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Volume 14, Issue 1 (2022)
3 2022, 14(1): 75-81 |
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Received: 2022/03/7 | Accepted: 2022/03/26 | Published: 2022/05/1
Received: 2022/03/7 | Accepted: 2022/03/26 | Published: 2022/05/1
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Mustafa R, Jasim H, Al-Salait S. Impact of Gene Expression of TLR4, TLR7, and TLR9 in Children with Acute Lymphocytic Leukemia in Basrah. 3 2022; 14 (1) :75-81
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1- Department of Microbiology, College of Medicine, University of Basrah, Basrah, Iraq
2- Department of Pathology, Al-Zahraa Medical College, University of Basrah, Basrah, Iraq
2- Department of Pathology, Al-Zahraa Medical College, University of Basrah, Basrah, Iraq
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Introduction
Leukemia is defined as a hematopoietic cell proliferation that invades the bone marrow and spreads to the circulation and other organs [1]. Lymphocytic and myeloid cells are produced by hematopoietic stem cells. B-cells, T-cells, and natural killer cells are lymphoid cells, while erythrocytes, megakaryocytes, neutrophils, eosinophils, basophils, macrophages, and monocytes are myeloid cells [2]. Hematopoietic lineages (Hb, platelets, and neutrophils) are normal, but they have an unfavorable effect on blood cell formation and immunity. As a result, people with acute leukemia are more sensitive to infectious complications [3].
Acute Lymphocytic Leukemia (ALL) is a malignancy of B or T lymphoblasts characterized by uncontrolled proliferation of abnormal, immature lymphocytes and their progenitors which ultimately leads to the replacement of bone marrow elements and other lymphoid organs resulting in a typical disease pattern characteristic of Acute Lymphocytic Leukemia [4]. Acute lymphoblastic leukemia (ALL) is the most prevalent malignancy in children, accounting for up to 25% of all malignancies in children under the age of 15 [5]. A higher risk of ALL is in childhood patients aged 1 to 10 years. It was observed that the majority of ALL pediatric ages range from 2 to 12 with percentages (38% and 36%) respectively [6-8]. Clinical outcome is related to the age of diagnosis, infants and adolescents have a worse prognosis [9]. The better prognosis noted in children aged 1 to 10 years is attributable to the presence of favorable cytogenetic characteristics, e.g. hyperdiploidy or translocation t (12;21) in leukemic blasts [10].
TLRs are integral membrane type I glycoproteins with 3 main domains: an extracellular domain with various numbers of leucine-rich repeat (LRR) motifs, a transmembrane domain, and a cytoplasmic domain (similar to that of the interleukin-1 receptor; IL-1R) known as the Toll/IL-1R (TIR) domain. TLRs have a direct role in the control of inflammatory reactions as well as the activation of innate or adaptive immune responses to eliminate pathogenic microorganisms and cancer debris [11]. TLRs which are found in bacteria, viruses, fungi, and parasites, are components of the immune system and recognize pathogen- and danger-associated molecular patterns [12]. Several of the TLR type (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11) are contained in the plasma membrane, while others are located in endosomes (TLR3, TLR7, TLR8, and TLR9). Two signaling pathways are dependent on MyD88 protein and signaling pathways that are not [13]. TLRs homo or heterodimerize in response to ligand binding, resulting in the recruitment of adaptor proteins-the most prevalent of which is MyD88, which is shared by all TLRs except TLR3 [14]. TLRs are associated with the transduction of molecular signals in immune processes such as induction and regulation of immunity, production of cytokines, and recognition of specific molecular patterns on the surface of microorganisms, but they are also involved in cancer development, as evidenced by previous studies.
Because there are few specific researches on TLR expression in Acute Lymphoblastic leukemia at different phases of progression, this study was tried to shed light on any possible association of gene expression of TLR4, TLR7, and TLR9 in pediatric patients with the pathogenesis of acute lymphocytic leukemia and compared to that in normal matched children.
