Which One of the Human Immune Genes are more Involved in Against HTLV-1 Infection?

Background: Human T-lymphotropic virus type 1 (HTLV-1) is a main member of type C retrovirus. This virus was rst isolated from cutaneous T-cell lymphoma cases. Among all infected individuals, only 5% of cases progress to acute form, and 4% of them to chronic form. Nevertheless, about 90% of patients remain asymptomatic. Adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are accounted as acute and chronic forms of disease respectively. Methods: The gene expression prole of CD4+ T cells in four groups, healthy donors, asymptomatic HTLV-1 carriers (ACs), HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis), and ATLL (Adult T-cell leukemia/lymphoma) based on GPL9686 platform was evaluated. Results: According to Gene enrichment analysis, it was determined that hub genes in present study are able effect on various pathways such as apoptosis, proliferation and T cell activation, Ras signaling pathway, integrin signaling pathway, P53 signaling pathway, CCKR, TLR, FGF, DNA Damage, MAPK signaling, integrated cancer, Caspase, NF-κB, BCL-2 family, survival complex, breast cancer, Pancreatic cancer. Conclusions: Overall, based on scientic results in the present study, it seems the immune system via stimulation of biological processes such as cell survival, proliferation, CTLs exhaustion, and apoptosis concomitant, caused immortalization of HTLV-1 infected CD4+ T cells.


Background
Like some oncogenic bacteria such as Helicobacter pylori (H. pylori), Human T-cell leukemia virus-I (HTLV-1) is among the oncogenic viruses, and in other words, is the most cariogenic virus (1). Despite the lack of attention, HTLV-1 is known as one of the most oncogenic viruses for human (2). Adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are accounted as two famous forms of infections which are caused by this virus (3). The virus identi ed by Galo group in 1979, but despite about 4 decades of research on the integrity of this virus, but still there is no a reliable therapeutic method for treatment of its infections (4). In addition, HTLV-1 virus is involved in the some autoimmune diseases (immune system attacks parts of own body by mistake) including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjogren's syndrome (SS) (5). In the patients involved with HAM/TSP disease, it seen a high production of pro-in ammatory cytokines and chemokines such as CXCL9, CXCL10, IFN-γ, and TNF-α (6). Nevertheless, the patients involved with ATLL, have high levels of immunosuppressive cytokines such as IL-10 and TGF-β (7). In the present study, we identi ed immune genes which are more affected than other genes following HTLV-1 infection. These gene include IL-10, IL-6, JAK, IL-15, SOCS1, TNFR6, FOXP3, PD-1, FADD, Ras, BCL2, Rb, RICTOR, RAC1, MAP2K4, BCL2A1, MAP1B, CXCR4, BRCA1, MSH2, ATM, and RIG1.

Methods
First, based on collected results from an international public database, The Gene Expression Omnibus Database. The database evaluated human immune-microarray with Accession number: GSE19080. Also, the gene expression pro le of CD4 + T cells in four groups, healthy donors, asymptomatic HTLV-1 carriers (ACs), HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis), and ATLL (Adult T-cell leukemia/lymphoma) based on GPL9686 platform was evaluated. From 38 samples, 8 samples were healthy, and other samples were ACs (11 samples), HAM/TSP (12 samples), and ATLL (7 samples). Evaluated cell lines and providing patient details was better in this study compared to other microarray studies. Each of data was received with CEL format, and their quality was con rmed by the R software MetaQC package. All data were classi ed in three category including ATLL, ACs, and healthy individuals. In the next step, data was calculated by affy package, based on Log2, differentially expressed genes (DEGs), and fold change (FC) of datasets (FDR-adjusted p value < 0.05) (8). After that, by searching on Google Scholar, Scopus, and PubMed databases, related articles were studied, and gene expression variations of immune system during HTLV-1 infection were investigated. According to review of the literatures, it was determined that, 65 immune genes had variations, signi cantly; therefore, these genes were evaluated. Then, DGE and Log FC of considered genes was calculated. According to pro le changes of considered genes, heatmap was constructed for healthy individuals, ACs, and ATLL groups. Following this step, a protein-protein interaction network (PPIN) including studied genes was provide through online server STRINGER. Finally, all 65 genes were evaluated in terms of Gene Enrichment and Gene ontology. Given that information, a signaling network was recommended based on the role of immune system changes in progression of HTLV-1 infection toward to ATLL.

Results
Studied genes in our research are listed in the Box 1, and other genes were deleted from study, due to lack of any signi cance in statistical test or lack of enough information.

