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 (B… Show more
“…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 This baseline dermal fibrosis is also present in Gata1 low mice (ARM, unpublished observations) and it is possibly exacerbated once the mast cells are activated by the mechanical stress induced by the mini-pumps implanted subcutaneously,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.…”
Section: Resultsmentioning
confidence: 99%
“…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 ).…”
Section: Resultsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-β, the megakaryocytes from the bone marrow of the Gata1low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-β1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-β1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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 ).…”
Section: Resultsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-β, the megakaryocytes from the bone marrow of the Gata1low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-β1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-β1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.
“…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.…”
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder with limited therapeutic options. Insufficient understanding of driver mutations and poor fidelity of currently available animal models has limited the development of effective therapies. Since GATA1deficient megakaryocytes sustain myelofibrosis, we hypothesized that they may also induce fibrosis in lungs. We discovered that lungs from IPF patients and Gata1low mice contain numerous GATA1negative immune-poised megakaryocytes that, in mice, have defective RNA-seq profiling and increased TGF-β1, CXCL1 and P-selectin content. With age, Gata1low mice develop fibrosis in lungs. Development of lung fibrosis in this model is prevented by P-selectin deletion and rescued by P-selectin, TGF-β1 or CXCL1 inhibition. Mechanistically, P-selectin inhibition decreases TGF-β1 and CXCL1 content and increases GATA1positive megakaryocytes while TGF-β1 or CXCL1 inhibition decreased CXCL1 only. In conclusion, Gata1low mice are the first genetic-driven model for IPF and provide a link between abnormal immune-megakaryocytes and lung fibrosis.
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