Background
Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified as a causative agent of human diseases, including adult T-cell leukemia/lymphoma (ATL) [
1‐
3] and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [
4,
5]. HAM/TSP is a chronic progressive inflammatory myelopathy, pathologically characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration by HTLV-1 infected cells [
6,
7]; it is therefore widely assumed that the immune response causes the inflammatory spinal-cord damage in HAM/TSP [
8]. It is well known that elevated production of interferon-gamma (IFN-γ) is an important immunological marker of HAM/TSP pathogenesis. Indeed, in HTLV-1 infected effector memory cells of HAM/TSP patients, intracellular expression of the viral transcriptional regulator Tax is associated with rapid upregulation of IFN-γ [
9]. HTLV-1-specific CD8+ cytotoxic T lymphocytes (CTLs) have the potential to secrete high levels of IFN-γ [
9,
10] and are abnormally abundant in cerebrospinal fluid and spinal cord lesions [
11,
12]. HTLV-1 primarily infects CD4+ T helper (Th) cells, which play a key role in adaptive immune responses [
13]. More precisely, the predominant viral reservoir of HTLV-1 in infected individuals is CD4 + CD25 + CCR4+ T cells [
14], which consist primarily of suppressive T cell subsets such as regulatory T cells (Tregs) and Th2 cells [
15]. Importantly, two viral transcriptional regulators, Tax and HTLV-1 bZIP factor (HBZ), regulate the expression of the Foxp3 gene at the mRNA level [
14,
16]; thus, the majority of HTLV-1 reservoir CD4 + CD25 + CCR4+ T cells consist of Foxp3+ Tregs [
17]. Furthermore, Tax induces transcriptional changes in HTLV-1 infected T cells through loss of expression of the transcription factor Foxp3 resulting in conversion of immunosuppressive Foxp3 + CD4 + CD25 + CCR4+ Tregs to IFN-γ-producing proinflammatory Foxp3-CD4 + CD25 + CCR4+ T cells [
15]. Furthermore, during HTLV-1 infection, the frequency of HTLV-1–negative Foxp3 + CD4+ T cells is positively correlated with the HTLV-1 proviral load (PVL) [
18,
19], the most important risk factor for developing HAM/TSP, and CTL activity is negatively correlated with the frequency of HTLV-1–negative Foxp3 + CD4+ cells [
19]. These findings suggested that T cell plasticity induced by HTLV-1 infection contributes to the pathogenesis of HAM/TSP.
EOS, which is encoded by the Ikzf4 gene, is a transcription factor that belongs to the Ikaros family and is primarily expressed in Tregs [
20]. It plays a critical role in maintaining the stability and suppressive functions of Tregs [
21]. Importantly, EOS forms a complex with Foxp3 that helps maintain the suppressive Treg cell phenotype, and downregulation of EOS in response to specific inflammatory signals induces a regulated transformation of Tregs into T helper-like cells which are capable of secreting proinflammatory cytokines [
20]. These findings suggested that dysregulation of EOS in HTLV-1 infected Tregs is involved in the inflammatory and neurodegenerative processes of HAM/TSP. We therefore investigated whether there was an alteration in the expression of EOS in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals with various clinical statuses, i.e. ATL, HAM/TSP, or asymptomatic carriers (ACs).
The results showed that there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals, irrespective of their clinical statuses. Furthermore, we found an inverse correlation between EOS mRNA levels and PVL in ATL, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP, suggesting a possible role for EOS in the pathogenesis of HTLV-1-associated diseases.
Discussion
The members of the Ikaros family of zinc finger (Ikzf) transcription factors have a crucial role not only in immune cell development and homeostasis, but also pathological conditions in human [
21,
44]. It has been reported that deregulation of IKAROS, the founding member of the family encoded by the Ikzf1 gene, results in leukemia in both mice and human [
45], whereas downregulation of EOS, encoded by the Ikzf4 gene, induces a regulated conversion of Tregs into T helper-like cells that are capable of secreting proinflammatory cytokines in response to specific inflammatory signals [
20]. As HTLV-1 can cause both leukemia (i.e., ATL) and inflammatory disease (i.e., HAM/TSP), and conversion of Tregs to T helper-like cells has been reported in patients with HAM/TSP [
15], we investigated in this study whether the expression level of EOS is associated with HTLV-1 infection and progression to disease.
Our data indicated that most HTLV-1 infected T-cell lines (i.e., 8 out of 10 tested) exhibited high expression of the EOS protein. In HTLV-1 infection, it is well established that both Tax and HBZ play a key role in the transactivation of viral and cellular genes for HTLV-1 replication and pathogenesis [
46]. To date, several genes including transcription factors and cell signaling mediators have been reported to be Tax targets [
39,
47,
48]. Consistent with these findings, our data showed that the expression of EOS protein was augmented after Tax induction in JPX-9 cells, and Tax, but not HBZ, physically interacts with the EOS protein. Tax-mediated expression of EOS was further supported by our previous microarray study, which indicated that the EOS gene was induced after induction of Tax but not HBZ in Jurkat T-cells [
39]. These findings suggest that EOS is one of the cellular targets of Tax. Interestingly, however, among those HTLV-1 infected T-cell lines which exhibited high expression of the EOS protein, gene expression (mRNA) levels of EOS were relatively low except for two T-cell lines (MT-2 and ILT-M1), thus did not correlate well with protein levels. These results suggest that stable expression of EOS is not only dependent on Tax-mediated regulation, but also on various cellular processes including protein degradation and/or epigenetic mechanisms, which might be altered in different HTLV-1 infected T-cell lines.
