Original Article

Angiopoietin 4 Decreases TLR Expression on Monocytes in Multiple Sclerosis Patients

Introduction

Multiple sclerosis (MS) is an inflammatory pathological condition, the main function of which is to disrupt myelin sheaths on neurons in the brain and spinal cords. This trauma affects the ability of many cells responsible for communication in the nervous system, leading to the emergence of many physical symptoms.^1-2 Angiopoietin 4 is a peptide containing 43 amino acids found in all cells except red blood cells. This peptide plays a vital role in repairing damaged skin, eyes, heart, and nerve tissues. Many more biological activities can be done via angiopoietin 4 such as the inhibition of endothelial cell migration, the reduction in inflammatory responses, the inhibition of apoptosis, and oxidative damage. This peptide has an important part in angiogenesis inhabitation, and reacting with actin monomers can be a neurologic factor.^3,4

Toll-like receptors (TLRs) are innate immune system receptors that were detected in Drosophila fly for the first time. These receptors can be found in many immune cells such as natural killer cells, mast cells, B cells, regulating T cells, macrophages, dendritic cells, neutrophils, basophils, monocytes, and non-immunity cells such as epithelial and endothelial cells.⁴ TLRs play a major role in inflammation, and their influence on autoimmune diseases has been approved.^5-7 It was thought that TLRs are restricted to microglia, astrocytes, and oligodendrocytes, but it has been confirmed that these receptors are not only in red blood cells, epithelial tissues, endothelial cells, and the brain but also in neurons. The importance of TLR2, TLR4, and TLR9 in message transmission on cellular and membrane surfaces is the main reason to choose these receptors. The purpose of this study was to learn/figure out the inhibitory effect of angiopoietin 4 on the expression of TLR2, TLR4, and TLR9.^1,8 Angiopoietin 4 may have an inhibitory effect on the expression levels of the monocytes in MS patients. However, the theoretical aspects of the relationship between angiopoietin 4 and TLR in MS have not been explained.

Materials and Methods

Sample Collection

Samples were collected from MS patients who were referred to the Imam Reza hospital neurology clinic. Thirty MS patients with moderate to severe stages were selected, ranging from 17 to 40 years. The patients used fingolimod and interferon beta as medicine. Blood samples were taken from these patients and monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by the magnetic-activated cell sorting method. MS patients who have been treated for two years using fingolimod and interferon beta were included and patients who were pregnant, patients who discontinued taking the medicine in any way, patients who have been recently diagnosed, and vulnerable people were excluded from the study. The patients who have used fingolimod and interferon beta as a medicine and MS patients who have been treated for two years using fingolimod and interferon beta were included.

Cell Culture

To evaluate the function of angiopoietin 4, four monocytes in the blood were isolated and cultured by the PBMC method. In addition to the magnetic-activated cell sorting method, the purified cells were examined microscopically for the required cell count, survival, and CD14 expression, which indicates the purity of the monocytes. Cells were cultured in Dulbecco’s Modified Eagle Medium. These cells were treated with 10 µL of angiopoietin 4 for 2, 4, 12, 24, and 48 hours.

The Isolation of mRNA and Real-Time Polymerase Chain Reaction

Total RNA was isolated from the monocyte cells from PBMC. The polymerase chain reaction (PCR) was performed with a 1 µL, 1 µL of 5 mM dNTP, 2.5 µL of Taq polymerase, 1.2 µL of 50 mM MgCl2, and 1 µL of each primer. The PCR condition was as follows: initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 40 minutes, and a final elongation cycle at 72°C for 10 minutes (Corbett 6000 Real-Time PCR). Beta-actin was used as a control.

Statistical Analysis

Data were summarized as mean ± standard deviation. The statistical analysis of the results was performed by the one-way analysis of variance, and P values less than 0.05 were considered significant. Statistical analysis was conducted using GraphPad Prism 7 to compare the expression of genes.

Results

Decreased Expression of TLR2, TLR4, and TLR9 by the Stimulation of Angiopoietin 4

The monocyte cells were treated with angiopoietin 4 as a time course (2,4,12,24, and 48 hours), and TLRs’ expression was determined by real-time PCR. The results showed that the expression of TLR2 decreased significantly 12 hours after stimulating with angiopoietin 4, and the same results were observed for both TLR4 and TLR9 (P < 0.0001). The B-ACTIN gene was used as an internal control in this test.

