The specificity and restriction of Foxp3 expression to a subset of T lymphocytes has provoked controversy in the scientific literature 30–33
. However, more recent studies indicate that Foxp3 is expressed in some epithelial cells and some tumor cells 24, 34
. The abnormal platelet levels observed and the hemorrhaging that occurs in IPEX syndrome and in Foxp3sf
mice prompted us to examine Foxp3 expression in the megakaryocyte lineage and determine if it plays a role in megakaryopoiesis and platelet production. Herein, we demonstrate that primary human megakaryocytes and megakaryoblastic cell lines express Foxp3 mRNA and protein, although to a lesser extent than in Treg
cells. Consistent with its putative role as a transcription factor, Foxp3 protein expression in megakaryocytes was predominantly nuclear which may account for the failure to detect Foxp3 in human platelets (). Nuclear localization of Foxp3 may also explain increased levels of Foxp3 protein expression following PMA treatment (), as PMA increases cellular DNA content in megakaryocytes by inducing endomitosis 35
. We also demonstrated that Foxp3 protein expression was greater in megakaryoblastic cell lines compared to primary human megakaryocytes (). However, the reason for, and the consequences of this increased expression are unknown. It has been speculated that Foxp3 plays an intrinsic role in malignant transformation and tumor survival 24, 36
. Interestingly, the Meg-01 cell line and the Dami cell line were derived from patients with megakaryoblastic leukemias that presented elevated bone marrow blast cells and thrombocytosis 10, 37
. Future studies will examine the importance of Foxp3 expression in megakaryoblastic cell lines.
Our findings indicate that Foxp3 plays an important role in megakaryopoiesis. Foxp3sf mice, which lack the full length, functional Foxp3 protein, had 4-fold fewer bone marrow megakaryocytes compared to C57BL/6J mice (). Further, megakaryocyte colony number was ~ 50% lower in Foxp3sf mice compared with C57BL/6J mice (). In addition, megakaryocyte mean colony size was lower in Foxp3sf mice compared with that in C57BL/6J suggesting that a reduction in progenitor proliferation contributes to reduced progenitor number (). These new findings demonstrate that the reduced colony number in Foxp3sf mice is due to a defect in megakaryocyte progenitor proliferation and could also be due to a potential role of Foxp3 in the generation of megakaryocyte progenitors from upstream bipotential or multipotential progenitors. We also observed that platelet count does not necessarily correlate with megakaryocyte number or megakaryocyte progenitor number. The platelet counts from individual mice exhibited more variability which may indicate differences in the ability of mature megakaryocytes to produce platelets. These data however, do not rule out the possibility that the reduced megakaryocyte number is a consequence of profound defects in T regulatory cells. Megakaryopoiesis may be impaired in vivo due to the wide range of autoimmune-associated symptoms which characterize the Foxp3sf mouse. However, Foxp3 knock-down human Meg-01 cells demonstrate a greatly reduced proliferative response, suggesting a direct role for Foxp3 in megakaryopoiesis ().
The precise mechanism by which Foxp3 regulates megakaryopoiesis remains unknown. Foxp3 functions in T lymphocytes, in part, as a transcriptional repressor by recruiting both histone acetyl transferases and histone deacetylases 38
. Foxp3 also functions as a passive transcriptional repressor by physically interacting with proteins such as nuclear factor-kappa B (NF-κB) 39, 40
and acute myeloid leukemia 1 (Aml1)/runt related transcription factor 1 (Runx1) 41
. Foxp3 may be playing a similar role in megakaryocytes by suppressing or activating transcription factors.
