Why does extramedullary hematopoiesis occur

These patients exhibit splenomegaly and an intense EMH 1 , 36 , This process is of pathophysiological relevance since the immune cells have a short life and are eliminated during infection. Several studies confirm HSPCs activation not only after bacterial infection, but also in polymicrobial, viral, and fungal challenges. Immune cells are known to detect pathogen-associated molecular patterns PAMPs by their Toll-like receptors TLRs , leading to an increased proinflammatory response.

HSPCs that express TLRs can respond directly to infection or inflammation, and differentiate into specific cell lineages. This HSPCs activation also promotes the recruitment of these cells to EMH sites, where they are capable of generating neutrophils and monocytes. Moreover, psychosocial stress has been reported to increase blood cell progenitors production in the BM and its mobilization to the spleen, where they establish persistent myelopoiesis 33 , Therefore, increased production of monocytes and neutrophils occurs in a wide range of diseases, from anxiety to atherosclerosis 25 — 27 , 33 , 42 — In fact, ectopic erythropoiesis in the spleen tends to improve anemia caused by stress 11 , 33 , Conversely, therapeutic stimulation of the nicotinic acetylcholine receptor alpha 7 reduces splenic monocyte mobilization and antagonizes atherogenesis Dysfunction or injury in the spleen causes significant loss of blood cells, either from the parenchyma or from arteries and veins that supply it 50 — In human adults, spleen size is up to g and ca.

The spleen displays several essential functions: hematological maturation of RBCs, efficient removal of abnormal cells by phagocytosis, iron recycling and removal of particles, such as opsonized microorganisms or cells coated with antibodies , immunological humoral and cell-mediated immunity and metabolic homeostasis 23 , 53 , It is integrated in the regulation of local immune responses but also in systemic immunity, thus participating in the development of certain inflammatory and chronic disorders that will be discussed 51 , 54 — The spleen has two compartments that are very different in their architecture, vascular organization, morphology, cell composition and physiological functions: the red pulp RP and the white pulp WP , separated by the marginal zone MZ.

The organ is surrounded by a capsule of dense fibrous tissue, elastic fibers and smooth muscle cells. The outermost layer of the splenic capsule is composed of mesothelial cells. Irregularly spaced trabeculae of smooth muscle and fibroblastic tissue emanate from the capsule in the splenic parenchyma and contain blood and lymphatic vessels, and nerves. Lymphatic vessels are efferent vessels through which lymphocytes migrate to splenic lymph nodes 57 — The RP constitutes a blood filter that removes foreign material, damaged erythrocytes for iron recycling , and platelets.

In rodents, it is a place of EMH 1 , 23 , It is composed of a three-dimensional mesh of splenic cords and venous sinuses. The highly active RP macrophages are phagocytic cells that remove old and damaged RBCs and particles that flow through the blood 51 , 55 , EMH is common in the RP of rodents, especially in fetal and neonatal animals 1.

It consists of lymphocytes, macrophages, dendritic cells DCs , plasma cells, arterioles and capillaries in a reticular framework similar to that found in the RP. The PALS is composed of lymphocytes and concentric layers of reticular fibers and flattened reticular cells The external PALS is formed by B and T lymphocytes, macrophages and, after antigenic stimulation, newly formed plasma cells.

They contain germinal centers, which are formed after antigenic stimulation and contain macrophages and apoptotic B cells. The MZ of the spleen is composed of a wide variety of cell types, some of which have a fixed position therein, such as macrophages in the MZ, metallophilic macrophages on the edge and, to a lesser extent, B cells in the MZ 52 , 58 , 59 , Therefore, continuous flow of blood-borne immunocompetent cells together with the sessile cell populations, make the MZ a dynamic area suited for antigens recognition and processing.

There is no other lymphoid organ in which such a unique combination of cells and functions can be found. Peripheral to the marginal sinus, is the thick outer ring of the MZ, with reticular fibroblasts, macrophages, DCs and B cells. Although not all the potential functions of the metallophilic macrophages of the MZ are known, they are important for the removal of foreign particles, bacteria and viruses.

These receptors are essential to detect pathogens and to lead to efficient phagocytosis. The antigenic fragments that are produced can be absorbed by DCs, which enter the spleen through the blood as part of a mobile immune surveillance system and present them to T cells, promoting the clustering and enrichment of antigen-specific T cells. Therefore, the MZ also acts as a lymphocyte trafficking site 52 , Cardiovascular diseases CVDs are the main cause of mortality worldwide, being atherosclerosis one of the underlying agents of these pathologies.

