]), leads to the constitution of a smaller number of HSC clones (38% of the initial pool). The expansion of these limited clones, that is already noticeable 3 weeks after the cessation of WD (early group in [Fig. 1E]), will inevitably promote the emergence of CH, regardless of genetic mutations. It is worth noting that even in a healthy hematopoietic system, small clones carrying mutations can exist.[15] If one of these clones harboring a mutation proliferate, it will then become over-represented in the context of WD-induced hematopoiesis.[14] Alternatively, as uncontrolled proliferation increases the likelihood of mutations, it raises the probability that one clone acquires driver mutations and dominates over others if there is a selective advantage for the variant.

The environment of the bone marrow niche plays an important role in hematopoiesis.[3] [4] The function of the arterial niche for the maintenance of HSCs has been controversial. A proportion of HSCs was shown to associate with arterioles and molecular factors produced by vascular endothelial cells and perivascular cells as well as a microenvironment low in reactive oxygen species were proposed to maintain HSC quiescence.[16] [17] [18] [19] In contradiction to these findings, it was also shown that quiescent HSCs were closely associated with sinusoids, while arterioles did not play a role in HSC maintenance.[7] [20] [21] Our findings suggest that a disturbed microenvironment centered around arterioles, and induced by a brief consumption of WD, results in the activation (differentiation and/or proliferation) of HSCs. In our study, laminin was utilized for the identification of bone marrow arterioles which showed a clear increase of this extracellular matrix protein upon WD stimulus. Interestingly, an altered production of laminin may be considered as an early hallmark of vascular remodeling.[22] Moreover, it was shown that bone marrow laminins serve as adhesive substrates[23] to HSCs and regulate their maintenance.[24] Potentially, additional changes in the activated bone marrow niches that occur in parallel of laminin expansion could influence the maintenance of HSCs. Nevertheless, it is conceivable that short-term WD activates bone marrow arterial endothelial and perivascular cells, which leads to an increase of laminin production in arterioles and consequently affects HSC biology. The exact reason behind the prolonged impact of WD on laminin levels after WD cessation remains uncertain. However, it is possible that the turnover of laminin, which relies on a delicate balance between synthesis and degradation processes,[25] might be influenced in this particular context. It is conceivable that cells surrounding arterioles could remain active for an extended period even after WD discontinuation and continue to produce laminin. Alternatively, the onset of laminin degradation, facilitated by enzymes like metalloproteinases and proteases, could be slower in this scenario.

In conclusion, our data show that a brief exposure to WD induces a rapid and sustained alteration of the bone marrow physiology. This may have deleterious effects in mounting an efficient immune response[26] and could have harmful consequences in the development of an inflammatory response.[13] [14] Further research is required to understand the regulatory pathways involved in the sustained remodeling of bone marrow niches and its consequences on hematopoiesis.

Conflict of Interest

None declared.

Authors' Contribution

M.B. designed and performed experiments, analyzed, and interpreted data; Z.M.-R. performed experiments, analyzed, and interpreted data; C.W. provided funding and intellectual input; R.T.A.M and J.D. provided funding, designed the research, interpreted data, and wrote the manuscript.

^* These authors contributed equally to this study.

• References

• 1 Libby P, Buring JE, Badimon L. et al. Atherosclerosis. Nat Rev Dis Primers 2019; 5 (01) 56
• 2 Rohde D, Vandoorne K, Lee IH. et al. Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease. Nat Cardiovasc Res 2022; 1 (01) 28-44
• 3 Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature 2014; 505 (7483) 327-334
• 4 Pinho S, Frenette PS. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 2019; 20 (05) 303-320
• 5 Baccin C, Al-Sabah J, Velten L. et al. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nat Cell Biol 2020; 22 (01) 38-48
• 6 Gomariz A, Helbling PM, Isringhausen S. et al. Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy. Nat Commun 2018; 9 (01) 2532
• 7 Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005; 121 (07) 1109-1121
• 8 Duchene J, Novitzky-Basso I, Thiriot A. et al. Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis. Nat Immunol 2017; 18 (07) 753-761
• 9 Pietras EM, Reynaud D, Kang YA. et al. Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions. Cell Stem Cell 2015; 17 (01) 35-46
• 10 Pelus LM, Fukuda S. Peripheral blood stem cell mobilization: the CXCR2 ligand GRObeta rapidly mobilizes hematopoietic stem cells with enhanced engraftment properties. Exp Hematol 2006; 34 (08) 1010-1020
• 11 Serbina NV, Pamer EG. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat Immunol 2006; 7 (03) 311-317
• 12 Zink F, Stacey SN, Norddahl GL. et al. Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly. Blood 2017; 130 (06) 742-752
• 13 Jaiswal S, Natarajan P, Silver AJ. et al. Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease. N Engl J Med 2017; 377 (02) 111-121
• 14 Heyde A, Rohde D, McAlpine CS. et al. Increased stem cell proliferation in atherosclerosis accelerates clonal hematopoiesis. Cell 2021; 184 (05) 1348.e22-1361.e22
• 15 Young AL, Challen GA, Birmann BM, Druley TE. Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults. Nat Commun 2016; 7: 12484
• 16 Itkin T, Gur-Cohen S, Spencer JA. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 2016; 532 (7599) 323-328
• 17 Kusumbe AP, Ramasamy SK, Itkin T. et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature 2016; 532 (7599) 380-384
• 18 Kunisaki Y, Bruns I, Scheiermann C. et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 2013; 502 (7473) 637-643
• 19 Xu C, Gao X, Wei Q. et al. Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow. Nat Commun 2018; 9 (01) 2449
• 20 Acar M, Kocherlakota KS, Murphy MM. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 2015; 526 (7571) 126-130
• 21 Chen JY, Miyanishi M, Wang SK. et al. Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 2016; 530 (7589) 223-227
• 22 Yousif LF, Di Russo J, Sorokin L. Laminin isoforms in endothelial and perivascular basement membranes. Cell Adhes Migr 2013; 7 (01) 101-110
• 23 Gu YC, Kortesmaa J, Tryggvason K. et al. Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells. Blood 2003; 101 (03) 877-885
• 24 Susek KH, Korpos E, Huppert J. et al. Bone marrow laminins influence hematopoietic stem and progenitor cell cycling and homing to the bone marrow. Matrix Biol 2018; 67: 47-62
• 25 Mouw JK, Ou G, Weaver VM. Extracellular matrix assembly: a multiscale deconstruction. Nat Rev Mol Cell Biol 2014; 15 (12) 771-785
• 26 Christ A, Günther P, Lauterbach MAR. et al. Western diet triggers NLRP3-dependent innate immune reprogramming. Cell 2018; 172 (1–2): 162-175.e14

Publication History

Received: 23 December 2023

Accepted: 14 July 2023

Accepted Manuscript online:
07 August 2023

Article published online:
21 September 2023