HTLV-1-host interactions on the development of adult T cell leukemia/lymphoma: virus and host gene expressions

The human lymphotropic virus type 1 (HTLV-1) is a member of the Retroviridea family, which infects about 10–20 million people in the world. Although, after a long period of time, a small percentage (3–5%) of infected individuals will progress to HTLV-associated myelopathy-tropical spastic paraparesis (HAM/TSP) or Adult T-cell leukaemia/lymphoma (ATLL), the large number of infected carriers remains asymptomatic [1].

The HTLV-1 endemic areas include, Southwestern Japan, the Caribbean basin, Central Africa, the Melanesian Islands, South America and the Middle East [2]. In Iran, at least in four provinces, including; Razavi Khorasan, Northern Khorasan, Golestan and East Azarbaijan HTLV-1 is endemic [3]. Furthermore, HTLV-1- associated diseases have been reported from some other provinces such as Alborz, Tehran and Fars [4].

The HTLV-1 activity and infectivity in asymptomatic carriers and HTLV-1 associated diseases is quantified by the number of infected peripheral blood mononuclear cells (PBMCs), which expressed as the proviral load (PVL) [5]. The increasing amount of PVL results in more expressions of viral genes, such as Tax and HTLV-1 bZIP factor (HBZ). These regulatory proteins have a close interaction through mutual transcriptional regulation interference in the infected T cells. HBZ as regulatory protein through inhibition of Tax can help to evade host immune responses. Therefore, the over-expression of HBZ is implicated on the development of malignancy in the late stages of cell transformation in HTLV-1-infected cells [6]. Moreover, previous studies have reported a correlation between HBZ mRNA expression and HAM/TSP disease severity [7, 8].

The Tax and HBZ may exert their oncogenic and pathogenicity effects by changing the outcome of signalling pathways, such as NFκB and phosphoinositide 3-kinase (PI3K), and apoptotic event [8, 9]. AKT or protein kinase B (PKB) is a primary mediator of the PI3K signalling pathway. AKT and other kinases in the PI3K pathway have some substrates that contribute to cell transformation, results in malignancy. AKT can inhibit apoptosis through phosphorylation and the inhibition of pro-apoptotic mediators such as Bcl-2-associated death promoter (BAD) and caspase-9 to contribute to the maintenance of the virus latent state and may facilitate transformation of infected cells [9, 10].

Likewise, BAD and BAX are members of the BCL-2 family proteins and are the main molecules in regulating cell survival and apoptosis [11]. HTLV-1 proteins modulate the levels of BCL-2 family members in favour of virus dissemination [12], and AKT inhibits the BAD activity. BAD as a pro-apoptotic agent is a target of AKT in such circumstances, and helps infected cells to escape cell death [9].

Interferons (types I, II, and III) are the most important antiviral agents in innate and adaptive immune responses. Type III IFNs, includes 3 subtypes, IFN-λ1, λ2 and λ3, known as interleukin-29 (IL-29), IL-28A, and IL-28B, respectively [13]. IFN-λ is known to inhibit replication in a range of viruses, including, hepatitis C and B virus (HCV and HBV), influenza virus, rotavirus, herpes simplex virus-1 and 2, encephalomyocarditis virus, vesicular stomatitis virus, cytomegalovirus, and West Nile virus [14]. This class of IFNs also can induced apoptosis and has direct growth inhibitory action, therefore, has direct antitumor effects and can augment major histocompatibility complex (MHC) class I antigens expression which may stimulate Th1 responses which increases antigen presenting in favor of anti-viral and anti-tumor activities of cytotoxic T cell and anti-angiogenesis [13].