Materials & Methods
A case-control study was conducted on pediatric patients with Acute Lymphocytic Leukemia who have been admitted to Al-Basra Children Teaching Specialty Hospital. Over a period from September 2020 through June 2021, 62 patients (42 newly diagnosed and 20 relapses) were enrolled, in addition to 60 matched normal control, aged 6 months to 16 years. Three ml of blood was collected from all participants in EDTA (Ethylene Diamine Tetra Acetic acid) tubes used for RNA extraction and then for molecular analysis. TLR4, TLR7, and TLR9 gene expression were done by Real Time q-PCR, qTOWER3 machine from Analytic-Jeana. The procedure included the RNA extraction step by using kits supplied by Wizbio solution (Korea), in addition, to the synthesis of the cDNA step by using the kits supplied by WizScript RT FDmix (Hexamer). Specific oligonucleotides sequence of primers for each TLR4, TLR7, and TLR9 gene and β-actin (housekeeping gene) were used (Table 1). Optimization and solving of the primers depended on the instruction supplied by the source. Then quantification was measured by using Real-Time qPCR. The conditions for amplification of TLRs genes were as in Table 2.
The data of the current study were analyzed by using Statistical Package of Social Science (SPSS) version 26. Analyzed the data of the current study by using Chi-square (X2) test to compare percentages. Numeric data were described by Mean±SD.
Table 1) Primers of TLRs genes used in the study
Table 2) Conditions of Real-Time-q PCR used for amplification of TLRs genes [16]
Findings
Gene expression of TLRs (TLR4, TLR7, and TLR9) of study groups was analyzed (Table 3) by using Real Time-qPCR, and the results were reported as ∆Ct (mean±SD), as shown in Table 3. The mean ∆Ct of TLRs reflects the high expression of a gene with the TLR7 being the most highly expressed gene (p<0.001). In general, the mean ∆Ct of TLRs were stated to be high in ALL patients than in the control group.
TLRs gene expression in newly diagnosed ALL patients compared to relapsed ALL patients was measured (Table 4). The mean ∆Ct±SD of TLR7 and TLR9 are high in both groups with no significant differences (P-value 0.686 and 0.400) respectively, while the mean ∆Ct±SD of TLR4 is higher in a newly diagnosed group (2.90524±1.02402) than relapsed one (6.49050±1.91184) with significant differences <0.001.
In real-time polymerase chain reaction for Toll-like receptors gene expression (Figure 1) shows the starting time of amplification as it was after 20 cycles/min. Also (Figure 1-E) shows the melting curve analysis of TLRs and the temperature of cycles (50C° for 1 minute). The intercalating dye was SYBR green.
Table 3) The Mean ∆CT*±SD of TLRs gene expression in ALL pediatric patients and control group measured by real time-qPCR (N=122)
Table 4) The Mean ∆CT*±SD of TLRs gene expression in both new cases and relapse ALL patients (N=62)
Leukemia is defined as a hematopoietic cell proliferation that invades the bone marrow and spreads to the circulation and other organs [1]. Lymphocytic and myeloid cells are produced by hematopoietic stem cells. B-cells, T-cells, and natural killer cells are lymphoid cells, while erythrocytes, megakaryocytes, neutrophils, eosinophils, basophils, macrophages, and monocytes are myeloid cells [2]. Hematopoietic lineages (Hb, platelets, and neutrophils) are normal, but they have an unfavorable effect on blood cell formation and immunity. As a result, people with acute leukemia are more sensitive to infectious complications [3].
Acute Lymphocytic Leukemia (ALL) is a malignancy of B or T lymphoblasts characterized by uncontrolled proliferation of abnormal, immature lymphocytes and their progenitors which ultimately leads to the replacement of bone marrow elements and other lymphoid organs resulting in a typical disease pattern characteristic of Acute Lymphocytic Leukemia [4]. Acute lymphoblastic leukemia (ALL) is the most prevalent malignancy in children, accounting for up to 25% of all malignancies in children under the age of 15 [5]. A higher risk of ALL is in childhood patients aged 1 to 10 years. It was observed that the majority of ALL pediatric ages range from 2 to 12 with percentages (38% and 36%) respectively [6-8]. Clinical outcome is related to the age of diagnosis, infants and adolescents have a worse prognosis [9]. The better prognosis noted in children aged 1 to 10 years is attributable to the presence of favorable cytogenetic characteristics, e.g. hyperdiploidy or translocation t (12;21) in leukemic blasts [10].