Box 1
The names of 65 interfering immune genes during HTLV-1  infection   IL-6  IL-4  IL-2  TGF-β  IL-10   STAT5  STAT3  JAK  CD4  IL-13   SOCS1  NFKB1  IRF4  IL-12  IL-15   RORϒt  FOXP3  Smad4  TNFR6  IFN-γ   Bak  Bad  Caspase 8  Caspase 3 PD-1 Regarding of assessments, it was cleared that in ATLL individuals compared with healthy ones, the expression of some of genes were upregulated, while other genes were dysregulated (Table 1). Expression gene pro le was almost exclusive in each of the three modes, healthy donors, ACs, and ATLL patients. Notably, between these gene, expression changes in some genes was clearer than others. These  As well as, the genes that had been dysregulated in their expression in healthy group were compared to ATLL and ACs groups are listed in Table 3. Table 3 Dysregulation of gene expression in healthy group compared to ACs and ATLL groups

Status
Gene manes The PPI network was constructed for prediction of role and relevance of immune system in progression toward to ATLL. This network included 71 nodes, 729 edges, clustering coe cient 0.66, and p value < 1.0e -16 ( Figure 2). The distance between each node was representative for interaction between that node with its adjacent nodes, and different colors are representative for this interaction based on different databases.
According to received data from STRING database, it was found that the PPI network effects on various biological processes such as cell communication, cytokine signaling, signal transduction, cell death, protein kinase, and microtubule rearrangement. In this network, some elements such as signal transduction factors, MAPK, signaling death, PI3K, mTOR, and NF-κB in center of network surrounded by interleukins. On the other, based on scienti c assessments it was clear that these processes occur at the nucleoplasm, cytosol, and cell membrane levels. In addition, 34 of these genes had an impact in pathogens cancer, and 14 of them in hostvirus interaction.
According to Gene enrichment analysis, it was determined that hub genes in present study are able effect on various pathways such as apoptosis, proliferation and T cell activation, Ras signaling pathway, integrin Based on performed assessments, it was known that the main transcription factors which activated under the effect of these genes, are including E2F1, NFKB1, TP53, IRF1, RELA, GLI1, STAT1/3, PML, CEBPB, SMAD3, and JUN (Figure 4a and b). Also, by searching in miRTar Base database, it was found that some microRNA molecules such as miR-574-3p, miR-34a-5p, miR-146a-5p, miR-21-5p, and mir-223-3p can to be caused dysregulation in network ( Figure 4c).
Finally, in order to determination of effects of immune gene changes on ATLL infection, a signaling network was drew based on received Gene ontology information from KEGG, Wiki pathway, and Panther databases.
This pathway drew with regards to hub genes and proteins that have the most interaction with them. The basis of this network was on signaling pathways such as NF-κB, PI3K-Akt, mTOR, MAPK, TP53, Ras, apoptosis, DNA damage, and cell cycle. Considering the results of this study, the components of immune system by affecting processes such as in ammation, apoptosis, DNA damage, cell proliferation, T cell activation, invasion, angiogenesis, cell survival, and cell migration cause the progression of HTLV-1 towards ATLL cancer ( Figure 5).
The results of Gene enrichment showed that some of microRNAs play pivotal role in regulation of our studied genes (hub genes). Between these elements, hsa-miR-574 is considered as an appropriate candidate for prediction of ATLL cancer (9)(10)(11). Also, based on Cui et al. studies, hsa-miR-574 can be effective on treatment of colorectal cancer (10). Based on Garcia et al. studies, it was found that hsa-miR-146a can via concomitant of BRCA1 expression has both preventive and therapeutic roles in breast and ovarian cancers (11). Another element is hsa-miR-21 can stimulate proliferation of tumor mass, and concomitant of activation of this molecule leads to tumor metastases (12). Using by Kaplan-Meier Plotter database, we assessed the important of microRNAs in prognosis of cancer, and according of collected results, the changes of these molecules can be accounted as suitable candidates for prognosis of tumors ( Figure 6).

