Serum substance P level as a marker for subclinical rheumatoid arthritis activity

ABSTRACT Background Regular assessments of disease activity are essential to prevent the progression of rheumatoid arthritis (RA). Aim of the work This study aimed to assess the utility of serum substance P (SP) level as a marker for RA activity and to establish the value of SP level for the diagnosis of subclinical RA activity. Patients and Methods The study included three groups each 30. subject Group 1 was with minimal or no disease, group 2 had moderate to severe disease activity and group 3 was healthy controls. RA patients were assessed using a Disease Activity Score-28 (DAS28) and power Doppler ultrasonography measurement of the synovitis total score. The serum substance P levels of study groups were assessed using ELISA technique. Results Statistically significant difference in serum SP level was detected in RA patients compared to controls (p ≤ 0.001) and between different disease activity groups (p ≤ 0.001). The best cut off SP value to distinguish patients in subclinical activity active disease patients was 555.5 pg/ml. This had a diagnostic sensitivity, specificity, and accuracy of 96.7%, 80% and 88.3 %, respectively. Conclusion Serum SP can be used as an indicator of RA disease activity even in those with subclinical activity.


Introduction
The autoimmune disorder rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovitis, progressive destruction, and joint deformity [1].When compared to other autoimmune illnesses, which affect 5% to 7% of the global population, RA prevalence ranges from 0.3% to 1% [2].Women are more likely than men to have rheumatoid arthritis (3:1 ratio) [3].The T lymphocytes cells are the major cells responsible for causing synovitis and hence can be implicated in the pathophysiology of RA [4].Rheumatoid arthritis commonly impacts the smaller joints in the hands and feet.RA can also affect the musculoskeletal system, which includes tendons, muscles, bones, and less frequently extra-articular sites [5].
Blood tests (anti-cyclic citrullinated peptide, rheumatoid factor, inflammatory markers), radiography, and ultrasonography all contribute to the diagnosis of RA.Early diagnosis is crucial because early action creates a 'window of opportunity'.Currently, therapy aims to restrict disability and prevent structural damage in addition to relieving symptoms [5].The overall strategy for treating RA patients relies on the timely and prudent application of a variety of therapeutic approaches, which depends on early diagnosis and the early use of disease-modifying antirheumatic medications (DMARDs) with the goal of remission or low disease activity [6].
Substance P (SP) is a neuropeptide containing 11 amino acids in its structure and is encoded by Tachykinin Precursor 1 (TAC1) gene on human chromosome 7 [7].It is widely present in both central and peripheral nervous systems [8].Also, SP is released from immune cells such as macrophages, lymphocytes, and dendritic cells [9].In addition to the role of SP in pain, the non-neuronal effects of SP include vasodilation, smooth muscle contraction, change in cardiovascular tone, blood cell production, pruritus, wound healing, cancer, etc. [8,10].
Asthma, inflammatory bowel disease, psoriasis, atopic dermatitis, fibromyalgia, sickle cell disease, and rheumatic disorders, such as RA, are among the pathological disorders where substance P levels are elevated [11].Since proinflammatory cytokines like IFN-α, TNF α and γ IL-1b are linked to the degeneration of cartilage and bone erosion in RA, the ability of SP to regulate the synthesis of pro-inflammatory cytokines in the synovium suggests that it played a key role in the development of RA [12].TNF is a well-known cytokine that is present in both the synovium and serum of RA patients.It is a strong inducer of proinflammatory cytokines and disturbs the physiological balance between proand anti-inflammatory mediators [13].A decrease in serum SP brought on by anti-TNF therapy is associated with a reduction in RA disease activity [14].Limited information is available about the variation of SP serum level in different stages of RA activity and its role as a diagnostic marker for RA.This study was aimed at investigating serum level of substance P as a marker of subclinical rheumatoid arthritis activity.

Patients
A case-control study was carried on 60 rheumatoid arthritis patients fulfilled the criteria of the American College of Rheumatology/European League against Rheumatism (ACR/EULAR) 2010 [15] attended to the outpatient clinics of the rheumatology & rehabilitation department at Mansoura University Hospital, and 30 healthy controls who attended to blood bank served as the control group.The study was conducted over a 12-month period, from November 2018 to October 2019.Both the patients and the controls were over the age of 18. Demographic data and clinical data including duration of illness, results of rheumatoid factor, c reactive protein and anticyclic citrullinated peptide laboratory tests were collected from patient's files.Demographic data of the studied groups are presented in table 1.According to the Disease Activity Score-28 (DAS28) score, rheumatoid arthritis patients were split into two groups based on their level of RA activity.Clinical evaluation of tender and swollen joints was performed.The DAS28 score, which incorporates the swollen joint count (SJC) and tender joint count, was used to measure the severity of the disease (TJC), the erythrocyte sedimentation rate (ESR) and the patient's and physician's global assessment of activity on the visual analogue scale (VAS).