TLRs are integral membrane type I glycoproteins with 3 main domains: an extracellular domain with various numbers of leucine-rich repeat (LRR) motifs, a transmembrane domain, and a cytoplasmic domain (similar to that of the interleukin-1 receptor; IL-1R) known as the Toll/IL-1R (TIR) domain. TLRs have a direct role in the control of inflammatory reactions as well as the activation of innate or adaptive immune responses to eliminate pathogenic microorganisms and cancer debris [11]. TLRs which are found in bacteria, viruses, fungi, and parasites, are components of the immune system and recognize pathogen- and danger-associated molecular patterns [12]. Several of the TLR type (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11) are contained in the plasma membrane, while others are located in endosomes (TLR3, TLR7, TLR8, and TLR9). Two signaling pathways are dependent on MyD88 protein and signaling pathways that are not [13]. TLRs homo or heterodimerize in response to ligand binding, resulting in the recruitment of adaptor proteins-the most prevalent of which is MyD88, which is shared by all TLRs except TLR3 [14]. TLRs are associated with the transduction of molecular signals in immune processes such as induction and regulation of immunity, production of cytokines, and recognition of specific molecular patterns on the surface of microorganisms, but they are also involved in cancer development, as evidenced by previous studies.
Because there are few specific researches on TLR expression in Acute Lymphoblastic leukemia at different phases of progression, this study was tried to shed light on any possible association of gene expression of TLR4, TLR7, and TLR9 in pediatric patients with the pathogenesis of acute lymphocytic leukemia and compared to that in normal matched children.
Materials & Methods
A case-control study was conducted on pediatric patients with Acute Lymphocytic Leukemia who have been admitted to Al-Basra Children Teaching Specialty Hospital. Over a period from September 2020 through June 2021, 62 patients (42 newly diagnosed and 20 relapses) were enrolled, in addition to 60 matched normal control, aged 6 months to 16 years. Three ml of blood was collected from all participants in EDTA (Ethylene Diamine Tetra Acetic acid) tubes used for RNA extraction and then for molecular analysis. TLR4, TLR7, and TLR9 gene expression were done by Real Time q-PCR, qTOWER3 machine from Analytic-Jeana. The procedure included the RNA extraction step by using kits supplied by Wizbio solution (Korea), in addition, to the synthesis of the cDNA step by using the kits supplied by WizScript RT FDmix (Hexamer). Specific oligonucleotides sequence of primers for each TLR4, TLR7, and TLR9 gene and β-actin (housekeeping gene) were used (Table 1). Optimization and solving of the primers depended on the instruction supplied by the source. Then quantification was measured by using Real-Time qPCR. The conditions for amplification of TLRs genes were as in Table 2.
The data of the current study were analyzed by using Statistical Package of Social Science (SPSS) version 26. Analyzed the data of the current study by using Chi-square (X2) test to compare percentages. Numeric data were described by Mean±SD.
Table 1) Primers of TLRs genes used in the study
Table 2) Conditions of Real-Time-q PCR used for amplification of TLRs genes [16]
Findings
Gene expression of TLRs (TLR4, TLR7, and TLR9) of study groups was analyzed (Table 3) by using Real Time-qPCR, and the results were reported as ∆Ct (mean±SD), as shown in Table 3. The mean ∆Ct of TLRs reflects the high expression of a gene with the TLR7 being the most highly expressed gene (p<0.001). In general, the mean ∆Ct of TLRs were stated to be high in ALL patients than in the control group.
TLRs gene expression in newly diagnosed ALL patients compared to relapsed ALL patients was measured (Table 4). The mean ∆Ct±SD of TLR7 and TLR9 are high in both groups with no significant differences (P-value 0.686 and 0.400) respectively, while the mean ∆Ct±SD of TLR4 is higher in a newly diagnosed group (2.90524±1.02402) than relapsed one (6.49050±1.91184) with significant differences <0.001.
In real-time polymerase chain reaction for Toll-like receptors gene expression (Figure 1) shows the starting time of amplification as it was after 20 cycles/min. Also (Figure 1-E) shows the melting curve analysis of TLRs and the temperature of cycles (50C° for 1 minute). The intercalating dye was SYBR green.
Table 3) The Mean ∆CT*±SD of TLRs gene expression in ALL pediatric patients and control group measured by real time-qPCR (N=122)
Table 4) The Mean ∆CT*±SD of TLRs gene expression in both new cases and relapse ALL patients (N=62)
Figure 1) Bloating and melting curve of TLRs
Discussion
The most complex and life-threatening disease is cancer which affects different aspects of life [17]. It has been documented an increase in childhood leukemia (children below the age of 15 years) [18] in Iraq and specifically, Basrah, is highly populated, the area is surrounded by oil fields and subjected to being a battleground by three wars; including the Iraq-Iran war in 1980, The US invasion in 1991, and the last US invasion in 2003. These reasons might be the risks for increasing childhood leukemia [19]. Many studies were conducted to shed light on many factors that might be explained the increasing incidence, risks factors, and immune-pathogenesis of the diseases [18, 0-21]. Consequently, in this study, the authors try to investigate any possible association of gene expression of specific cell receptors with the pathogenesis of leukemia.