To lower the host immune pressure, HBZ overexpresses to suppress the expression of Tax as a virus activator and dominated antigen to escape the immune response. Therefore, the persistence and pathogenesis of HTLV-1 depends on the host immune response such as Th1, cytotoxic T lymphocytes (CTLs), natural killer cells (NK-cells) and Th17 [15]. Regulatory T cells (Tregs) that express the forkhead box P3 (FOXP3) transcription factor, play a critical role in the maintenance of the immune system homeostasis. An immunosuppressive micro-environment enables HTLV-1-infected cells to escape the host immune responses. Besides, the most HTLV-1-infected cells have been suggested to be the Treg lymphocytes. Many reports have been conducted on the role of Tregs in HTLV-1-associated disease progression [16]. Moreover, viral replication can be enhanced by proinflammatory responses of Th17. In case of HTLV-1 associated diseases the roles of Treg and Th17 cells differ depending on the infection stage and host immune status [17–19]. In the context of IFNs and transcription factors, it is better to elucidate the role of these central arms of immune responses through their main transcription factors FOXP3 and RAR-related orphan nuclear receptor (RORγt).

In the present study, the host-virus interactions in the manifestation of ATLL and HAM/TSP were investigated by assessment of HTLV-1-PVL and HBZ as viral elements, AKT1 as a regulator of the cell cycle progression and cell survival, BAD as proapoptetic agent and finally RORγt, FOXP3 genes as the main transcription factor in Th17 and Treg cells, and IFN-λ3 as an antiviral agent in the innate immune responses.

It is well known that only 3–5% of infected subjects develop ATLL or HAM/TSP [1], therefore, HTLV-1 infection alone may not be sufficient for developing its associated diseases. Furthermore, over many years after the discovery of HTLV-1, the pathogenicity of this virus in developing ATLL or HAM/TSP in a small proportion of infected individuals, yet to be understood. Therefore, viral–host interactions in context of gene expressions are assumed to be the main effective factors in HTLV-1 associated diseases development and progression. Thus, it is more likely that in addition to viral factors, the host genetic abnormalities and inappropriate epigenetic responses should be considered for understanding of the mechanism of the oncogenesis of ATLL or developing autoimmunity in HAM/TSP.

Although, the previous studies have suggested a possible important role for PVL in the development of ATLL and HAM/TSP, the association between PVL and disease development remains unclear. According to Iwanaga et al. study, the role of high PVL level remains uncertain because the majority of ACs with a high PVL level remained asymptomatic [20]. In the present study, the level of PVL is significantly higher in ATLL patients than in ACs, but the level of PVL is not statistically higher in HAM/TSP patients than in ACs. Of course, the increasing trend of PVL in ATLLs is due to the presence of a huge number of malignant infected cells in ATLL patients. Collectively, in our previous studies and many other authors significant differences in PVL were observed between HTLV-1 associated diseases and ACs [4, 21–23].

In addition to PVL, Tax is produced in the early phases of infection to establish viral replication and induce T cell transformation, proliferation and DNA damage. HBZ is subsequently expressed to inhibit Tax expression to escape the host immune pressure for virus elimination by Tax specific CTLs. Therefore, it produces as a second oncogenic signal for the maintenance of the leukemic cells. HBZ is expressed in nearly all malignant cells of the ATLL patients, and its expression is correlated positively with PVLs and negatively with tax/rex and gag/pol expression [24]. In the present study, a significant association was also observed between PVL and HBZ expression gene since it is possible with an increase of PVL, HBZ increment happens in infected cells.

Our findings showed that HBZ expression surprisingly showed an 800-fold higher increase in ATLL patients compared with ACs and HAM/TSP patients. Thus, these results are consistent with those of previous studies on the role of HBZ in the progression toward leukemogenesis, and support the critical role of HBZ in HTLV-1 oncogenesis. In the most cases, HBZ is the only HTLV-1 molecule that is expressed and may be involved in the maintenance of most ATLL cells which has an important role in leukemogenesis. Taken together, these studies have suggested that, although HBZ has growth-promoting activity in ATLL, it does not participate in transformation [25, 26].

Our findings show no significance difference in HBZ expression between HAM/TSP patients and ACs. This finding is consistent with previous studies that were shown more effective role of Tax than HBZ increment in progression toward HAM/TSP [4, 27].

