Study of plasma levels of soluble triggering receptor expressed on myeloid cells-1 in rheumatoid arthritis and its correlation with disease activity and tumor necrosis factor-α

Aim of the work The triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell surface receptor expressed mainly on monocytes and neutrophils. It acts as an amplifier of inflammatory response in acute and chronic inflammatory states. The aim of this work was to study the plasma-soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in rheumatoid arthritis (RA) patients and its correlation with disease activity and tumor necrosis factor-α (TNF-α). Patients and methods This study included 80 patients with RA and 20 age-matched and sex-matched controls. All were subjected to demographic, clinical, laboratory, and radiological studies using the 28-joint Disease Activity Score, erythrocyte sedimentation rate, complete blood count, and radiograph of both hands. Plasma levels of sTREM-1 and TNF-α were measured with enzyme-linked immunosorbent assay. Results RA patients had significantly higher sTREM-1 and TNF-a levels compared with controls (206.32±125.75 and 17.83±11.88; P<0.001) and (190.82±69.46 and 54.75±9.46; P<0.001). In RA patients, sTREM-1 levels were found to be positively correlated with 28-joint Disease Activity Score, erythrocyte sedimentation rate, and TNF-a level (r=0.408, P=0.001; r=0.287, P=0.010; r=0.749, P=0.001). sTREM-1 level was significantly increasing as patients had increasing disease activity (F-test=20.62; P=0.001). Conclusion RA patients had higher sTREM-1 and TNF-a level compared with controls, and sTREM-1 level was correlated with disease activity, suggesting that sTREM-1 plays a role in the inflammatory process associated with TNF-a, and it may be a useful disease activity marker in RA. TREM-1 may be a safe therapeutic strategy for RA, as blocking TREM-1 signaling was found to suppress inflammatory responses without affecting the immune system to fight bacterial infection.


Introduction
Rheumatoid arthritis (RA) is a form of autoimmune chronic inflammatory arthritis characterized by synovial hyperplasia that leads to joint damage, and its clinical manifestations are not limited to arthritis but also include various systemic problems [1].
Numerous inflammatory cells such as macrophages, monocytes, T cells, B cells, and neutrophils are involved in the inflammatory process of RA, and various cytokines and chemokines play important roles in the pathogenesis of the disease [2]. Identification of cytokines and chemokines associated with RA may reveal insights on pathogenesis, and they could be used as markers of disease activity of RA or to monitor treatment response [1].
Triggering receptor expressed on myeloid cells (TREM-1) is described as a member of the immunoglobulin superfamily and is expressed on neutrophils, monocytes, and macrophages [3]. TREM-1 has been identified as a transmembrane receptor, and the membranous domain of TREM-1 binds to DNA activation protein of 12 kDa, an immune receptor tyrosine-based activation motif containing the adapter molecule [4].
TREM-1 serves as a critical amplifier of inflammatory signaling and is upregulated by various stimuli such as proinflammatory cytokines and microbial Toll-like receptor ligands [5].
In addition to its membrane-bound form, a soluble form of TREM-1 soluble triggering receptor expressed on myeloid cells (sTREM-1) has been detected in the blood of patients with RA. It has been reported that sTREM-1 levels are elevated in patients with RA compared with controls [6], and that synovial fluid sTREM-1 levels are greater than those in gouty arthritis, nonseptic/nonrheumatoid inflammatory arthritis, and noninflammatory arthritis [7].
Our aim was to measure the plasma sTREM-1 level in patients with RA to evaluate its usefulness as a marker of disease activity in RA patients compared with other disease activity measures and tumor necrosis factor-α (TNF-α) as the proinflammatory cytokine.

Patients
Eighty RA patients who fulfilled the 2010 revised American College of Rheumatology/European League Against Rheumatism classification criteria [8] were selected from the Physical Medicine, Rheumatology and Rehabilitation Outpatient Clinic, Menoufia University Hospitals, in the years 2013-2014. Only patients older than 16 years were selected. They were classified into two groups: group 1, comprising RA patients with disease duration less than 6 months (early RA), and group 2, comprising RA patients with disease duration more than or equal to 6 months (established RA). Written consent was obtained from all patients and controls after a full explanation of the study. This study was approved by the Ethical Committee of the Faculty of Medicine, Menoufia University.