Cancer cells acquire immune cell characteristics, allowing them to control the immune response to benefit their development and survival [22]. The connection between B cell precursor (BCP) ALL and childhood infections was postulated decades ago, but the pathogenic processes are still controversial. Pathogens' immediate transformation of B cell precursors is improbable. Instead, immune cells that have been activated and enlarged in response to those infections may interact positively with B cell precursors, promoting leukemia. The cells are ideal candidates for such leukemia-supportive immune cells because of their involvement in the immune system and existence in the bone marrow. Multiple TLRs are expressed in both normal HSCs (Hematopoietic Stem Cells) and bone marrow niche stromal cells. Although TLR signaling may be required for the formation of a normal immune response, evidence indicates a link between increased TLR expression and signaling and hematological diseases and malignancies such as lymphoid neoplasms [23].
According to previous research TLRs are expressed by different hematological malignant cells, including ALL cells. TLR4 and TLR9 could orchestrate a signal that helps cancer cells to bypass the immune responses by increasing the expression of immunosuppressive cytokines and anti-apoptosis proteins [24]. TLR4 expression was shown to be deeply linked to tumor invasiveness [25].
There is controversy about TRL4 expression in acute leukemia, as some studies showed over-expression [26-27], while the results of Sánchez-Cuaxospa et al. [23] and Pehlivan et al. [28] demonstrated lower expression levels compared to normal control. In this study, TLR4 expression is higher in ALL patients (newly diagnosed and relapsed) compared to the control group (4.0618±2.16754, 11.3295±1.45013) respectively with p<0.001.
TLR4 is linked to IL10 production as an anti-TLR4 antibody can cause blockade in IL10 production. It is well-known as an immunosuppressive cytokine with a possible role in the pathogenesis and progression as higher levels in cancer patients are associated with a worse prognosis [29]. Increased IL10 production can lead to inhibition of CD8+ T cell-dependent antitumor immunity in the tumor microenvironment [30]. In solid tumors, TLR4 over-expression is associated with carcinogenesis and early metastasis according to Morais et al. [31]. Cytoplasmic TLR4 was shown to be strongly associated with greater tumor grade, found that TLR4 expression is greater in initial tumors than in local recurrent cancers.
The present study demonstrated the mean ∆Ct±SD level of TLR-7 expression in ALL patients (-5.2200±3.05991) and in normal control (10.0243±1.38014), with a significant difference (p<0.001). Also, the mean ∆ct level of the newly diagnosed patient compared with relapsed ones (-5.11048, -5.45000) respectively, with no significant difference (p=0.686).
As shown previously the expression of TLR7 was high mean ∆Ct value in relapsed patients than in newly diagnosed ones, this is differently supporting the role of worse disease prognosis and progress. TLR-7 appears to play a dual or contentious function in cancer formation, according to a large study [32]. The present study confirmed the finding of most authors who demonstrated high expression of TLR7 in a patient with ALL in comparison with normal control. It has been also indicated that the co-stimulation of TLR7 could induce chemo-resistance in human lung cancer cells [33].
Acute Myeloid Leukemia (AML) researchers investigated the quantitative expression of TLRs in patients with newly diagnosed or recurrent AML and determined that TLR-7 had the greatest level of TLR expression in patients than the control [34-35]. This finding supported that TLR-7 may play a role in the immune escape from acute leukemias (AML and ALL). Han et al. [36] found that Patients who exhibited very high TLR7 expression in tumor cells of oral squamous cell carcinoma had poor differentiation and prognosis.
These findings suggest that TLR-7 targeting may inhibit the growth and induce apoptosis of leukemic cells, providing new insights into the biology and therapy of acute leukemias. Other studies done by Basith et al., [37] and Rybka et al. [38] found that the level of TLR7/β-actin mRNA expression was elevated in AML patients compared to normal cases with statistically significant (p<0.05).