The deposition of cholesterol-rich lipoproteins in the arterial wall initiates the atherosclerotic process, triggering leukocyte recruitment and chronic inflammation.

Aforetime, atherosclerosis was thought to be only the consequence of the progressive lipid accumulation in the blood vessels. The current view is that it is a much more complex process involving both lipids and immune cells Figure 1 29 , 70 — Recently, a strong association between leukocytosis and CVDs has been demonstrated, thus highlighting innate immunity as a hallmark in the onset and progression of these pathologies 29 , 71 , While inflammation associated with atherosclerosis may contribute to this relationship, there is also evidence that leukocytosis directly increases atherosclerosis and thrombosis 26 , 27 , 70 , 74 , The numerous risk factors for CVDs, including obesity, smoking, sedentary lifestyles and metabolic syndrome which in turn includes individual components, such as dyslipemia and low HDL , are associated with leukocytosis 42 , Figure 1 Conditioning factors in extramedullary hematopoiesis EMH -especially in the spleen- leading to atherosclerotic disease.

Different mechanisms lead to EMH, the generation of new leukocyte progenitors and their role in the progression of the atherosclerotic disease. EMH is caused by truncated normal hematopoiesis or inadequate bone marrow BM function. The concurrence of EMH with alterations in lipid metabolism and cholesterol-efflux pathways synergize in the aggravation of the proatherogenic pathways.

In addition, the mechanisms of proliferation and differentiation of HSPCs remain partly unknown, but epigenetic modifications on the HSPCs appear to be important in the memory of trained innate immunity 77 , Several findings indicate that extramedullary anatomical sites complement the hematopoietic function of the BM producing circulating inflammatory cells that infiltrate atherosclerotic lesions 2 , 72 , 74 , 79 , Preclinical studies of atherosclerosis in mice determined that the spleen contains a reservoir of monocytes, which further yield Ly-6C high monocytes to the growing atheroma layer.

These monocytes express pro-inflammatory interleukins such as pro-IL1 and have proteolytic capacity, contributing to the remodeling of the atheroma and favoring plaque instability. In addition to this, they exhibit increased production of reactive oxygen species, promoting the oxidation of LDL, which results in macrophages loaded with lipids foam cells 55 , Regarding the ability of HSPCs to produce different cell lineages, it has been shown the existence of specific signals that rule the selection processes, but also memory epigenetic modifications that accelerate and condition innate immune function responses 29 , 45 , 77 , 78 , Neutrophilia and monocytosis have been specifically associated with CVDs and with atherosclerotic plaque burden in both prospective and cross-sectional studies.

In animal models, monocytosis has been associated to atherosclerosis 6 , 29 , 42 , 44 , 45 , 72 , 73 , More than 30 years ago a relationship between dietary hypercholesterolemia, monocytosis and atherosclerosis was observed in pig and rabbit models 85 , In hypercholesterolemic pigs, an increased colony forming units in the BM was observed, which was also corroborated lately in other experimental models Increased HSPCs elevate monocyte production in the BM and other extramedullary tissues, resulting in the accumulation of cells that worsen the atherosclerotic disease due to lipid accumulation.

Additional work has highlighted that the disturbance of cholesterol pathways may set this HSPC-dependent monocytosis 29 , 44 , 82 , Ly-6C hi monocyte subset is thought to differentiate into an inflammatory phenotype 55 , In advanced atheromata, in contrast to previous views on macrophage proliferation, macrophages can proliferate inside the lesions, through an Scavenger Receptor Class A SRA -dependent pathway 71 , 93 , Furthermore, cholesterol homeostasis appears to support hematopoietic stillness and quiescence of HSPCs.

These cells show enhanced expression of Abca1 , Abcg1 and Apoe , all of them essential cholesterol-efflux genes. These hypercholesterolemic-primed progenitors also produce myeloid cells that enter the atheroma and increase the lesion size 79 , Not only is the quantity of circulating monocytes important to these processes, but also the physiological sites of generation as well, because spleen-produced monocytes appear to have a pro-atherogenic phenotype 29 , Not only has systemic hypercholesterolemia been associated with monocytosis, but also with severe neutrophilia as well 27 , Current evidence also suggests that neutrophilia may promote early lesion development.

In addition to this, cholesterol-related pathways control the proliferation of progenitor hematopoietic stem cells 25 , 81 , 82 , Therefore, there is a particular link between metabolism and inflammation whose study could contribute to the development of different strategies for the treatment of CVDs Monocytes circulate in the blood and patrol the vascular endothelium.