Apart from these viral factors in the immortalisation of host-infected cells, survival and apoptotic signalling pathways are pivotal in promoting malignancy, such as PI3K pathway and BCL-2 family. The present study focuses on the AKT1 molecule from the PI3K signalling pathway and the pro-apoptotic factor from the intrinsic programmed cell death pathway as an antagonist for BAD. For instance, AKT inactivates BAD through phosphorylation and therefore inactivates its ability to induce apoptosis for promoting cell survival. Conversely, the dephosphorylation of BAD leads to the targeting of BAD to the mitochondrial membrane in which BAD interacts with anti-apoptotic proteins, such as BCL-2 and BCL-xL, to induce apoptosis. Nakahata et al. showed that the PI3K/AKT signalling pathway has a critical role in the induction of ATLL and other malignancies [28]. However, the exact mechanisms by which this signalling pathway is activated in the infected cells of HAM/TSP patients remain unclear [29]. In our study, AKT1 was evaluated as the primary mediator of the PI3K signalling pathway, and the findings showed that AKT1 expression in the ATLLs is 12–18 times more than the ACs and HAM/TSP patients. These findings showed that the PI3K/AKT1 pathway may have stronger effects on ATLL than on HAM/TSP. In the present study, a significant association was found between HBZ and AKT1 genes expression, thus HBZ increment can impact on AKT1 expression and its subsequent effects on maintenance of HTLV-1 infected cells and ATLL progression. Also, lower HBZ gene expression in HAM/TSP patients than ACs may affect low expression of AKT1 in HAM/TSP patients compare with ACs.

Signalling pathways such as NF-κB and PI3K/AKT1 can somehow regulate proteins from the BCL-2 family. Nicot et al. found that the anti-apoptotic BCL expression is high and that the level of pro-apoptotic proteins Bax, BAD and Bak are not considerably changed in HTLV-1 infected T cells [30]. In the present study, the expression indices for BAD were not significant between ATLL patients and ACs. Conversely, the AKT1 expression had a significant difference between these groups in Jeong et al. study. Therefore, AKT1 may inhibit the BAD pro-apoptotic molecule and contribute to cell survival [8]. Previous studies have shown that CTL-induced apoptosis of HTLV-1-infected T cells is a possible mechanism for the elimination of HTLV-1-infected cells [31, 32]. Therefore, according to our findings (i.e. a significant difference in BAD expression between HAM/TSPs and ACs) and previous studies, BAD which involves in cell death, preventing the BAD apoptotic pathway may exacerbate as well as induce the HTLV-1 associated diseases.

Although, ATLL cells display an activated helper/inducer (CD4⁺ and CD25⁺) T-cell phenotype, they are not regarded as having helper functions. Instead, ATLL cells demonstrate a strong immunosuppressive activity in vitro [33]. Kohno et al. cleared that FOXP3 and/or GITR mRNA is expressed in almost all of the primary ATLL samples [34], and Chen et al. reported the first evidence that ATLL cells have a Treg-like regulatory function [35]. In our study, the FOXP3 expression level in ATLL cells was significantly higher than that in ACs and HAM/TSP patients. Consistent with this finding Satou et al. showed in HBZ-transgenic mice CD4⁺ CD25⁺ Treg cells increased and also they showed HBZ expression directly induced FOXP3 gene transcription in T cells, however these cells functionally impaired while their proliferation was enhanced, thereby impairing the suppressive function of Treg cells [6].

In the present study, a significant correlation between HBZ and FOXP3 expression were observed, therefore HBZ increment could impact on expression and function of FOXP3. This finding may indicate that the suppressive activity of Treg is part of the immunodeficiency condition in ATLL patients.