Helminen et al. [32] discovered that TLR7 expression is increased in both epithelial and stromal compartments of human pancreatic cancer, leading them to hypothesize that TLR7 signaling is essential for carcinogenesis. Only one result is inconsistent with our finding and belongs to Sánchez-Cuaxospa et al. [23] that showed a low level of TLR7 in a patient with a newly diagnosed patient with ALL compared with the normal control group. TLR expression may differ depending on the stimulus, environment, cell type, and subset, and probably age group. The TLR expression profile appears to be also influenced by the location of the cells [23].
TLR9 has heterogeneity in signaling on B cells and its expression in B-cell malignancies may influence the degree of TLR9-mediated B-cell activation [23]. In addition, the research found that neonatal naive B cells express more TLR9 than adult naive B cells [39].
The Present study demonstrates the high level of TLR9 in newly diagnosed patients with ALL. The mean ∆Ct was found to be in newly diagnosed cases (4.3168±1.17849) compared with the mean ∆Ct (10.9862±1.43895) in the control group. So, there was a statistically significant difference in TLR-9 mRNA expression levels between these two groups with p<0.001. The mean ∆Ct is slightly higher in new cases than the relapsed cases (4.22905±1.00882 and 4.5010±1.48662) respectively. These findings were consistent with prior research by Ignatz-Hoover et al. [40] who showed high expression of TLR9 in leukemic newly diagnosed patients.
Alharbi et al. [41] showed that increased TLR9 expression inside cervical cancer cells may be directly related to tumor invasion and carcinogenesis by inhibiting TLR9 expressing immune cells from detecting tumor/viral antigen, therefore manipulating the immune response to malignancies. Furthermore, Smith et al. [42] and indicated that TLR9 has a role in tumor resumption mediated by MyD88/NF-B activation, which promotes IL-6 production. TLR9 also promotes Jak/STAT3 signaling, which controls tumor-promoting inflammation and angiogenesis [43]. Although TLR9 gene expression is identical in Memory B cell-related marginal zone lymphomas and diffuse large B cell lymphomas (DLBCLs), they have varied responses to TLR9 activation [44-45].
According to Sánchez-Cuaxospa et al. [23], TLR9 expression in PBMCs from pediatric patients with ALL was low when compared to those from healthy children. TLRs are expressed at varying levels in people with neoplasia. The patients' ages likely contribute to these disparities because age is a significant factor in the relationship between innate and acquired abilities adaptive immunological responses in healthy and diseased people. There is a lot of variation amongst different types of people. Reports on the research of patients and cell lines; in certain situations, it may be linked to disease activity. Similar but not identical findings from Morsi et al., [35] demonstrated no statistically significant difference between the mean level of TLR-9 mRNA expression in AML patients and that in normal cases. In another study, patients with AML expressed TLR9 in monocyte-derived DCs, and has been found that TLR9 was expressed at a lower level in the patient group [26]. The results of the current study suggest that at the presentation of ALL patients, leukemic cells exposure the TLRs expression to escape immune surveillance by providing wrong signaling in the wrong direction. The more the ability to display the receptors, the worse the clinical outcome [36].
We were unable to collect a larger number of samples due to the limited time for the study. Intracellular and surface membrane expression of these TLRs has not been attempted due to limited access to required material and equipment.
Conclusion
TLR4, TLR7, and TLR9 gene expression are higher in ALL patients, whether newly diagnosed or relapsed than in the control group. TLRs expression might be part of the immune-evasion mechanism developed by the malignant cells that play an important role in leukemogenicity and disease progression.
Acknowledgments: The authors would like to thank staff members of Basrah Children's Hospital and the Children’s parents for their kind help.
Ethical Permissions: The approval of the research's Ethical Committee of the College of Medicine/University of Basrah and Basra Health Authority was obtained before conducting this study at Basrah Children Specialty Hospital. Written consents were taken from the parents, or at least one of them, for patients’ participation in the study to enroll their children in this study.
Conflicts of Interests: The authors declare no conflicts of interests.
Authors’ Contributions: Mustafa R.A. (First Author), Introduction Writer/Main Researcher (40%); Jasim H.A. (Second Author), Methodologist/Statistical Analyst (40%); Ali Al-Salait S.Kh. (Third Author), Discussion Writer/Methodologist (20%).
Funding/Sources: None declared.