Under inflammatory conditions, they accumulate at the target sites and mature to macrophages or DCs. In the arteries, they differentiate into macrophages, which accumulate oxidized lipoproteins in the atheroma layer and give rise to foam cells, contributing to the necrotic nucleus of the lesions. Although monocytes are thought to arise exclusively in the BM, HSPCs easily move from their niches in the BM, accumulating in the periphery where they differentiate.

This EMH phenomenon gives rise to erythrocytes, platelets, granulocytes and DCs, but its regulation is a controversial issue 4 , 55 , The spleen also contains proliferating myeloid cell progenitor cells that give rise to its progeny in vivo.

Using murine models of atherosclerosis and fate-mapping approaches, progenitor and hematopoietic cells have been shown to progressively move from BM to splenic RP. In addition, plasma lipids are closely related to monocytosis, exhibiting a direct correlation with total cholesterol and an inverse correlation with high-density lipoprotein HDL in plasma 26 , 45 , Recently, the role of HDL in promoting the outflow from cholesterol-loaded foam cells, has been identified as a better marker of prognostic atherosclerosis than others Reconstituted HDL rHDL infusions or ApoA-I transgenic models protected the athero-prone mice from the disease by preventing the formation of cholesterol-loaded cells, the adhesion of inflammatory cells and the activation of the endothelium 25 , However, there are contrasting studies that show that HDL facilitate the removal of cholesterol from cells, which in turn regulates HSPCs and the ontogeny of leukocytes, especially monocytes 44 , Deletion of the cholesterol efflux genes also resulted in an increase in monocytosis and neutrophilia, leading to an exacerbated progression of atherosclerotic lesions.

Moreover, it also promoted leukocyte infiltration in other tissues spleen, liver and intestine and enhanced EMH. When HSPCs reach EM sites like the spleen, this immune secondary organ becomes an active hematopoietic site; hence, it constitutes a huge provider of myeloid cells that might eventually interact with the atheroma lesion.

Thus, there is a connection between hypercholesterolemia, impaired cholesterol-flow pathways, monocytosis and atherosclerotic disease. These results identify an emerging role of cholesterol-efflux in the control of HSPCs 32 , Beyond lipids and cholesterol impact on EMH and atherosclerosis, the influence of lipid rafts on hematopoiesis homeostasis and CVD has been studied. Highly ordered cholesterol and sphingolipid-rich regions of the plasma membrane lipid rafts , contain receptors involved in HSPCs hematopoiesis e.

Many mechanisms modulate the distribution and composition of lipid rafts, but efficient cholesterol efflux is the most important one and, consistently, cholesterol pathways contribute to hematopoiesis homeostasis Changes in the cholesterol-efflux in the cell membrane, as can happen in various pathologies, augment lipid raft content, leading to distend receptor occupancy and increasing downstream signaling.

Stimulated lipid rafts contribute to protein dimerization, phosphorylation, or crosslinking, activating intracellular signaling pathways. Furthermore, in certain diseases, such as diabetes and obesity, exogenous fatty acid production causes an increase in lipid rafts and in inflammation ABC transporters are found in lipid rafts and their deficiency improves the formation of lipid rafts in HSPCs and myeloproliferation 32 , This mechanism reduces inflammatory signaling downstream of TLR4 Oxysterols, molecules derived from oxygenated cholesterol, participate in the synthesis of bile acids and are important mediators in hematopoietic and immune pathways This situation is common during pregnancy, in which cholesterol plasma levels are increased During pregnancy, HSPCs proliferation and EMH are induced in order to maintain the rapid increase in the maternal blood volume 81 , The spleen plays an important role in lipid metabolism: elevated LDL, altered lipid values, and atherosclerosis are common consequences of surgical removal of the organ in animal models and in humans — Conversely, partial splenectomy and conservative procedures reduce these changes in lipid metabolism Different studies support this view: higher cholesterol, triglycerides and phospholipid levels, and diminished HDL have been reported after splenectomy in rabbits and other animals; however, some researchers reported conflicting results , In humans, splenectomy was related to increased LDL levels and alterations in serological lipid profiles , , , Moreover, individuals with hypersplenism and splenomegaly i.

However, as there are contradictory conclusions in these studies , the contribution of the spleen to the pathophysiology of inflammation and atherogenesis remains an open issue.

This is due to the fact that the synthesis of lipoproteins and their excretion occurs mainly in the liver, and LDLR activity is modulated by many factors, including circulating PCSK9 levels , Furthermore, splenectomy inhibits the pro-inflammatory activity of Kupffer cells in the liver, which are responsible of bacterial and lipoproteins clearance , Moreover, neutralizing the proinflammatory activity of Th1 cells by activation of spleen memory B cells, as well as by the splenic reservoir of T reg cells, has been shown in several pro-atherogenic animal models to be effective against atherosclerosis 54 , — Finally, the spleen is an essential immune tissue that filters the blood and responds to non-self-antigens and to oxLDL by generating anti-oxLDL antibodies, conferring protection against atherosclerosis — Figure 2 Different outcomes in splenectomized patients.