Exclusion criteria
Patients with a history of diabetes, hypertension, congestive heart failure, pregnancy, or concomitant renal, hepatic, cardiac, or infectious processes were excluded from the study.

Controls
Twenty healthy volunteers matched for age and sex with the patient group were enrolled in the study as controls.

Methods
All patients were subjected to the following: (1) Demographic data recording.
The locomotor system was examined and local examination of joint tenderness and swelling was carried out. Chest, heart, and abdominal examination to detect extra-articular manifestations of RA was carried out for all patients. (3) Disease activity assessment: this was achieved using the Disease Activity Score (DAS28) with three variables [erythrocyte sedimentation rate (ESR), number of swollen joints, and number of tender joints] [9].
Data obtained were tabulated and analyzed with SPSS (SPSS Inc., Chicago, Illinois, USA) statistical package, version 18, on an IBM compatible computer. Quantitative data were expressed as X ± SD . Qualitative data were expressed as number and percentage and analyzed by applying the χ 2 -test. Comparisons between groups were made using the unpaired t-test. Pearson's correlation (r) was used to detect the association between quantitative variables. P values greater than 0.05 were considered nonsignificant, values less than 0.05 were considered significant, and values less than 0.001 were considered highly significant.

Results
The clinical data of the studied RA patients are shown in Table 1. The study revealed a statistically highly significant difference between RA patients and controls regarding the level of TREM-1 and TNF-α ( Table 2 and Fig. 1). There was no statistically significant difference in serum levels of sTREM-1 and TNF-α between RA patients with positive RF and those who were seronegative (Table 3) or between patients with positive ACCP and those with negative ACCP (Table 4). There was a statistically highly significance difference (P<0.001) regarding serum sTREM-1 and TNF-α levels in RA patients according to disease activity (Table 5 and Fig. 2). Pearson's correlation revealed a statistically highly significant positive correlation between sTREM level and DAS28 (r=0.408, P<0.001), TNF-α (r=0.749, P<0.001), and ESR (r=0.287, P<0.010). There was a statistically significant negative correlation between sTREM level and hemoglobin (Hb) (r=−0.228, P<0.435), whereas there was no statistically significant correlation between sTREM level and age, disease duration, leukocytic count, platelet count, and radiological findings (Table 6 and Fig. 3). Pearson's correlation revealed a statistically highly significant positive correlation between TNFα level, DAS28 (r=0.520, P<0.001), sTREM (r=0.749, P<0.001), and ESR (r=0.317, P<0.004) and statistically significant negative correlation between TNF-α level and Hb (r=−0.345, P<0.002).
There was no statistically significant correlation between TNF-α level and age, disease duration, leukocytic count, platelet count, and radiological findings (Table 7 and Fig. 4).

Discussion
RA is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints, which leads to progressive destruction of cartilage and bone. It is now clear that a large number of inflammatory cytokines such as TNF-α, interleukin-1b (IL-1b), IL-6, IL-12, IL-23, and IL-17A play an important role in persistent inflammation and progressive destruction of cartilage and bone [16].
TREM-1 is known as a cell surface receptor mainly expressed on monocytes and neutrophils; it is expressed in acute and chronic inflammatory conditions and is upgraded by various Toll-like receptor ligands [2].
TREM-1 is expressed in many diseases, including infections, and in acute and chronic noninfectious inflammatory disorders, such as inflammatory bowel disease and ankylosing spondylitis. More specifically, TREM-1 is upregulated in RA synovium, and its activation induces many proinflammatory cytokines in patients with RA [5].  RA, rheumatoid arthritis; TNF-α, tumor necrosis factor-α; TREM-1, triggering receptor expressed on myeloid cells-1. *P<0.05 is significant. **P<0.001 is highly significant.
Therefore, our aim was to study the plasma level of sTREM-1 in RA patients and its correlation with disease activity and TNF-α as a proinflammatory cytokine.
This study showed that patients with RA have significantly higher plasma sTREM-1 and TNF-α levels compared with controls. This was in agreement with the results of Kuai et al. [6], El Bakry Samah et al. [2], and Sung et al. [5], who found that the average amount of soluble TREM-1 in RA patients was higher than that of controls. The data showed that sTREM-1 has a major role in RA.
In this study, sTREM-1 correlated significantly with disease activity. Serum levels of sTREM-1 were significantly higher in patients with elevated ESR and CRP. sTREM-1 levels were elevated in patients with severe disease activity (DAS>5.1) compared with those with mild disease activity (DAS<3.2). This was in agreement with the findings of Kuai et al. [6], El Bakry Samah et al. [2], and Sung et al. [5], who found that sTREM-1 levels were found to be significantly elevated in patients with RA and correlated significantly with disease activity. These results give an index that sTREM-1 contributes to the inflammatory process of RA, and that plasma sTREM-1 levels may be used as an activity marker in RA.
In this study, RA patients had higher TNF-α levels, which were correlated with plasma sTREM-1 levels; this is supported by the work of Collins et al. [7], who have assessed the TREM-1 synovial expression in patients with certain types of inflammatory and   DAS, Disease Activity Score; RA, rheumatoid arthritis; TNF-α, tumor necrosis factor-α; TREM-1, triggering receptor expressed on myeloid cells-1. *P<0.05 is significant. **P<0.001 is highly significant.

