Migration inhibitory factor in spinal tuberculosis: -173G/C polymorphisms, and transcript and protein levels in a northern province of China

Abstract The aim of this study was to elucidate the possible association between migration inhibitory factor (MIF)-173G/C gene polymorphisms and transcript and plasma levels of MIF in spinal tuberculosis (TB) patients. Clinical data were collected from 254 spinal TB patients and 262 healthy controls participating in the study. The genotype of the MIF-173G/C gene was amplified by polymerase chain reaction and genotyped by DNA sequencing technology. The level of mRNA expression was determined by real-time polymerase chain reaction and MIF plasma levels were measured by a solid-phase enzyme-linked immunosorbent assay. The frequency of the C allele and GC+CC genotype in MIF-173G/C was over-represented in spinal TB patients. The mean MIF mRNA level in spinal TB patients and patients with the GG and GC+CC genotype were significantly lower than controls; however, our study also indicated that the MIF concentration in spinal TB patients and patients with the GG and GC+CC genotypes were significantly higher than controls. Spinal TB patients with the GG genotype had higher MIF plasma levels than patients with the GC+CC genotype. The C-reactive protein level and erythrocyte sedimentation rate was correlated with the MIF plasma level. In summary, the association between the MIF-173G/C genetic polymorphism, reduced transcript and increased plasma levels of MIF in spinal TB patients, and MIF may play an important role in the occurrence, development, and damage of spinal TB in the northern Province population of China.


Introduction
Tuberculosis (TB) is a common disease in developing countries, and spinal TB is 1 of the most serious forms of extra-pulmonary TB, with its severe disability and high morbidity rate. [1] Macrophage migration inhibitory factor (MIF) is a soluble protein secreted by the activation of T cells, which was first described by Bloom in 1966. [2] MIF regulates the balance of Th 1 and Th 2 cells during the host inflammatory reaction and immune responses, inhibits macrophage migration, and promotes accumulation of macrophages in areas of inflammation, infiltration, proliferation, activation, and secretion of IL-1, IL-2, IL-6, IL-8, TNF-a, and IFN-g. [3] MIF has a close relationship with macrophages and TB. MIF also aggravates inflammatory injuries through the interaction with inflammatory factors; spinal TB is 1 type of inflammation.
Two types of functional polymorphisms have been identified in the MIF promoter [À173G/C and À794CATT (5-8)] microsatellite polymorphism. [4] Several recent reports have indicated that MIF is associated with the destruction of intervertebral discs and 2 kinds of polymorphisms are correlated with TB, [5,6] while there is no research data reporting the correlation with spinal TB. The aim of this work was designed to elucidate whether or not there is an association between the MIF-173G/C genetic polymorphism, transcript and serum levels of expression, and spinal TB in Shandong, a northern province in China.

Study population
This study was approved by the Ethics Committee of Weifang and Liaocheng in China. This study included 254 patients living in Shandong province, who were diagnosed with spinal TB and underwent surgery in the Weifang People's Hospital and Liaocheng People's Hospital between January 1, 2015 and April 30, 2020. The diagnosis of spinal TB was made based on post-operative histopathologic sections, and the etiologic examination showed that pus-positive, acid-fast staining or positive results of pus isolation and culture of Mycobacterium TB. Other diseases, such as acquired immune deficiency syndrome (AIDS), tumors, pulmonary TB, and ankylosing spondylitis, were excluded. The control group included 262 healthy subjects and subjects without pulmonary TB, spinal TB, and other forms of extra-pulmonary TB. The following data were collected for the 2 groups: gender; age; duration of symptoms; pain intensity (visual analog scale score); smoking habit; C-reactive protein (CRP) level; and erythrocyte sedimentation rate (ESR). All data for the 2 groups are listed in Table 1.

Genomic DNA and RNA extraction
Genomic DNA and total RNA were isolated from blood samples using TRIzol (Life Technologies, USA) according to the manufacturers' instructions. The extracted products were stored at -80°C. DNA and RNA qualities and concentrations were determined with a Nanodrop2000 micro-volume spectrophotometer (Thermo Scientific). RNA integrity was measured by 1% agarose gel electrophoresis and staining with ethidium bromide.

Polymerase chain reaction (PCR)
The primer sequences were designed using the UPL Assay Design Centre web service, as follows: forward primer, 5 0 -CTG ACTT CTC GGA CAC CAC T-3 0 ; and reverse primer, 5 0 -AAG GGT AAG GGG CCA TCT TC-3 0 . Amplified fragment size was 352 bp. DNA was amplified in the PCR machine through PCR thermal cycling with the following conditions: initial denaturation at 95°C for 10 minute, followed by 30 amplification cycles of 95°C for 30 second; annealing temperature (given separately) and extension at 60°C for 30 s; 72°C for 1 minute; and a final extension at 72°C for 10 minute with annealing at 4°C. The PCR products were sent directly to the Sangon of Shanghai (China) for sequencing. The sequencing primers were the same as the sequencing primers of PCR.