The Effect of Angiopoietin 4 on the Expression of TLR2, TLR4, and TLR9 in Mononuclear Cells of MS Patients Using Fingolimod and Interferon

To investigate the effect of both proteins on the expression of TLR2 and TLR9, their efficiency on the receptor pathways of the innate immune system has been studied. The results indicated that the expression of TLR2 and TLR4 receptors decreased significantly with the time course of stimulation, which reached its lowest level in patients using fingolimod compared to beta interferon (Figure 1).

Close

Figure 1. The Expression of TLRs by treating the monocyte cells with angiopoietin 4 (A) TLR2 expression decreased significantly after 24 hours of treatment with angiopoietin 4; (B) TLR4 expression decreased significantly after 12 hours of treatment with angiopoietin 4; (C) TLR9 expression decreased significantly after 12 hours of treatment with angiopoietin 4; and (D) The expression of TLR2 and TLR4 with inpatient who used the fingolimod and interferon drugs (The TLR9 results did not work properly). Note. TLR: Toll-like receptor

Discussion

MS is common in people living far from the equator, although there are some exceptions. These cases include ethnic groups that are less likely to get infected such as American Indians, Maori New Zealand, and Inuit Canada. Some groups which live close to the equator are at risk of getting infected such as Palestinians. The reason for this pattern of geographical prevalence is not clear. Several studies showed that people who have traveled to different parts of the world before the age of fifteen have the same rate of infection as the new region, but if the migration occurs after the age of fifteen, the rate of infection will be equal to the rate of the country in which the person was born. These facts indicate that environmental factors during childhood can affect people’s infection with MS.

Diseases such as MS with its complexities have posed a great challenge to researchers. MS is an inflammatory autoimmune disease that originates from the nervous system and affects most of the young people who make up the community of the workforce. Although many cells such as dendritic cells, mast cells, macrophages, and the like can affect this pathological condition, myelin-specific T CD4 autoreactive cells play a more important role in the pathogenesis of this disease.⁸ Th17, which secrete IL17, plays a key role in the progression of the disease, but regulatory T cells are associated with reducing inflammation.⁹ The history of MS indicates that this disease has been treated by different doctors in various ways about a century ago.⁸

Perhaps the most important reason for these different treatments has been doctors’ thinking about the cause of the disease, but over time, in the twentieth century, studies revealed that MS was an autoimmune disease. Some studies have demonstrated that although the number of T lymphocytes is normal in MS patients, they are functionally defective, and Treg cells in MS patients have less power to suppress IL-17 production compared to healthy individuals. Some studies showed a decrease in the number of immune cells in MS patients.⁶ Recent studies displayed that TLR2 expression increases in patients with MS.^10-14 Increased expression is associated with the dysfunction of lymphocyte regulatory cells that can lead to disease.^15-20 In another study, several studies found that… found that TLR9 expression significantly increased in MS patients compared with the healthy control group.^21-28 A study by Papatheodorou et al showed that although the abundance and inhibitory function of innate immune cells decreases in the peripheral blood of the patient with MS, the abundance of these cells in the cerebrospinal fluid increases.²⁹ In a study done by Zheng et al, it was proved that the percentage of the Th17 cells in the blood samples of patients with active MS was about 7 times higher than that in the healthy control group.³⁰ In Hamid and colleagues’ study, by examining the TLR4 gene in pulmonary patients, it was found that an increase in the stimulation of this gene resulted in the increase in the symptoms and severity of the disease.³¹ Based on practical background, Tao et al and Wasko et al concluded that this gene activates pro-inflammatory cells by studying the effect of TLLR2 and TLR9 on brain inflammation following stroke.^32,33 The exact cause of this disease is not fully understood, but the overproduction of pro-inflammatory cytokines and metalloproteinase by synovitis, macrophages, and monocytes plays an important role in the development and exacerbation of this disease.¹⁵

Conclusion

Based on the experiments such as real-time PCR and previous studies, we concluded that the expression of TLR2 and TLR4 receptors decreased significantly with the time course of stimulation over time, which reached the lowest level after stimulation with angiopoietin 4. Each type of disease has its difficulties and problems. The responsibility of all people, including medical students, professors, and medical staff, is to free the world from disease and suffering. Different treatments and different ways of thinking have led us to be hopeful and try to keep our beloved ones and people safe and free from any illness.

Competing interests

Authors declare that they have no competing interests.

Ethical Approval

The study was approved by the Ethics Committee of Islamic Azad University, Tabriz (IR.IAU.TABRIZ.REC.1399.088).

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