The hepatic extramedullary hematopoiesis during experimental murine Schistosomiasis mansoni

Abstract

Many years ago, our research group has demonstrated extramedullary hematopoiesis in the peripheral zone of murine hepatic schistosomal granulomas. In the present study, we revisit this phenomenon using new technical and conceptual approaches. Therefore, newborn mice were percutaneously infected by Schistosoma mansoni cercariae and euthanized between 35- and 60-days post infection. Liver samples were submitted to histopathology and immunohistochemical analyses. Cells under mitosis and/or expressing Ki67 demonstrated the proliferation of hematopoietic cells both around the parasite's eggs trapped in the liver and around hepatic vessels. After 50 days post infection, proliferating cells at different levels on differentiation were located preferentially in the peripheral zone of the granulomas, around the vessels and inside the sinusoids. The presence of acidic and sulfated glycoconjugates, reticular fibers and the absence of fibronectin characterized the microenvironment for attraction and maintenance of hematopoiesis. Some neutrophils secreted MMP9 from the earliest points of infection, indicating degradation of the extracellular matrix in regions of histolysis and a possible chemoattraction of hematopoietic stem cells to the liver. Fall-3+ cells and Sca-1+ cells indicated that early hematopoietic progenitors could be mobilized to the liver. Groups of vWF+ megakaryocytes suggest chemoattraction of these cells and/or migration, proliferation, and differentiation of very immature progenitors to this organ. The increase of blood vessels and extramedullary hematopoiesis in this environment, where markers of immature hematopoietic and endothelial cells have been identified, points to the possibility of the presence of progenitors for endothelial and hematopoietic cells in the liver during the infection. There is also the possibility of concomitant migration of more differentiated hematopoietic progenitors, that proliferate and differentiate in the liver, and the occurrence of angiogenesis caused by inflammation or release of ovular antigens that stimulate the activation and proliferation of endothelial cells. Altogether, these data increase knowledge about a murine model that is of interest for investigating the pathology of the schistosomiasis and also the dynamics of hematopoiesis.

Keywords: angiogenesis; extramedullary hematopoiesis; granuloma; hematopoietic environment; schistosomiasis.

Figure 1

Figure 1 Evolution of hepatic granulomatous lesions over the days of infection. HE-stained sections demonstrate: (A) a small inflammatory infiltrate (*) composed of eosinophils and macrophages around the egg (E) at 35 dpi; (B) inflammatory infiltrate (*) greater than that observed at the previous point of infection, located between the necrotic hepatocytes (arrow) around the egg (O); (C) Larger granuloma with deposition of fibrous extracellular material, but still disorganized at 45 dpi; (D, E) Granulomatous lesion found in the acute phase of infection, at 50 and 60 dpi, is organized into three zones – central (CZ), medial (MZ) and peripheral (PZ), with proliferation of hematopoietic cells in the peripheral zone. Bars, A: 20 µm, B – E: 50 µm.

Figure 2

Morphological aspect of hepatic perivascular lesions in mice infected with S. mansoni. HE-stained sections demonstrate: (A) Inflammatory infiltrate composed of myeloid cells, eosinophils (arrowhead), macrophage cells (yellow arrow) and neutrophils (black arrow) in the hepatic portal space of mice at 40 dpi. (B) hematopoietic cells of the myeloid lineage, highlighting a cell in mitosis (arrow), in the periphery of a vein at 45 dpi; (C) Hepatic portal space with many mature and immature hematopoietic cells at 60 dpi; and (D) highlighting cells in mitosis (arrow) in the periphery of vessels in the hepatic portal space at 60 dpi. Bars, (A, B, D) 20 µm, (C) 50 µm.