Figure 2
TREM-1 and TNF-α level in RA patients according to disease activity. RA, rheumatoid arthritis; TNF-α, tumor necrosis factor-α; TREM-1, triggering receptor expressed on myeloid cells-1. noninflammatory arthritis. They have reported an increase in TNF-α and sTREM-1 levels in septic arthritis and RA compared with gouty arthritis and noninflammatory arthritis.
Although the biologic agents used to block inflammatory cytokines have the ability to suppress the progression of inflammation and joint destruction in RA patients [17], about one-third of RA patients fail to achieve complete remission and there are serious side effects of bacterial infection and reactivation of tuberculosis with those biologic treatments [18]. Targeting of TREM-1 signaling may be a therapeutic modality in RA [19]. Further, it was hypothesized that modifiying the actions of TREM-1 may be a safe therapeutic strategy for RA, as blocking TREM-1 signaling was found to suppress inflammatory responses without affecting the ability of the immune system to fight bacterial infection [20].
In this study, there were no correlations between sTREM-1 and TNF-α level in RA patients according to RF status. This was in agreement with the findings of Kuai et al. [6], El Bakry Samah et al. [2], and Sung et al. [5], who found that the average amount of soluble TREM-1 and TNF-α level in RA patients was not correlated with RF status.
In this study, there were no correlations between sTREM-1 and TNF-α level in RA patients according to ACCP status. This was in agreement with the results of Kuai et al. [6], El Bakry Samah et al. [2], and Sung et al. [5], who found that the average amount of soluble TREM-1 and TNF-α level in RA patients was not correlated with ACCP status.
In this study, there were no correlations between sTREM-1 level and age, disease duration, platelets, and radiological findings. This was in agreement with El Bakry Samah et al. [2], who found that there were no significant correlations between sTREM-1 level and age, disease duration, morning stiffness, radiological narrowing, and erosion scores of the hand joints (P>0.05).
In this study, there were no correlations between sTREM-1 level and white blood cells (WBC). This was in agreement with the results of El Bakry Samah et al. [2], who found no significant correlations between sTREM-1 level and WBC and platelets. However, this is in contradiction to the results of Sung et al. [5], who found that sTREM-1 levels were significantly correlated with serum WBC and neutrophil counts. He found that TREM-1 is expressed on neutrophils, monocytes, and macrophages [21], and therefore plasma   sTREM-1 might be released by macrophages/ monocytes or neutrophils in RA patients.
In this study, there was a negative correlation between sTREM-1 and TNF-α level with Hb. It is reported that the blunting of bone marrow response to erythropoietin may be mediated by IL-1, TNF-α, and transforming growth factor β, which affect erythropoietin mRNA transcription [22]. It has also been demonstrated that IL-1, which is increased during inflammation, inhibits erythroid colonyforming units, which later develop into erythroblasts [23].
The present study demonstrated that RA patients had higher plasma sTREM-1 and TNF-α levels compared with controls, and plasma sTREM-1 levels were correlated with disease activity indices, including DAS28, ESR, and CRP, suggesting that plasma sTREM-1 could play a role in the inflammatory process associated with TNF-α, and it may be a useful disease activity marker in RA. Targeting of TREM-1 signaling may be a therapeutic strategy in RA, as blocking TREM-1 signaling was found to suppress inflammatory responses without affecting the ability of the immune system to fight bacterial infection.

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Conflicts of interest
There are no conflicts of interest.