Upregulation of circulating soluble programmed death-1 molecule and its correlation with the disease activity of rheumatoid arthritis patients in Ethiopia

doi:10.21203/rs.3.rs-4281432/v1

Background: Rheumatoid arthritis is an autoimmune disease characterized by the destruction of joints and, if left untreated, leads to functional impairment. Various elements heightened the inflammation and progress of the disease. Although the pioneered role of soluble programmed death-1 is controversial, it is believed to be correlated to the disease activity of Rheumatoid arthritis. So this study aimed to assess plasma soluble programmed death 1 (sPD-1) level and its correlation with disease activity in rheumatoid arthritis patients at Tikur Anbesa Specialty Hospital, Addis Ababa, Ethiopia.

Method: The hospital-based cross-sectional study was undertaken from February 2021 to June 2021. Participants were enrolled through consécutive sampling. Sociodemographic data was collected using structured questionnaires, while clinical data was collected using a data collection sheet. The disease activity of the Rheumatoid arthritis patients was measured with disease activity score-érythrocytes sédimentation rate (DAS28-ESR) score. Plasma soluble programmed death one concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit. A median variation in the concentration of soluble programmed death one between Rheumatoid arthritis patients and healthy controls were evaluated using the Mann-Whitney U-test, while Spearman’s correlation coefficient was measured to test the correlation between predictor and outcome variables.

Results: This study included 50 rheumatoid patients with a mean age of 48.08 ± 9.391 and 50 healthy controls with a mean age of 38.30 ± 13.386. In patients with RA, soluble PD-1 levels were (median; 1023 pg/mL, IQR; 850.5 - 1304.54) compared to healthy controls (median; 531.9 pg/mL, IQR: 364 - 809) increased significantly (p<0.001). Soluble PD-1 has a strong positive correlation with DAS28 (r=0.7157, p<0.001), ESR (r=0, 5657, p<0.001), and number of swollen and tender joints (r=0.5533, p<0.001). Plasma-soluble PD-1 levels were significantly lower in patients in remission compared to active RA patients.

Conclusion: soluble PD-1 Is significantly higher in RA patients and positively correlates with DAS28. This result demonstrates that sPD-1 is associated with RA disease activity and is a biomarker of RA disease activity.

Disease activity

DAS28

programmed death-1. soluble Programmed Death-1

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the joints but also affects other organs and tissues of the body (1–3). RA affects approximately 1% of the world's population, and women are more likely to be affected by RA than men. Typically, people between the ages of 30 and 60 years are affected (4, 5).

Rheumatoid arthritis is a chronic autoimmune disease caused by a complex interaction of genetic, environmental, and immune factors (6–8). In genetically susceptible individuals, environmental triggers such as infection, smoking, or stress can activate the immune system and lead to an inflammatory response (8).

The pathogenesis of RA involves the activation of multiple immune cells, including T cells, B cells, and macrophages. These cells release pro-inflammatory cytokines and chemokines, leading to synovial inflammation and joint destruction (9). In addition, autoantibodies such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA) contribute to the pathogenesis of RA by targeting self-antigens and promoting the formation of immune complexes(10). Dysregulation of immune cells and their interactions with other cells in the joint play a crucial role in the development and progression of RA (11).

Programmed death 1 (PD-1) is a type 1 transmembrane glycoprotein that is mainly expressed in immune cells such as T cells, B cells, monocytes, and natural killer cells (12, 13). It plays a vital role in regulating immune responses. When PD-1 interacts with its ligands programmed death ligands 1 and 2 (PD-L1 and PD-L2), it initiates inhibitory signals in immune cells, leading to downregulation of immune responses, thereby inhibiting the activation and proliferation of immune cells, protecting tissues from autoimmune attack and inflammatory response (14). This mechanism is a critical checkpoint to prevent excessive immune responses and maintain immune homeostasis.

However, in rheumatoid arthritis (RA), there is evidence that the PD-1 signaling pathway is dysregulated, leading to increased T-cell activation and reduced tolerance to self-antigens (15–17).

In recent years, the role of soluble PD-1 in the pathogenesis of rheumatoid arthritis has been a topic of considerable controversy and concern. Soluble PD-1 (sPD-1) is a truncated form of the PD-1 receptor that is released into the bloodstream (18). Soluble PD-1 is thought to be a decoy receptor that binds to PD-L1 and PD-L2 ligands and prevents them from interacting with membrane-bound PD-1 on T cells (12). This results in reduced T cell apoptosis and increased T cell activation and proliferation, leading to chronic inflammation and tissue damage in RA (19). In addition, sPD-1 can also directly regulate the functions of other immune cells such as B cells and dendritic cells.

The study examined the association between sPD-1 levels and disease activity in patients with RA. These studies found that higher levels of sPD-1 were associated with increased disease activity, including increased numbers of swollen and tender joint count, as well as increased levels of inflammatory markers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) (12). This suggests that sPD-1 could serve as a potential biomarker of RA disease activity and could be used to assess disease progression and treatment response.