The most complex and life-threatening disease is cancer which affects different aspects of life [17]. It has been documented an increase in childhood leukemia (children below the age of 15 years) [18] in Iraq and specifically, Basrah, is highly populated, the area is surrounded by oil fields and subjected to being a battleground by three wars; including the Iraq-Iran war in 1980, The US invasion in 1991, and the last US invasion in 2003. These reasons might be the risks for increasing childhood leukemia [19]. Many studies were conducted to shed light on many factors that might be explained the increasing incidence, risks factors, and immune-pathogenesis of the diseases [18, 0-21]. Consequently, in this study, the authors try to investigate any possible association of gene expression of specific cell receptors with the pathogenesis of leukemia.
Cancer cells acquire immune cell characteristics, allowing them to control the immune response to benefit their development and survival [22]. The connection between B cell precursor (BCP) ALL and childhood infections was postulated decades ago, but the pathogenic processes are still controversial. Pathogens' immediate transformation of B cell precursors is improbable. Instead, immune cells that have been activated and enlarged in response to those infections may interact positively with B cell precursors, promoting leukemia. The cells are ideal candidates for such leukemia-supportive immune cells because of their involvement in the immune system and existence in the bone marrow. Multiple TLRs are expressed in both normal HSCs (Hematopoietic Stem Cells) and bone marrow niche stromal cells. Although TLR signaling may be required for the formation of a normal immune response, evidence indicates a link between increased TLR expression and signaling and hematological diseases and malignancies such as lymphoid neoplasms [23].
According to previous research TLRs are expressed by different hematological malignant cells, including ALL cells. TLR4 and TLR9 could orchestrate a signal that helps cancer cells to bypass the immune responses by increasing the expression of immunosuppressive cytokines and anti-apoptosis proteins [24]. TLR4 expression was shown to be deeply linked to tumor invasiveness [25].
There is controversy about TRL4 expression in acute leukemia, as some studies showed over-expression [26-27], while the results of Sánchez-Cuaxospa et al. [23] and Pehlivan et al. [28] demonstrated lower expression levels compared to normal control. In this study, TLR4 expression is higher in ALL patients (newly diagnosed and relapsed) compared to the control group (4.0618±2.16754, 11.3295±1.45013) respectively with p<0.001.
TLR4 is linked to IL10 production as an anti-TLR4 antibody can cause blockade in IL10 production. It is well-known as an immunosuppressive cytokine with a possible role in the pathogenesis and progression as higher levels in cancer patients are associated with a worse prognosis [29]. Increased IL10 production can lead to inhibition of CD8+ T cell-dependent antitumor immunity in the tumor microenvironment [30]. In solid tumors, TLR4 over-expression is associated with carcinogenesis and early metastasis according to Morais et al. [31]. Cytoplasmic TLR4 was shown to be strongly associated with greater tumor grade, found that TLR4 expression is greater in initial tumors than in local recurrent cancers.
The present study demonstrated the mean ∆Ct±SD level of TLR-7 expression in ALL patients (-5.2200±3.05991) and in normal control (10.0243±1.38014), with a significant difference (p<0.001). Also, the mean ∆ct level of the newly diagnosed patient compared with relapsed ones (-5.11048, -5.45000) respectively, with no significant difference (p=0.686).
As shown previously the expression of TLR7 was high mean ∆Ct value in relapsed patients than in newly diagnosed ones, this is differently supporting the role of worse disease prognosis and progress. TLR-7 appears to play a dual or contentious function in cancer formation, according to a large study [32]. The present study confirmed the finding of most authors who demonstrated high expression of TLR7 in a patient with ALL in comparison with normal control. It has been also indicated that the co-stimulation of TLR7 could induce chemo-resistance in human lung cancer cells [33].
Acute Myeloid Leukemia (AML) researchers investigated the quantitative expression of TLRs in patients with newly diagnosed or recurrent AML and determined that TLR-7 had the greatest level of TLR expression in patients than the control [34-35]. This finding supported that TLR-7 may play a role in the immune escape from acute leukemias (AML and ALL). Han et al. [36] found that Patients who exhibited very high TLR7 expression in tumor cells of oral squamous cell carcinoma had poor differentiation and prognosis.
These findings suggest that TLR-7 targeting may inhibit the growth and induce apoptosis of leukemic cells, providing new insights into the biology and therapy of acute leukemias. Other studies done by Basith et al., [37] and Rybka et al. [38] found that the level of TLR7/β-actin mRNA expression was elevated in AML patients compared to normal cases with statistically significant (p<0.05).