Discussion
CD4 + T cells play a critical role in HTLV-1 infection and HTLV-1 preferentially infects these cells (13). Despite four decades of researches on HTLV-1, many ambiguity remain about the pathogenicity mechanism and key proteins involved in diverse pathological pathways. In this study, we investigated the gene expression pro le cells (14). It has been shown that the proliferation of HTLV-1 CD4 + T cells is due to JAK/STAT signaling pathway in response to some interleukins, speci cally IL-2, IL-6, and IL-15 (15)(16)(17). The Janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway plays critical roles in orchestrating of immune system, especially cytokine receptors (18). The activation of this pathway leads to lymphocyte proliferation. Interleukin-6 (IL-6) is a multifunctional cytokine with a pleiotropic effect on in ammation, immune response, and hematopoiesis (19). al. (27) showed that the level of IL-15R elevated 5-10-fold in the HTLV-I-infected cell samples. Also, in the present study, it was concluded that the expression level of IL-10, TGF-β, and FOXP3 in ATLL cases had increased compared to other groups, ACs and healthy donors. It seems that, increase in expression of IL-10,

TGF-β, FOXP3 and PD-1 caused to induction of production of T regulatory cells, immune-suppression and
CTLs exhaustion. ATLL is associated with high levels of IL-10 and TGF-β. These immunosuppressive cytokines could promote a protumoral micro-environment. In one study demonstrated that the levels of IL-10 increased in sera from ATL patients compared with sera from healthy subject. Moreover, IL-10 is constitutively produced by ATL cells and HTLV-I-infected cell lines (28). In recent study it has been shown that the antiin ammatory cytokine IL-10 and its downstream signals through the STAT3 and IRF4 pathways, potentially act as a switch for proliferation in HTLV-1-infected cells (29). It is suggested that an importance function of IL-10 in ATL may be inhibitory activities on macrophages and Th1and also as a means of escaping host defenses. It has been implicated that the negative regulatory programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway has an important role in the induction of CTL exhaustion during chronic viral infection and tumor (30)(31)(32). According to previous studies, suppression of immune responses and CTLs exhaustion lead to progression of disease to ATLL (33). In one study, Kozako et  and ATM leads to DNA replication errors and cancer growth (35,36). Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene that encodes a nuclear phosphoprotein, which is involved in repair damaged DNA and maintain genome stability. It has been shown that the expression of BRCA1 gene in ATLL patients is signi cantly reduced compared to normal individuals due to the effects of TAX protein and it is rationally in favor of increasing the tumor state (37). ATM (ataxia telangiectasia) gene like BRCA1 recognizes DNA damage or broken DNA strands. During DNA damages several phosphoproteins of the DDR pathway (H2AX, ATM, CHK1-2, P53, BRCA1) activated that lead to arrest the cell cycle transiently or lead to apoptosis and senescence but it has been demonstrated that the Tax inhibits the DDR machinery by sequestrating key signaling pathway components (38). So, in infected people, the expression level of the ATM gene is higher than normal individuals, which is as follows: ATLL > asymptomatic carriers > normal peoples (39). DNA mismatch repair protein Msh2 also known MSH2 is a tumor suppressor gene which involved in many different forms of DNA repair (40). In line with other studies (41), in the present study, it appears that the expression of this gene is decreased in AC and ATLL. Apoptosis play an critical role in destroying malignant cells (42). It has been demonstrated that in HTLV-1 infection, the virus through Tax, enhances expression of family inhibitor of apoptotic BCL2 and Bcl-xL (43,44) and suppress expression Bax gene which have role in inducing apoptosis (45). In this regard, our result showed that the expression level of these genes and antiapoptosis markers (i.e. BCL2, CytC, and BCL-XL) had been upregulated in ATLL. Moreover, in normal individuals compared to asymptomatic carriers with a LogFC = 0.66, indicating an increase in BCL activity in normal individuals and consequently suppression of apoptosis. Various studies showed that BCL2A1 overexpressed in hematological malignancies and solid tumors and may contribute to tumor progression (46). In addition, BCL2A1 by decrease in apoptosis and MDM2 by containment of TP53 caused stimulation of CDKs and protection of cell survival. Regarding of assessments in present study, it was cleared that the expression of BCL2A1 were upregulated in ATLL individuals compared with healthy ones. FADD (Fas associated via death domain) is a cytoplasmic protein that has an important role in apoptosis (47). It has been demonstrated that HTLV-1 transactivator protein Tax inhibits Fas-mediated apoptosis by induction of c-FLIP through activation of NF-κB, as a negative regulator of apoptosis by binding to the DED of FADD (48).
Our result indicated that the expression of the FADD is higher in normal people than ATL patient (log FC: 1.99) and therefore the level of apoptosis in ATL patient is decreased. Another protein that affected by Tax is Ras. Ras family proteins have a key role in control intracellular signaling networks. Moreover Ras regulated different processes such as actin cytoskeletal integrity, cell differentiation, cell proliferation, apoptosis, and cell migration (49). It has been reported that Ras family proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis (50). HTLV-1 infected cells increased RAS expression follow TAX expression, which increase sensitivity to apoptosis by using the antagonist Farnesyl thiosalicylic acid (51). In our study the expression of the RAS gene have been shown an increase in asymptomatic carrier as compared to normal individual and in normal individuals it was less expressed in lymphoma patients. Accordingly, during HTLV-1 infection the expression of TAX as follow as Ras were increased, leading to the proliferation and transformation of cells into malignancies and resistance to apoptosis (52). One of the hallmarks features of tumors is tissue invasion and chemokines and their receptors play a key role (53). Moreover, It is well known that chemokines and their receptors have a role in pathogenesis of HTLV-1 such as: in ammation in the central nervous system (CNS) in cases of HAM/TSP, T cell immortalization and tissue in ltration (54). Several chemokine receptors has been identi ed in ATLL including: CCR4 (55), CCR7 (56), CCR8 (54), and CXCR4 (57). One of chemokine receptor which is overexpressed in HTLV-1 infection is CXCR4 which interaction with stromal cell-derived factor 1 (SDF1)/CXCL12 play a paramount role in the migration of ATL cells (56). In this study our result indicated that the expression of CXCR4, FGF, EGF, RAC1, and MAPKs were increased in ATLL group and con rmed this hypothesis. Moreover, interestingly, it has been demonstrated that the absence of Rapamycin-insensitive companion of mTOR (RICTOR) increased the expression of CCR2, CCR4 and CXCR4, which associated with the homing and migration of T-cells (58). RICTOR is a key component of the PI3K/AKT/mTOR signal transduction pathway an essential component of the mTORC2 of mTOR complex (59). It is indicated that the RICTOR down-regulation suppresses cell proliferation and tumor formation (60,61). Log Fc in RICTOR gene in this study shown 1.61 fold change in normal individual compare to ATLL patient that mean dysfunction in RICTOR in ATLL, leading to an error in T cell proliferation and by affecting the expression of chemokine receptor genes in cells and it may affect the homing and migration of virus-infected cells (58,62). P53 and Rb are the main tumor suppressors, which lead to inhibition of proliferation (63,64). HTLV-1 oncogenes can induce malignancy through their effects on gene expression of cell cycle checkpoints such as P53 and Rb in the host cell (65). It has been demonstrated that HTLV-1 Tax protein hinders the function of p53 and Rb protein in host cells and lead to deregulate cellular division (66,67). In line with other studies, in this study, we found decrease in expression of these genes in ATLL group. So, HTLV-1 oncogenes by their effects on function and expression of p53 and Rb induce oncogenesis in host cells.

