Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis

Abstract

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-β1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-β1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-β1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-β1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-β1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.

Keywords: Autoimmunity; Inflammation; Myelofibrosis; TGF-β1.

Figure 1. The bone marrow and spleen of PMF patients present unique alteration signatures of TGF-β signalling

Hierarchical clustering of normalized gene expression in bone marrow and spleen from non-diseased volunteers (controls, C, n=3) and PMF patients. Results were obtained with BM from three JAK2V617F-mutated PMF patients and spleen from four JAK2V617F-mutated (in black) and two JAK2V617F-mutated PMF patients.

Figure 2. Quantitative RT-PCR validation that the alteration signatures of bone marrow and spleen of PMF patients predict activation of non-canonical (up-regulation of JUN, GADD45B, EVI1 and STAT1) rather than canonical (down-regulation of BMP4, BMP7, SMAD7, STAT1, and up-regulation of COL1A1), TGF-β signaling

Results are expressed in relative units considering the expression in the corresponding organs from non diseased volunteers (Ctrl) as 1 and are presented as mean (±SD) of values observed with BM from three PMF patients and three non-diseased individuals and with spleen from five PMF patients and three non-diseased individuals (the same samples analyzed in Figure 1). To allow appreciation of the difference in gene expression between BM and spleen, Figure S1 presents results from both BM and spleen expressed in relative units considering values in BM from non diseased volunteers as 1.

Figure 3. Plasma from PMF patients contains levels greater than normal of cell-free mitochondrial DNA (A) and anti-mitochondrial antibodies (B) and normal levels of antinuclear antibodies (C)

Results are expressed as individual values (symbols) and as mean (±SD) of values (columns) from non-diseased volunteers (Ctrl), essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. The same individuals were analyzed in A–C. The numbers of individuals included in each cohort is indicated in parenthesis. The yellow area in C indicates the range of optical density observed in the standards provided by the Kit as negative controls. See Material and Methods for further details.

Figure 4. A model for establishment of the autoimmune process possibly leading to fibrosis in PMF

Neutrophils presenting extracellular traps (NET) of autologous DNA trigger autoimmune reactions resulting in fibrosis in cystic fibrosis (Reviewed in ref 33). Using this mechanism as a foundation, we propose here that NET formed by mitochondrial DNA released by MK dying of para-apoptosis during the process of pathological emperipolesis with neutrophils (refs 10, 34, 35) may trigger the autoimmune reaction leading to fibrosis in PMF. Legend: Neu = neutrophil; MK = megakaryocyte; nu = para-apoptotic nucleus; mDNA = mitochondrial DNA.