Orange boxes : benefits of a fully functional spleen. Red boxes : summary of the changes that splenectomy exerts on the lipid profile and metabolism. Blue boxes : risks associated to splenectomy. Findings The CT scan of the chest revealed well-defined bilateral paraspinal masses in the lower thoracic regions with no calcification Figure 1.

Focal areas of low density were also seen in the masses. There were no erosive or extrinsic pressure changes on the adjacent vertebrae or ribs. The vertebrae, however, showed increased trabeculation Figure 2. There was hepatosplenomegaly. A large hypodense lesion was seen in the spleen Figure 3.

The diagnosis of EMH was confirmed by fine-needle biopsy of the paraspinal masses. Discussion Extramedullary hematopoiesis is the production of blood elements outside the bone marrow cavity. Until 20 weeks of prenatal life, blood-cell production occurs in the yolk sac and in the reticuloendothelial RE system. It was recently demonstrated that niche-constituting cells within human bone marrow include critical elements, for example mesenchymal stromal cells MSCs , blood vessels, perivascular cells, macrophages and endosteal cells 7.

Investigations into the various mechanisms underlying the interaction between these elements and HSCs have also been conducted. HSCs occur in the vicinity of, but not necessarily in direct contact with, osteoblastic bone-lining cells that constitute the endosteal niche regions.

However, the sinusoidal endothelium also interacts with HSCs, and form the vascular niche 6. Initially it was suggested that the endosteal and vascular niches may control distinct HSC populations; however, more recent data suggests that their effects may be more complicated due to functional crosstalk between cells within the two regions 8 , 9. Furthermore, the vascular niche has been suggested to regulate HSCs in a distinct mechanism or potentially harbor HSCs with various self-renewable properties 10 — Certain mesenchymal cells, including chemokine C-X-C motif ligand 12 CXCL12 -abundant reticular cells CAR cells 13 , nestin-expressing cells 14 and non-myelinating Schwann cells derived from the neural crest 15 , have been reported to function as bone marrow niches.

Furthermore, CXCLdeficient embryos exhibited a marked decrease in HSC populations and hematopoietic function was inhibited; therefore indicating that CXC12 has a critical role during the early stages of bone marrow hematopoietic cell colonization For example, it was demonstrated that stromal cells express CXCL12 and induce B-cell migration in the spleen A previous study suggested that hematopoiesis in bone marrow cannot be maintained by a vascular niche alone, as indicated by osteoblast deficient mice The spleen is a frequent site of EMH during the postnatal period; although, it is only a minor factor in the embryonic stages of hematopoiesis.

The mechanisms underlying the interaction between HSCs and the stromal cells of the spleen during EMH will be discussed subsequently. Conversely, hepatic EMH in adults is associated with various pathological states, including hepatic disorders, sepsis, transplantation, hepatic tumors, for example hepatoblastoma; adenomas and hepatocellular carcinomas 2 , 23 , EMH is most often observed within the hepatic sinusoids.

As with the spleen or liver, EMH may occasionally occur in the lymph nodes and is frequently associated with underlying hematopoietic neoplasms, for example myeloproliferative neoplasms MPNs The high clinical significance of bone marrow transplantation has attracted researchers to focus on the developmental origin of HSCs and niches The yolk sac, in the initial stages of embryonic development, is the initial site of primordial development in the human hematopoietic system, and occurs in the third week of development Vascular elements are initially formed within the yolk sac, as the mesodermal cells begin to accumulate Subsequently, clusters of primitive mesodermal cells emerge in close association with these vascular structures, termed blood islands, and the mesenchymal cells in these blood islands develop into hematopoietic cells and begin to circulate on approximately day 20 of development The development of the rudimentary liver occurs simultaneously to these events HSCs are not generated directly, but instead circulating hematopoietic precursor cells populate the fetal liver These cells proliferate and differentiate in the hepatic parenchyma, forming the primary site of hematopoiesis on approximately day 30 The aorta-gonads-mesonephros region also produces HSCs.