Figure 3

Localization of proliferating cells in the periphery of blood vessels in the liver by Ki67 labeling. (A) Some Ki67-positive cells are seen into the sinusoids at 35dpi; (B) cells of the neutrocytic (white arrow) and monocytic (yellow arrow) lineage that express Ki67 are located among other myeloid cells around a blood vessel at 40 dpi; (C) Ki67-positive neutrophils (white arrow) and monocytes (yellow arrow) within a sinusoid at 45 dpi; (D) cells of the eosinophilic (red arrow), neutrocytic (white arrow) and monocytic lineages (yellow arrow) in different stages of differentiation that expressing Ki67 are located inside of sinusoid at 60 dpi. At this point of infection also is possible to observe some hepatocytes expressing the molecule which indicates cell proliferation (arrowhead). Bar: 20 µm. Blue: DAPI, Red: Evan’s blue, Green: Ki67.

Figure 4

Localization of proliferating cells in schistosomal granuloma in the liver by Ki67 labeling. (A) Cells of the neutrocytic and monocytic lineage which express Ki67 are arranged among the other inflammatory cells that form the 40-day infection granuloma (B) A greater number of hematopoietic cells, mainly of the neutrocytic and monocytic lineage, are stained with Ki67 and are concentrated in the periphery of the granuloma at 45 dpi; (C, D) At 50 dpi in addition to cells of the monocytic and neutrocytic lineages, cells of the eosinophilic lineage expressing Ki67 are also observed. These cells are in the peripheral zone of the mature granuloma; (E, F) The proliferate Ki67-labeling hematopoietic cells are localized in the peripheral zone of granuloma at 60 dpi, as was observed in the previous point of infection analyzed. Bars, (A) 20 µm, (B, C, E) 50 µm, (D, F) 10 µm. Blue: DAPI, Red: Evan’s blue, Green: Ki67.

Figure 5

General localization of proliferating cells of murine schistosomal liver by Ki67 labeling, during acute phase of the disease. A Hematopoietic cells proliferating in the schistosomal liver of mice are mainly and almost restrictedly located in the periphery of the large vessels, inside some sinusoids and in the peripheral zone of mature granulomas. E, Egg; BV, Blood Vessel; MZ, Medial Zone; PZ, Peripheral Zone. Blue, DAPI; Red, Evan’s blue; Green, Ki67.

Figure 6

Extracellular matrix elements found in hepatic schistosomal granuloma. (A) Reticular fibers, highlighted in black by Gomori’s reticulin staining, extend throughout the immature granuloma; (B) The reticular fibers, evidenced by Gomori’s reticulin staining, in the 60 dpi granuloma form a looser mesh-like in the peripheral zone (PZ) of the granuloma, forming compartments (arrow) that house hematopoietic; (C) Types I and III collagen fibers are evidenced by Sirius red staining and are present among most of the cells that are part of the granuloma of 45 dpi, with the exception of the closest to the egg (E); (D)The collagenous fibers identified by Sirius red staining present in the mature granuloma have the conformation of a network with the looser weft in the peripheral zone (PZ) of the structure. In this region, these thin fibers form small compartments that house isolated hematopoietic cells or in small groups; (E) Sulfated acid glycoconjugates can be identified by Alcian Blue staining in the full extent of immature granulomas; (F) Alcian blue staining is concentrated in medial zone and is absent in the peripheral zone of mature granulomas; (G) In immature granuloma, fibronectin expression occurs more concentrated in the region closest to the egg and more diffused among cells that are distant from the parasitic element; (H) Fibronectin expression in mature granuloma can be seen in the medial zone (MD) and is absent in the peripheral zone (PZ). Bars (C–E): 50 µm, (A, B, F, G, H) 20 µm. Stains: (A, B) Alcian Blue pH 1,0, (C, D) Picrosirius, (E, F) Gomori’s Reticulin. (G, H) White: DAPI, Red: Evan’s blue, Green: Fibronectin.