Additionally, no such significant difference was found for FOXP3 expressions in HAM/TSP patients and ACs. Yamano et al. demonstrated that FOXP3 expression in HAM/TSP patients was lower than that in CD4⁺CD25⁺ T cells from healthy individuals [36]. Oh et al. also reported HAM/TSP patients have lower of FOXP3 expression in PBMCs than that in ACs and healthy individuals. Consequently, they suggested that impaired FOXP3 expression could contribute to the development of inflammatory disease during HTLV-1 infection [37]. The later study is consistent with our result that FOXP3 expression in inflammatory HAM/TSP reactions remains unchanged. This incapability of their suppressive ability to overcome such a situation results in the progression to inflammatory reactions such as HAM/TSP [38]. Since there was a positive association between HBZ and FOXP3 gene expression therefore, low HBZ expression could impact on low FOXP3 expression in HAM/TSP patients but not ATLL.

It has been demonstrated that the inappropriate production of IL-17 supresses the differentiation of Th1 cells, consequently inhibiting the production of IL-2 and IFN-γ. This suppression results in decrement of CTLs functions in favour of viral dissemination. However, the indirect evidence showed that Th17 sub-population may be necessary for preventing certain viral dissemination. Therefore, the role of Th17 in viral infection can differ depending on the virus, disease stage, and host immune background [18].

In the present study, RORγt was assessed as master regulator of Th17 which had high expression in ATLL patients than HAM/TSP and ACs groups. Moreover, high expression level of RORγt mRNA in protein level was confirmed by western blotting. According to what was earlier said about the role of Th17 in viral infections high level of RORγt in infected cells through inhibition of Th1 cells may cause HTLV-1 associated diseases. Zhao et al, reported HBZ enhances signalling of TGF-β [39], since TGF-β induce differentiation of Th17 [7, 40] therefore, HBZ increment may impact on RORγt high-level expression in ATLL patients. Additionally, RORγt expression has increased, consistent with inflammatory reactions in the skin and other ATLL patient’s organs.

The IFNλ3 was considered after observing the presence of specific polymorphisms near the IFNλ3 gene is associated with an increased response to treatment or spontaneous clearance of those that developed chronic hepatitis C [41, 42]. Moreover, Kotenko et al. showed viral elements induce IFNλ mRNA in various cell lines [43]. Also, we observed high IFNλ3 mRNA expression in ATLL and HAM/TSP patients than ACs, however, no significant difference was found between ATLL and HAM/TSP patients. Furthermore, consistent with Kotenko’s study in our study a significant association between viral factors (PVL, HBZ) and IFNλ3 gene expression was found. Besides, significant association between AKT1 expression and IFN λ3 was observed expression in both ATLL and HAM/TSP patients, thus this finding indicates high expression of AKT1 in HTLV-1 infected cell may induce high IFNλ3 mRNA expression in ATLL and HAM/TSP patients. In the present study, the elevated IFNλ3 level was observed in HTLV-1 but in infected individuals could not prevent to disease development.

This study has some limitations; the incidence of ATLL even in our endemic region is rare for large sampling. Furthermore, to obtain high and stable RNA extraction for gene expression using quantitative real-time PCR, the PBMCs were treated by TriPure. Therefore, the protein extraction from TriPure for western blot assay did not come to the proper results for all of studied protein, except AKT1 and RORγt.

Our last bioinformatics study using networking also showed that PI3K/AKT/mTOR, in cell cycle and anti-apoptotic pathways and BCRA-1 as DNA damage repair signalling pathways are main players in ATLL development and progression [44]. The present results, our previous study on LAT, BIM, c-FOS and RAD51 [45] and recent bioinformatics findings demonstrated that in contrast to HAM/TSP patients, AKT/mTOR and DNA repairing pathways such as BRCA1 and RAD51 are pivotal signalling pathways in the maintenance and progression of ATLL which should be targeted for therapy in ATLL malignancy. On the other side, HTLV-1 HBZ should be inhibited to guarantee the therapy and recurrence of the disease. However, more studies are needed to elucidate the additional mechanisms in the small proportion of HTLV-1 infected subjects that progresses to ATLL.