Preparation of cDNA and real-time PCR
Reverse transcription was performed using the PrimeScript RT reagent kit with a gDNA Eraser (TaKaRa, Japan) with 1000 ng of total RNA. The real-time PCR reaction used an ABI7500 realtime PCR system (Life Technologies). The reaction mixture consisted of 10 ml of Fast Start Universal SYBR Green Master (Roche, Germany), 0.6 ml of each upstream primer, 6.8 ml of RNase-free water, and 2 ml of cDNA. The RT-PCR reaction cycling conditions were as follows: 10 minute at 95°C; followed by 15 s at 95°C (40 cycles); and 1 minute at 60°C. To control the RT-PCR standard, beta actin was used as the internal control. The primer sequences were designed using the UPL Assay Design Centre web service, as follows: forward primer, 5 0 -AGA ACC GCT CCT ACA GCA AG-3 0 ; and reverse primer, 5 0 -TAG GCG AAG GTG GAG TTG TT-3 0 . All RT-PCR products used ethidium bromide under a UV light trans-illuminator after electrophoresis on 1.5% agarose gels. MIF transcript levels were calculated using the 2 −DDCT method. [7][8][9] All RT-PCR steps were repeated 3 times. The results of the analysis used ABI 7500 realtime PCR software (version 2.0.1).

Serum MIF levels
The MIF blood serum levels in all subjects were measured by testing with ELISA kits (R&D), according to the manufacturer's instructions. A standard curve was plotted, and the MIF concentration was calculated.

Statistical analysis
Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL). The general conditions of the 2 groups was compared using a t-test and chi-square test. The Kolmogorov-Smirnov 1-sample test was used to assess the normality of the distribution of the transcript levels and blood serum expression values for the genes studied. A t-test was used to compare the concentration of serum and mRNA expression of MIF between the 2 groups. Accordingly, a non-parametric test was used to compare the mRNA and blood serum expression of the MIF between groups and within the group in different genotypes and in spinal TB subjects with different clinicopathologic features. Hardy-Weinberg equilibrium was examined for each group. Data are expressed as the mean ± SD. The odds ratio (OR) at the 95% confidence interval (CI) and Pearson P-value were calculated. A P-value < .05 was considered statistically significant.

MIF-173 G/C polymorphism analysis
We evaluated the frequencies of MIF polymorphisms in spinal TB patients and controls, as shown in Table 2. The sequencing analysis results of the MIF-173GC polymorphism are shown in Figure 1. Both the genotype and allele frequency distributions were in agreement with the Hardy-Weinberg equilibrium.

Comparison of MIF transcript level and serum concentration in patients and controls
The MIF and beta-actin amplification curves showed that the cycle threshold values were within an acceptable range (Fig. 2). The specificity of the primers was verified through melting curves.
The standard curves were plotted as the Ct versus the concentration of the total RNA from 10 times the gradient serial dilution. The t-test showed that the transcript level of MIF in spinal TB patients was significantly lower than controls (0.36 ± 0.14 vs 1.22 ± 0.47, P < .001); however, the serum concentration of MIF in spinal TB patients was significantly higher than controls, as shown in Table 3 (11.38 ± 3.79 ng/mL vs 5.76 ± 2.69 ng/mL, P < .001).

Comparison of MIF transcript level and serum concentration in patients and controls with different genotypes
The transcript level of MIF was significantly lower in spinal TB patients than in controls with GC+CC (0.36 ± 0.16 vs 1.17 ± 0.65, P < .001) and GG genotypes (0.35 ± 0.13 vs 1.32 ± 0.41, P < .001), and the serum MIF concentration was significantly higher in spinal TB patients than in controls with GC + CC (10.43 ± 3.29 ng/mL vs 5.82 ± 2.61 ng/mL, P < .001) and GG genotypes (11.89 ± 4.31 ng/mL vs 5.79 ± 2.75 ng/mL, P < .001).
Our results also showed that the serum concentration of MIF in spinal TB patients with the GG genotype (n = 123) was significantly higher than spinal TB patients with the GC+GC genotype (n = 131; 11.89 ± 4.31 ng/mL vs 10.43 ± 3.29 ng/mL, P < .001; Table 3).

Comparison of MIF transcript and serum levels in patients with different clinicopathologic features
We further evaluated the association between stratification analysis of MIF with clinicopathologic factors in the spinal TB group. Patient clinical characteristics, such as gender, age, smoking habit, pain intensity, duration of symptoms, ESR, and CRP level, are shown in Table 1. The serum level of MIF was significantly higher in patients with high CRP levels (>30 mg/L) compared to patients with low CRP levels (<10 mg/L; P < .001), and was significantly higher in patients with a high ESR (>40 mm/h) than patients with low a ESR (<20 mm/h; P < .001; Fig. 3). Other patient characteristics did not significantly affect the serum levels of MIF in the present study. There was no significant difference in the MIF mRNA level based on clinicopathologic factors.

