Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice

Abstract

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-β1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

The CXCR1/CXCR2 Inhibitor Reparixin Alters the Development of Myelofibrosis in the Gata1low Mice

“…This alternative hypothesis is supported by the observation that, since GATA1 regulates the differentiation of dermal mast cells, derma of Gata1 low mice contains great numbers of these cells ( 37 ). In addition, wild-type CD1 mice, the background in which we harbor the Gata1 low mutations express a systemic pro-inflammatory signature which determines chronic dermatitis with dermal fibrosis ( 32 ). This baseline dermal fibrosis is also present in Gata1 low mice (ARM, unpublished observations) and it is possibly exacerbated once the mast cells are activated by the mechanical stress induced by the mini-pumps implanted subcutaneously, reducing the efficiency of the dermal absorption and of the plasma levels of the drug.…”

“…Since the hypomorphic Gata1 low mutation is characterized by reduced expression of GATA1 in MKs and the BM from these mice express high levels of TGF-β ( 32 , 44 ), we hypothesized that this fibrosis may be sustained by increased numbers of niche-poised MKs with low levels of GATA1 due to the increase in BM TGF-β and that the reduced levels of TGF-β induced by Reparixin would reduce the degree of marrow fibrosis by reducing the frequency of this niche-poised MKs present in BM. To test this hypothesis, we first analyzed the content of GATA1 in MKs from BM sections of mice treated either with vehicle or with Reparixin ( Figure 7 ).…”

“…Femurs were fixed in formaldehyde (10% v/v with neutral buffer), treated for 1 h with a decalcifying solution (Osteodec; Bio-Optica, Milan, Italy) and paraffin embedded. Spleens were fixed in formaldehyde as previously described ( 32 ) and embedded in paraffin. Paraffin-embedded tissues were cut into consecutive 3 μm sections and stained either with Hematoxylin–Eosin (H&E; Hematoxylin cat no.…”

“…Mice carrying the hypomorphic Gata1 low mutation express the same MKs alterations observed in MF patients and develop progressive MF closely resembling human disease, namely, a BM failure syndrome and development of extramedullary hematopoiesis ( 28 – 30 ). Previous studies have demonstrated that MKs from the BM of Gata1 low mice express not only high levels of TGF-β1 ( 31 ), but also high levels of mCXCL1, the murine equivalent of hCXCL8 ( 32 ). Using these data as a foundation, in the current study we have evaluated whether MKs from Gata1 low mice express CXCR1 and CXCR2 and tested whether treatment with Reparixin affects the development of MF in this mouse model.…”

GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis

“…The causative role of this abnormal MK in the etiology of the disease has been validated by experiments demonstrating that mice expressing either the driver mutations or low levels of Gata1 ( Gata1 low mice) only in MK 36 develop myelofibrosis with age 37 . The abnormalities of human and murine GATA1 hypomorphic MK include a content greater than normal of TGF-β 38 , CXCL1, the murine equivalent of IL-8 39 , and P-SEL 40 , three of the proinflammatory proteins that have been implicated also in the development of IPF.…”