Serum Substance P levels were measured by
ELISA [18]: For every subject in all the three groups, 5-ml venous blood samples were withdrawn under complete aseptic condition by nurses in outpatient clinics of rheumatology & rehabilitation department and the control samples were taken from blood bank.Then, serum was separated by centrifugation 2000-3000 RPM for 20 min and kept frozen in test tubes at −20c and this step was performed at (Immunological lab of medical microbiology and immunology department at faculty of medicine Mansoura University) until analysis of SP using ELISA kit (Shanghai Korain Biotech company, China) according to manufacture instructions.

Ultrasound imaging
Ultrasound examination of each patient was performed on the same day of sample collection by a sonographer trained in power Doppler US.The time required to scan a patient completely was 20 min using EDAN U 2 ultrasound device (Shenzhen, China) with linear array transducer (8-13.4MHz) a frequency of 13 MHz was used.Power Doppler ultrasound was used to assess degree of synovitis and obtain synovitis total score by scanning of the most clinically affected 7 hand and foot joints (wrist, 2 nd and 3 rd metacarpophalangeal joint (MCP), 2 nd and 3 rd proximal interphalangeal joint (PIP), 2 nd and fifth metatarsophalangeal joint (MTP) [19].Synovitis total score ranged from 0 to 39 and calculated by the equation described before [20].The scoring system had grades of 0-3 [21].
The study was approved by the institutional review board at faculty of medicine Mansoura Univerisy.Informed consent was obtained from all patients included in the study.

Statistical analysis
Data were analyzed using SPSS program (Standard version 21).The normality of data was assessed using Kolmogorov-Smirnov test.Number and percentage were used to describe qualitative data.Continuous variables were presented (min-max), mean ± standard deviation (SD) for parametric data and median for nonparametric data.The two groups were compared with student t-test for parametric data and Mann Whitney test for non-parametric.More than two groups were compared by ANOVA test (parametric) and kruskal-Wallis test (non-parametric).Continuous parametric data were tested for correlation using Pearson correlation, whereas continuous non-parametric data were correlated using Spearman correlation.By using a receiver operating curve (ROC) sensitivity and specificity at different cutoff point were identified.Results were deemed significant for all of the aforementioned statistical tests when the p value was less than 0.05.

Results
The study enrolled 60 RA patients divided into 2 groups according to RA activity as Regarding serum substance P level, there was a significant higher serum substance P level in group 1 and 2 compared to the healthy controls (P a : ≤0.001 and P b : ≤0.001 respectively).In addition, upon comparing patients in remission and low disease activity to patients in moderate and high disease activity regarding serum SP level (group 1 and group 2), there was a statistically significant difference between them (P c value≤0.001) (Table 3).
Correlation between serum SP level in RA patient groups and clinical, laboratory parameters and synovitis total score was tested.Significant positive correlation between SP levels and synovitis total score, TJC, DAS28 and VAS was detected in group 1 and 2 patients.Moreover, significant positive correlation was detected between SP serum level and age (p = 0.046) in patients of group 1 only.In addition, serum SP level had significant positive correlation with SJC (p = 0.005) in group 2 patients only ( active RA from those with subclinical activity.The best cutoff value of serum SP in diagnosing RA cases was 99.0 pg/ml and area under the curve (AUC) was 0.988.This had a diagnostic sensitivity, specificity and accuracy of 98.3%, 90% and 95.5%, respectively (Table 5 and figure 1).

NPV: negative predictive value
The best cutoff value of serum SP in differentiating patients with active RA from those with subtle disease activity was 555.5 pg/ml.The AUC was 0.924.This had a diagnostic sensitivity, specificity and accuracy of 96.7%, 80% and 88.3% respectively (Table 6 and Figure 2).