In PBMCs of HTLV-1 infected individuals, EOS mRNA levels was considered low when compared with NCs, regardless of clinical statuses, i.e., ATL, HAM/TSP, or ACs. However, since ex vivo data obtained using patient samples may provide more valuable insight into the clinical significance of EOS with respect to the risk of HTLV-1 associated diseases, we assume that this is the most fascinating and important finding of the present study. In ATL patients, there was an inverse correlation between EOS mRNA levels and HTLV-1 PVL, suggesting lower levels of EOS mRNA expression in ATL cells through an as yet undetermined mechanism. ATL is divided into four clinical subtypes, i.e., acute, lymphoma, chronic, and smoldering [
24], and patients with acute, lymphoma, and chronic type (with unfavorable prognostic factors) are categorized as suffering from aggressive ATL, whereas those with the chronic type without unfavorable prognostic factors, and the smoldering type are categorized as suffering from indolent ATL [
49]. Treatment of ATL is usually determined based on these clinical subtypes and prognostic factors, and the presence of an aggressive or indolent ATL is critical when making clinical decisions. As our data showed significantly lower EOS mRNA levels in patients with aggressive ATL than in patients with indolent ATL, EOS mRNA levels may serve as an additional diagnostic marker for aggressive or indolent ATL.
In contrast to ATL patients, we found a positive correlation between EOS mRNA load and PVL in HAM/TSP patients. Moreover, consistent with our previous study [
43], we also found a positive correlation between HBZ mRNA load and PVL. As a consequence, the expression levels of EOS mRNA showed a significant positive correlation with HBZ, but not with Tax, expression. Since we previously reported that the expression levels of HBZ but not Tax mRNA positively correlated with disease severity in HAM/TSP patients [
43], it is conceivable that HBZ, which has a bimodal function at the mRNA and protein levels, increases the level of EOS mRNA in PBMCs of HAM/TSP patients. However, there was conversely a significant decrease in EOS mRNA level in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses, i.e., ATL, HAM/TSP, or ACs. Since the EOS gene is regulated by various cellular processes, including protein degradation and/or epigenetic mechanisms, one possible explanation for the decreased levels of EOS mRNA in PBMCs is the differing methylation status of the EOS regulatory regions between HTLV-1 infected individuals and NCs, especially the Treg-specific methylation region (TSDR). Namely, if the demethylation rate is lower in PBMCs from HTLV-1 infected individuals than in NCs, decreased demethylation may be associated with decreased expression. Indeed, Anderson et al. have already reported that the demethylation rate of the Foxp3 TSDR is lower in PBMCs and CD4 + CD25+ T cells from HAM/TSP patients than in uninfected normal controls, and decreased TSDR demethylation is associated with decreased functional suppression via Tregs in HAM/TSP patients [
50].
It is well established that HTLV-1 infection induces an abnormal frequency and phenotype of Foxp3 + CD4+ T cells in infected individuals [
51]. In particular, HTLV-1–negative Foxp3 + CD4+ Tregs are negatively correlated with CTL activity [
19], and positively correlated with HTLV-1 PVL [
18,
19]. These findings suggest that HTLV-1–negative Foxp3 + CD4+ Tregs are the primary determinant of the efficiency of T cell–mediated immune control of HTLV-1. Both Tax and HBZ are associated with this process. Namely, HBZ RNA promotes T cell proliferation [
52] and induces Foxp3 expression [
16], which results in the increased frequency of HTLV-1–negative Foxp3 + CD4+ Tregs, whereas Tax induces the conversion of immunosuppressive Foxp3 + CD4 + CD25 + CCR4+ Tregs to IFN-γ-producing proinflammatory Foxp3-CD4 + CD25 + CCR4+ T cells via transcriptional changes in HTLV-1 infected T cells through loss of Foxp3 [
15]. Importantly, in addition to neurological symptoms, some HAM/TSP cases also exhibit autoimmune-like disorders, such as uveitis, arthritis, T-lymphocyte alveolitis, polymyositis, and Sjögren syndrome [
53], and both Tax and HBZ are associated with the inflammatory process of HAM/TSP via regulation of Treg and T helper cells [
54]. Because increased HBZ levels increased the risk of developing HAM/TSP [
55], it may be possible that HBZ induces Foxp3, which reduces CTL activity, which, in turn, increases the HTLV-1 PVL.
Meanwhile, downregulation of EOS impaired and reduced Foxp3-mediated gene repression, thereby leading to Treg dysfunction and autoimmunity. EOS-deficient (EOS−/−) mice developed more severe experimental autoimmune encephalomyelitis, which is the most commonly used experimental model for the human inflammatory demyelinating disease called multiple sclerosis, which is pathologically similar to HAM/TSP and presents with an increased abundance of effector T cells in the periphery and central nervous system [
56], Furthermore, EOS reduces Foxp3 acetylation and enhances K48-linked polyubiquitylation [
57], EOS interacts with Foxp3 and mediates its gene silencing activity [
20], and selective deletion of EOS in Tregs leads to loss of suppressive function and development of systemic autoimmunity [
58]. Although further studies are required to determine the in vivo significance, these findings suggest the possibility that HTLV-1 contribute to development of inflammatory conditions observed in HAM/TSP, by altering Treg status via modulating both Foxp3 levels and biological activities of EOS.
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