Helminen et al. [32] discovered that TLR7 expression is increased in both epithelial and stromal compartments of human pancreatic cancer, leading them to hypothesize that TLR7 signaling is essential for carcinogenesis. Only one result is inconsistent with our finding and belongs to Sánchez-Cuaxospa et al. [23] that showed a low level of TLR7 in a patient with a newly diagnosed patient with ALL compared with the normal control group. TLR expression may differ depending on the stimulus, environment, cell type, and subset, and probably age group. The TLR expression profile appears to be also influenced by the location of the cells [23].
TLR9 has heterogeneity in signaling on B cells and its expression in B-cell malignancies may influence the degree of TLR9-mediated B-cell activation [23]. In addition, the research found that neonatal naive B cells express more TLR9 than adult naive B cells [39].
The Present study demonstrates the high level of TLR9 in newly diagnosed patients with ALL. The mean ∆Ct was found to be in newly diagnosed cases (4.3168±1.17849) compared with the mean ∆Ct (10.9862±1.43895) in the control group. So, there was a statistically significant difference in TLR-9 mRNA expression levels between these two groups with p<0.001. The mean ∆Ct is slightly higher in new cases than the relapsed cases (4.22905±1.00882 and 4.5010±1.48662) respectively. These findings were consistent with prior research by Ignatz-Hoover et al. [40] who showed high expression of TLR9 in leukemic newly diagnosed patients.
Alharbi et al. [41] showed that increased TLR9 expression inside cervical cancer cells may be directly related to tumor invasion and carcinogenesis by inhibiting TLR9 expressing immune cells from detecting tumor/viral antigen, therefore manipulating the immune response to malignancies. Furthermore, Smith et al. [42] and indicated that TLR9 has a role in tumor resumption mediated by MyD88/NF-B activation, which promotes IL-6 production. TLR9 also promotes Jak/STAT3 signaling, which controls tumor-promoting inflammation and angiogenesis [43]. Although TLR9 gene expression is identical in Memory B cell-related marginal zone lymphomas and diffuse large B cell lymphomas (DLBCLs), they have varied responses to TLR9 activation [44-45].
According to Sánchez-Cuaxospa et al. [23], TLR9 expression in PBMCs from pediatric patients with ALL was low when compared to those from healthy children. TLRs are expressed at varying levels in people with neoplasia. The patients' ages likely contribute to these disparities because age is a significant factor in the relationship between innate and acquired abilities adaptive immunological responses in healthy and diseased people. There is a lot of variation amongst different types of people. Reports on the research of patients and cell lines; in certain situations, it may be linked to disease activity. Similar but not identical findings from Morsi et al., [35] demonstrated no statistically significant difference between the mean level of TLR-9 mRNA expression in AML patients and that in normal cases. In another study, patients with AML expressed TLR9 in monocyte-derived DCs, and has been found that TLR9 was expressed at a lower level in the patient group [26]. The results of the current study suggest that at the presentation of ALL patients, leukemic cells exposure the TLRs expression to escape immune surveillance by providing wrong signaling in the wrong direction. The more the ability to display the receptors, the worse the clinical outcome [36].
We were unable to collect a larger number of samples due to the limited time for the study. Intracellular and surface membrane expression of these TLRs has not been attempted due to limited access to required material and equipment.
Conclusion
TLR4, TLR7, and TLR9 gene expression are higher in ALL patients, whether newly diagnosed or relapsed than in the control group. TLRs expression might be part of the immune-evasion mechanism developed by the malignant cells that play an important role in leukemogenicity and disease progression.
Acknowledgments: The authors would like to thank staff members of Basrah Children's Hospital and the Children’s parents for their kind help.
Ethical Permissions: The approval of the research's Ethical Committee of the College of Medicine/University of Basrah and Basra Health Authority was obtained before conducting this study at Basrah Children Specialty Hospital. Written consents were taken from the parents, or at least one of them, for patients’ participation in the study to enroll their children in this study.
Conflicts of Interests: The authors declare no conflicts of interests.
Authors’ Contributions: Mustafa R.A. (First Author), Introduction Writer/Main Researcher (40%); Jasim H.A. (Second Author), Methodologist/Statistical Analyst (40%); Ali Al-Salait S.Kh. (Third Author), Discussion Writer/Methodologist (20%).
Funding/Sources: None declared.