Discussion
Rheumatoid arthritis is an autoimmune, multisystemic inflammation disease that causes substantial functional decline over time, radiographic damage, recurrent job incapacity, and early mortality [22].Assessment of the illness's activity is crucial for the best management of RA since rheumatoid flares and activity are linked to disease progression [23,24].The primary goal of novel therapy approaches for RA, which include the currently available synthetic and biological DMARDs, is remission or at least low disease activity [25].Remission, however, may not always signify a total absence of inflammation.Power Doppler ultrasound is one of the more sensitive instruments that is needed to assess the course of the disease [26].These methods can be used to identify patients who are at risk of structural progression despite an apparent clinical response or to help with drug withdrawal decisions [20].Inflammatory and immunological modulators like Substance P are one of the tools that have been suggested [14].Inflammation, apoptosis, and the production of chemokines and proinflammatory cytokines are all significant  aspects of how substance P controls the immune system [7].
Our study was aiming at evaluation of the possible use of SP as a marker for RA activity.Serum substance P level was evaluated in age and sex matched groups including two groups of RA patients (low and active disease) and healthy control.
Our study confirmed the findings of numerous earlier findings [27][28][29] by demonstrating that women are more likely than men to have RA.This can be related to the fact that females have strong immune systems and respond forcefully to vaccination and infection.Female hormones have also a significant role as estrogen and prolactin can stimulate the growth of B cell autoantibodies [30].
Comparing serum SP in RA patients and healthy controls, significant higher serum substance P levels was found in RA patients (p ≤ 0.001).This result matches with previous studies [31][32][33].
In our study, patients with remission and low disease activity (group 1) and patients with moderate and severe disease activity (group 2) had statistically different serum SP levels (p value ˂0.001).So even in patients who appear to be in remission, serum SP levels may be a sign of RA activity.Furthermore, our research revealed a significant positive correlation between serum SP levels and DAS28 score (p ≤ 0.001).These results are consistent with those previously obtained [34].On contrary, Lisowska et al. 2015 [18] observed insignificant correlation between the DAS28 score and serum SP level.
In our study, serum SP and VAS were significantly correlated in groups 1 and 2 (p = 0.015 and ≤0.001, respectively).The VAS has been validated as a subjective tool for measuring both acute and ongoing pain [35].Additionally, for groups 1 and 2, a significant positive correlation between blood SP level and TJC was found (p values of 0.002 and ≤0.00, respectively).This contrasts with the finding of Origuchi et al. [14] which showed an insignificant correlation between serum SP level and TJC.TJC has a multifactorial base and may not always indicate continuous inflammation (e.g.joint degeneration as in the case of secondary osteoarthritis or subluxation causing pain even if the RA inflammation is no longer present) [36].In comparison to clinical examination, power Doppler ultrasound is a contemporary imaging tool that allows for direct visualization of joint structures and is more effective at detecting synovitis and disease activity in RA [37].In order to differentiate between inflammatory and non-inflammatory arthritis, synovitis is evaluated using the synovitis total score [38].We found a significant positive correlation between synovitis total score and serum SP levels (P value≤0.001).
The best serum SP cutoff value for diagnosing RA cases and differentiating patients with active RA from those with subclinical activity    was determined using ROC curve analysis.For diagnosis of RA cases, the optimum serum SP cutoff value was 99.0 pg/ml and AUC was 0.988.This exhibited 98.3% diagnostic sensitivity, 90% diagnostic specificity, and 95.5% diagnostic accuracy.The best cutoff value of serum SP in differentiating patients with active RA from those with subtle disease activity was 555.5 pg/ml, and the AUC was 0.924 with a diagnostic sensitivity, specificity and accuracy of 96.7%, 80% and 88.3%, respectively.In this regard, serum SP had higher sensitivity, specificity and accuracy when compared to other markers like serum calprotectin [39].
To the best of our knowledge, this study is the first to specify a cutoff value for serum SP for RA diagnosis or activity assessment.In light of all prior findings, we recommend considering serum SP as a new candidate for RA diagnosis and activity assessment to increase the ability to recognize patients with subclinical disease activity and enable an earlier start to RA treatment.In addition, we advise future research to establish the function of SP in RA therapy monitoring and SP antagonist use.

Conclusion
Even in patients with subclinical activity, serum SP can be utilized as a marker of RA disease activity.
According to our knowledge, this study is the first study that defines a cut-off value of serum SP either to diagnose or to evaluate the activity of RA.According to all previous results, we recommended considering serum SP as a new diagnostic tool in RA to improve the ability of identifying patients in subclinical disease activity and this allows the earlier initiation of RA treatment.Also, we recommend further investigations to determine the role of SP in monitoring RA therapy and use of SP antagonists in RA treatment.

Conclusion
Serum SP can be used as a marker of RA disease activity even in those with subclinical activity.

Figure 2 .
Figure 2. Receiver operating characteristics (ROC) curve of substance P for prediction of disease activity.

Figure 1 .
Figure 1.Receiver Operating characteristics (ROC) curve of substance P in differentiating cases from control group.

Table 1 .
Demographic data of the studied groups.

Table 2 .
Clinical, laboratory, and radiographic data of patients Note: TJC: Tender joint count, SJC: Swollen joint count, VAS: Visual analogue scale.

Table 3 .
Clinical, laboratory, and radiographic data of patients.

Table 4 .
Clinical, laboratory, and radiographic data of patients.

Table 6 .
Validity of serum substance P level (pg/ml) in differentiating active RA disease from subclinical activity.
Note: AUC: area under the curve.CI: confidence interval.PPV: positive predictive value.

Table 5 .
Validity of substance P level (pg/ml) in diagnosing RA cases.AUC: area under the curve.CI: confidence interval.PPV: positive predictive value.