Genetic lesions in megakaryocytes that cause thrombocytopenia often cause abnormal platelet function. Foxp3sf
mice have reduced platelet counts and our new findings demonstrate that they exhibit striking activation abnormalities. TGF-β serum levels were significantly lower in Foxp3sf
mice. Since platelet-derived TGF-β is a cytokine mainly involved in wound healing and tissue repair, we speculate that reduced TGF-β could potentiate the dermatitis and the skin lesions which characterize the IPEX disease and the Foxp3sf
mice. In addition, TGF-β can suppress T cell responses. TGF-β reduces T cell proliferation by inhibiting IL-2 production and upregulating cell cycle inhibitors 42
and inhibits the differentiation of Th1 to Th2 by down-regulating T-bet and GATA-3 43
. TGF-β also inhibits the activation of macrophages and reduces the ability of dendritic cells to present antigens to T cells 42
. Interestingly, we found that Foxp3sf
serum had elevated levels of CD40L and two arachidonic acid metabolites, TXB2
and 12(S)- HETE, despite having fewer platelets. TXB2
is a more stable metabolite of thromboxane A2
), a cyclooxygenase-derived product generated by platelets which induces irreversible platelet aggregation and vascular smooth muscle contraction. 12(S)-HETE is a 12-lipoxygenase-derived product that is produced abundantly in platelets during activation. These data suggest that the Foxp3sf
platelets produce more arachidonic acid metabolites during activation. As described in the Introduction, CD40L activates immune and structural cells, as well as platelets. The majority of the circulating soluble CD40L originates from platelets and CD40L levels are elevated during inflammatory disease states 44–46
. Collectively, these new data suggest that the platelet phenotype in Foxp3sf
mice contributes to the inflammation observed in the ‘scurfy’
mice also had increased mean platelet volumes (). There are many intrinsic and reactive reasons why platelet volume is elevated in disease states. For example, Gata-1 knock out mice have deficiencies in megakaryocyte maturation and as a result, their platelets have elevated volumes 47
. The peripheral platelet destruction from circulating anti-platelet antibodies increases platelet volume in immune thrombocytopenic purpura (ITP) patients because a higher percentage of platelets are younger 48, 49
. Therefore, the increased platelet size observed in Foxp3sf
mice may indicate both impaired platelet production and peripheral platelet destruction or in contrast that the elevated mean platelet volume may be compensating for the decrease in platelet number.
To determine if a similar platelet phenotype was observed in IPEX, the human correlate of scurfy, we examined the platelets of an IPEX patient. IPEX is rare disease involving various Foxp3 mutations and can result in death at an early age 29
. About 50% of IPEX patients are reported to be thrombocytopenic and hemorrhage is one of the most common causes of death in untreated patients 9
. Gastrointestinal bleeding has occurred in a case with normal platelet counts, suggesting inadequate platelet function 50
. The IPEX donor evaluated herein was aged 17 years and had a G to A transition (1150G>A) in exon 11, resulting in a substitution of Ala to Thr at residue 384, within the DNA binding domain of Foxp3 20
. His platelets demonstrated striking abnormalities in spreading () and a reduced ability to release CD40L and TGF-β in response to potent platelet activators (). Our IPEX donor also demonstrated a profound elevation in plasma levels of CD40L (). These data indicate that the IPEX patient did not respond normally to platelet activators and possibly that his platelets already released internal stores of CD40L in vivo
We also demonstrated that the IPEX patient had 6 times more platelet factor 4 (PF4) in the supernatants of both unactivated and activated platelets compared with the normal donor. This suggests that the IPEX platelets contain higher levels of PF4 or that the release of PF4 from alpha granules is enhanced. Since IPEX platelets release less TGF-β and CD40L than the normal platelets, we speculate that IPEX platelets contain higher levels of PF4. The increased release of PF4 may implicate platelets in the symptomology of IPEX. IPEX disease is characterized by severe atopic dermatitis and recently, plasma levels of PF4 were shown to be elevated in patients with atopic dermatitis and in a mouse model of atopic dermatitits 51–53
. In addition, PF4 is a negative regulator of megakaryopoiesis suggesting that elevated PF4 may be a mechansism for inhibiting megakaryocyte proliferation 54
. Collectively, these new findings demonstrate that the defect in Foxp3 observed in IPEX influences platelet function.
Our study adds considerable new information to the ongoing discussion of the presence of Foxp3 in cell types other than Tregs. Overall, we have shown that Foxp3 deficiency results in a lesion of megakaryocyte proliferation that is associated with platelet dysfunction. These new findings support the concept that genetic disorders that cause thrombocytopenia also cause abnormal platelet function such as occurs in myelodysplasias. Therefore, we have elucidated an underlying mechanism of megakaryopoiesis that contributes to the pathophysiology of IPEX syndrome and the ‘scurfy’ disease.