Conclusions
Overall, based on scienti c results in the present study, it seems the immune system via stimulation of biological processes such as cell survival, proliferation, CTLs exhaustion, and apoptosis concomitant, caused immortalization of HTLV-1 infected CD4 + T cells. As well as, this system via angiogenesis stimulation and cellular growth factors causes Tissue invasion phenomenon. In addition, this system through stimulation of angiogenesis and cellular growth factors in malignant tissues, and has an important role to progression of HTLV-1 to ATLL cancer. Also, immune system targeting by microRNA molecules as a new and effective approach, is accounted for development of new generation drugs against ATLL.

Abbreviations
Human T-lymphotropic virus type 1: HTLV-1 There is no any con ict of interest among the all authors.

-Funding
We have not received any funding for this research.  PPI network as a mathematical representations of interactions between immune genes in the cell. In this network, some elements such as signal transduction factors, MAPK, signaling death, PI3K, mTOR, and NF-κB in center of network surrounded by interleukins. These interactions occur at the nucleoplasm, cytosol, and cell membrane levels.

Figure 3
Gene enrichment analysis. It was determined that hub genes in our studies were effective on various pathways. These pathway is noted in the text.  The components of immune system effect on processes such as in ammation, apoptosis, DNA damage, cell proliferation, T cell activation, invasion, angiogenesis, cell survival, and cell migration. Eventually, this interaction lead to the progression of HTLV-1 towards ATLL cancer.

Figure 6
Gene enrichment results about the properties of miRNA molecules. The hsa-miR-574 molecule is predicted as an appropriate candidate for treatment of ATLL cancer. The has-miR-164a concomitant of BRCA1 expression,