Figure 7

Extracellular matrix elements found at the periphery of large hepatic vessels during Schistosoma mansoni infection. (A, B) the arrangement of reticular fibers, highlighted by Gomori’s reticulin, was resembling to the observed in samples stained by picrosirius in both of point of infection; (C), (D) At different times of infection, types I and III collagen fibers, identified by picrosirius, are arranged in a similar morphology among the hematopoietic cells that surround the large hepatic vessels. They form compartments like those seen in the periphery of mature granulomas, which harbor groups of hematopoietic cells; (E) Absence of Alcian Blue staining, which identifies sulfated acid glycoconjugates, among hematopoietic cells present in the periphery of large hepatic blood vessels (BV), such as portal veins (PV), at earlier points of infection; (F) and during the acute phase of the disease; (G, H) Anti-fibronectin immunostaining did not identify the expression of the molecule among the hematopoietic cells that surrounded the hepatic blood vessels at the two points of infection (45 and 60 dpi). Bars, (A–G) 20 µm, (H) 50 µm. Stains: (A, B) Alcian Blue pH 1,0, (C, D) Picrosirius, (E, F) Gomori’s Reticulin. (G, H) White: DAPI, Red: Evan’s blue, Green: Fibronectin.

Figure 8

Identification of expression of MMP9 in murine liver granulomas by immunofluorescence. (A) Neutrophils expressing MMP9 were immunolabeling in an inflammatory infiltrate between necrotic hepatocytes (arrow) at 40 dpi; (B) The expression of MMP9 in granulomas found in mice at 45 dpi was concentrated in neutrophils located in de periphery of structure; (C, D) Neutrophils at different points of infection express MMP9 preferentially in the peripheral zone of mature granulomas at 50 dpi. Bars, (A–C) 20 µm, (D) 10 µm. DAPI: blue (A, B), white (C, D), Evan’s blue: red, MMP9: green.

Figure 9

Identification of expression of MMP9 in the periphery of large blood vessels in mice infected by S. mansoni by immunofluorescence. (A) Some neutrophils expressing MMP9 are arranged randomly among others hematopoietic cells in the periphery of a vessel with no apparent parasitic element at 45 dpi and (B) 60 dpi. Bars, (A) 20 µm, (B) 50 µm. DAPI: blue (A), white (B), Evan’s blue: red, MMP9: green.

Figure 10

Identification of immature hematopoietic cells by immunofluorescence in livers of mice infected by S. mansoni. (A, C) Fall 3 immunostaining identified cells with characteristics of immature hematopoietic cells (loose chromatin and protruding nucleolus) close to a central vein (CV); (B) These cells Fall 3-positives also were found in the periphery of 45 dpi granuloma; (D, E) The immunostaining identified the expression of Sca-1 in a blood vessel (arrow) placed on medial zone some cells located in the peripheral zone of 50 dpi granuloma. Bars (A, B, D, E) 20 µm, C: 12 µm DAPI: red (A–C), white (D, E), Evan’s blue: blue (A–C) and red (D, E), Fall 3: green (A–C), Sca-1: green (D, E).

Figure 11

Expression of vWF in livers of mice infected by S. mansoni, identified by immunofluorescence. (A) Detection of more than one megakaryocyte among the hepatocytes in the same area of analysis at 45 dpi; (B) Blood vessel vWF-positive with inflammatory cells in the periphery situated between the endothelium and the hepatocytes at 45 dpi; (C) (C) Megakaryocytes (arrow) are still found in the liver of infected animals during the acute phase of the disease, close to the peripheral zone of the granulomas, but still in contact with hepatocytes, (D) and close to a vessel with surrounding hematopoietic cells at 50 dpi; (E) central vein (CV), vessels associated with the periphery of the granuloma and some sinusoids express the protein at 60 dpi. Bars, A: 50 µm, (B, C, E) 20 µm, D: 10 µm. DAPI: white, Evan’s blue: red, vWF: green.

Figure 12

Identification of blood vessels in livers of mice infected by S. mansoni, immunostaining by CD31 and Lyve 1. (A) Sinusoids that are observed among the other cells that form the granuloma at 45dpi express CD31; (B) CD31 immunostaining identified a blood vessel with sinusoidal morphology (arrow) in the medial zone of the granuloma and other vessels with broad lumen (arrowhead) in the peripheral zone of the same granulomas at 60 dpi; (C) Identification of lymphatic vessels (arrowhead) located in the periphery of mature granulomas at 50 and (D) 60 dpi. Bars, 20 µm. DAPI: blue, Evan’s blue: red, CD31: green (A, B), Lyve-1: green (C, D).