Discussion
TB is a curable infectious disease, but can result in death. The active TB prevalence rate in China was 5% in 2010, and every 3% to 5% of active TB cases involves dissolved bone lesions, most of which occur in the spine. [10] Spinal lesions may lead to pain, nerve compression symptoms and paraplegia. Some studies have shown that spinal TB patients have different pathologic and predisposing factors. [11] Further studies with spinal TB in other groups of patients will help clarify this question.
MIF is widely expressed in various cells, and is considered to be a versatile cytokine. MIF is not only a T cell-derived cytokine, but could also be released from other cells, such as monocytemacrophage cells and anterior pituitary cells. MIF is an upstream inflammatory amplification factor which can amplify the inflammatory response and can counteract the effects of glucocorticoids, activate immune or inflammatory cells, and promote the expression of MMP-9, NO, IL-6, IL-8, TNF-a, IL-1b, IFN-g, and PGE2. [3] Early reports indicate that MIF may be associated with susceptibility to TB and intervertebral disc lesions. [5,6] In recent years. it has been reported that the MIF concentration may be associated with the severity of inflammatory damage and inhibition of biological function, and can also promote the repair and regeneration of the injured site. [12] To obtain more detailed information and investigate the association between MIF and spinal TB, we studied the relationship between MIF and spinal TB with respect to gene variants, and transcript and serum levels of MIF.
Gene polymorphism plays an important role in the occurrence and development of TB. Several TB B susceptibility genes have been discovered, 1 of which is MIF. In recent years, it has been shown that MIF gene polymorphisms can affect host susceptibility to TB. [13,14] In fact, some researchers have found that MIF-173G/C polymorphism may be involved in the development of pulmonary TB in Asians and Caucasians and the frequency of MIF-173CC homozygote and C allele in pulmonary TB group are significantly higher than that in healthy control group. [15] Liu et al have found that the frequency of MIF-173CC homozygote and C allele in pulmonary TB group in Southwest China were significantly higher than that in healthy control group. Similarly, according to our data, the MIF C minor allele and MIF C carriers are more frequent in spinal TB patients than controls. Interestingly, our study confirmed that the MIF transcript level in spinal TB patients is significantly lower than healthy controls; however, our findings showed that the MIF plasma levels were significantly higher in spinal TB patients compared with controls. Both Li and Liu et al have found that the level of serum MIF in TB group in Southwest China was also significantly higher than that in healthy control group, [16,17] which was consistent with our study results. In addition, Xiang et al have found that the level of MIF plasma in Asian patients with pulmonary TB was significantly higher than that in the control group, but there was no significant difference among Africans. [15] We also showed that the GG genotype may be associated with a higher serum concentration of MIF in spinal TB patients. Based on a review of the correlation between the expression of mRNA transcription and protein levels in many cases is not good. The mRNA and protein levels are a coupled process, but are not necessarily a consistent trend or miRNA may play a important role in this process. Therefore, a decrease in the level of mRNA transcription does not necessarily reduce the level of protein concentration expression. [18] In addition, we also noticed a major difference in the MIF concentrations reported in different studies, owing to the following factors: ELISA kits; specimen preservation methods; populations investigated; and varying degrees of disease severity.
The ESR and CRP levels are reliable parameters when evaluating the treatment and prognosis of spinal TB. [19,20] Our results indicated that the level of MIF serum protein are increased in spinal TB patients with high ESR and CRP levels. Therefore, when MIF enters the systemic circulation of TB patients, which causes an inflammatory reaction, the blood ESR and CRP levels Table 3 Comparison of migration inhibitory factor transcript levels and serum in patients and controls with different genotypes.  increase. Thus, we speculate that the blood ESR and CRP level are related to the expression of MIF, and may affect inflammation and the immune response in patients with spinal TB.
In conclusion, our study showed that the increased levels of MIF, as well as the MIF genetic variant, are associated with spinal TB. Furthermore, the C minor allele of the MIF-173GC genetic variant appears to be a risk factor for the development of spinal TB in the Shandong Province population of China. We believe that our report about the relationship between MIF polymorphisms and the transcription and plasma levels are the first reported in the Chinese spinal TB population. This study was relatively limited with 1 small cohort. The reason the levels of MIF mRNA transcription and protein expression do not exhibit a consistent trend warrants further study. The mechanism MIF plays in the development of spinal TB remains unclear. We will perform the study in a larger patient group. Further experimental studies are needed to confirm our findings.