Bone marrow hematopoietic elements commence formation at approximately eight weeks of development, which is relatively late in embryogenesis By week 11, HSCs and the bone marrow niche environment are established in the marrow cavity By contrast, the spleen performs a minor function as a site of erythropoiesis Expansion of the hematopoietic space outside of the bone marrow has been observed in numerous benign hematological disorders, including thalassemia, sickle cell anemia, hereditary spherocytosis, congenital dyserythroblastic anemia and idiopathic thrombocytopenic purpura In response to reduced red blood cell RBC numbers, a homeostatic mechanism induces an increase in the synthesis of RBCs, typically via the production of erythropoietin If the reduction in RBCs is sufficiently severe, hematopoiesis will occur in the extramedullary spaces If granulocyte or platelet production is also insufficient, a similar state occurs Not only benign conditions, but also neoplastic disorders, have been described by the term EMH 32 — Neoplastic myeloid proliferation in the extramedullary spaces has been identified in association with MPNs, myelodysplastic syndromes, granulocytic sarcoma and other myeloid neoplasms 32 — Cellular proliferation may consist of trilineage marrow elements similar to those of benign EMH 32 — The most common sites of EMH associated with neoplastic disorder are the spleen, lymph nodes, skin, bone, small intestine, orbit, breast, cervix, nasal sinus, mediastinum and brain 26 , 28 , Several sites of extramedullary hematopoiesis EMH have been reported in the literature.

The common ones are spleen, liver, lymph nodes, and thymus. Rarely, EMH has been reported in the cardiac tissue, breasts, renal tissue, adrenal glands, pleura, retroperitoneal tissue, skin, prostate, broad ligaments, peripheral and cranial nerves, and the spinal canal [ 3 — 5 ]. Reports indicate that the dura mater may have hematopoietic capacity during fetal life which becomes re-activated in pathologic states [ 6 ]. Other theories include extrusion of hematopoietic tissue from the verterbral body or the proximal rib ends [ 7 , 8 ].

Development of hematopoietic tissue in the intercostal veins with subsequent embolization has also been suggested [ 9 ]. The occurrence of spinal cord compression secondary to EMH is extremely rare and very few cases have been reported in the literature.

The first case was reported by Close et al. The most common cause for spinal EMH is thalassemia. Other rare causes include pernicious anemia, myeloproliferative neoplasm such as polycythemia vera, sideroblastic anemia, pyruvate kinase deficiency, and myelofibrosis disorders [ 11 — 16 ]. Symptomatic spinal cord compression secondary to EMH showed a preference for localization in the middle and lower thoracic spine.

Some authors related this to the narrow spinal diameter at this level [ 17 — 19 ]. Here we report a case of EMH presented as cytopenia and cord compression. The patient is a 51 year-old female who presented with progressively worsening bilateral lower extremity numbness and gait difficulty for 2 months.

Her medical history was significant for stage I breast cancer which was treated with wide-excision lumpectomy followed by radiation therapy.

No hormonal therapy nor chemotherapy was administered. On physical examination, her vital signs were stable and there were no focal cardiac, respiratory or abdominal findings. There was no icterus or palpable lymphadenopathy. There was no palpable hepatosplenomegaly. Her neurological examination revealed intact motor strength and normal tone throughout.

There were no cranial nerve or cerebellar deficits. There was diminished sensation to light touch and pin prick at T5-T6 level. Liver and renal function tests were also normal.

The patient had relatively low reticulocyte count, normal iron studies, folate and vitamin B12 levels. Hb electrophoresis revealed Hb A2 alpha 2 delta 2 3. Urine and serum protein electrophoreses were unremarkable. Bone marrow karyotyping showed 46 XX. Alpha-globin gene analysis for alpha thalassemia was negative. MRI of the entire spine revealed abnormal signals of the cervical, thoracic and lumbar vertebrae. A heterogeneous predominantly T2 hyperintense and T1 isointense enhancing lesion 9.

In addition, there were large bulky bilateral enhancing paraspinal lesions from T6 extending to T12 There was a soft tissue presacral mass ajacent to S5 measuring approximately 2. The differential diagnosis at this point was metastatic disease with unknown primary, likely breast vs. The patient underwent a bone marrow biopsy which revealed hypercellular bone marrow with erythroid hyperplasia. Flow cytometry study was negative for leukemia and lymphoma. Bone marrow core biopsy was negative for metastatic disease.

For relief of neurological symptoms and final tissue diagnosis, patient underwent T4-T9 total laminectomy and removal of the thoracic epidural tumor. Intra-operative frozen section revealed suspicious lymphoproliferative process. Final pathology of the resected mass revealed dense hematopoietic tissue with erythroid hyperplasia, scattered megaloblastic forms and megakaryocytes, entrapped bony spicules, intervening fibrous septa and adjacent scant adipose tissue Fig.

The tissues were positive for hemoglobin A and CD