Another study examined the association between sPD-1 and disease activity in patients with early RA. The researchers found that higher sPD-1 levels were associated with higher disease activity scores, including the Disease Activity Score of 28 joints (DAS28). They also observed a decrease in sPD-1 levels after effective treatment with disease-modifying anti-rheumatic drugs (DMARD), suggesting that sPD-1 levels may reflect changes in disease activity over time (20).

While these recent findings provide insights into the potential role of sPD-1 in rheumatoid arthritis, further research is needed to fully understand its mechanisms of action and clinical implications. Therefore, this study aimed to investigate the plasma levels of sPD-1 and their association with disease activity in RA patients. There is no study done in our country on the role of sPD-1 in rheumatoid arthritis. So it will used as a starting point for further studies.

Study design, setting, and period

A cross-sectional study was conducted at Tikur Anbesa Specialized Hospital, Addis Ababa, Ethiopia from February 2021 to June 2021.

Study participants

All RA-suspected individuals visiting the rheumatology clinic during the study period were used as the source population. Patients fulfilling the revised RA classification criteria (21) were considered as the study population. In addition, healthy individuals were recruited from the hospital staff, and other healthy individuals took care of patients in the hospital as a control for the comparison group.

Inclusion and exclusion criteria

Volunteer study participants whose age is above 18 and who fulfill revised 2010 ACR RA disease classification criteria were included in the study. Study participants with known viral infections such as human immunodeficiency virus (HIV) and hepatitis b virus (HBV) and chronic diseases such as diabetes mellitus and cancer were excluded. In addition, other rheumatic diseases such as SLE are also excluded.

Sample size and sampling techniques

Consecutive sampling technique was used and 50 RA patients visiting the Rheumatology clinic were recruited for the study between February 2021 and June 2021. Fifty healthy controls were included in the study for comparison purposes.

Study variables

The outcome variable was the plasma concentration of soluble PD-1. Whereas predictor variables are sociodemographic and behavioral characteristics such as age, gender, family history of the disease, and smoking habit. In addition, clinical data of RA such as number of tender and swollen joints, general health assessment, ESR level, RF seropositivity, DAS28-ESR, and disease activity stage.

Data collection and laboratory methods

Socio-demographic and clinical data

A structured questionnaire was prepared. After obtaining written informed consent, sociodemographic characteristics and behavioral factor-related data of RA patients were collected through direct face-to-face interviews at the Rheumatology clinic of TASH. Rheumatologists examined RA patients. Individual RA patients’ clinical data such as the number of tenders and swollen joints, patient general health assessment, and disease activity scores were collected using RA clinical data collection sheets.

Blood sample collection and processing

Four milliliters of venous blood were collected from both RA patients and healthy control using a syringe method and transferred to an EDTA test tube following the appropriate blood collection procedure. The whole blood was centrifuged; plasma was separated and stored at -80^oC until analysis was done.

Determination of soluble program death-1 (sPD-1)

The Human PD-1 Duo Set® ELISA Development System (DY 1086) was used to measure the concentration of sPD-1 in plasma. The kit utilizes a sandwich ELISA method. First, capture antibodies were added to a microtiter plate and incubated overnight. The plate was then washed with phosphate buffer saline (PBS) with Tween, and a blocking agent (1% Bovine serum albumin BSA in PBS) was added and incubated for 1 hour. After another round of washing, the standard and sample were added and incubated for 2 hours. Detection antibodies were then added and incubated for another 2 hours. Streptavidin-horseradish peroxidase (HRP) was added and incubated for 20 minutes, followed by a washing step. Substrate solution (a mixture of hydrogen peroxide (H₂O₂) and tetramethylene benzidine (TMB)) was added and incubated for 20 minutes. Finally, stop solution sulphuric acid (H2SO4) was added and the optical density was measured at 450 nm with an ELISA reader. A standard curve was generated, and the concentration of sPD-1 in the sample was determined using the standard curve.

Data processing and analysis

Statistical analysis was performed by using GraphPad Prism statistical analysis software. Categorical descriptive data was presented by using frequencies and percentages, and numerical continuous data was presented as the mean ± standard error of the mean (SEM) for normally distributed data, and median and interquartile range (IQR) for non-normal distribution. Mann Whitney U test was used to analyze the sPD-1 levels median difference between RA patients and healthy control and to analyze the relationship of the categorical predictor variable with the outcome variable. Correlation analysis of continuous variables was performed using Spearman’s correlation coefficient analysis. Kruskal- Wallis Test was used to test the difference in the level of plasma sPD-1 on disease activity of RA patients and healthy control. In all tests, the level of significance was a two-sided p-value of less than 0.05.

Sociodemographic characteristics of study participant

The research involved 50 individuals diagnosed with rheumatoid arthritis, consisting of 11 males (22%) and 39 (78%) females. The participants had an average age of 48.08 ± 9.391 years, ranging from 30 to 66 years old. Additionally, 50 healthy individuals were included as a control group, with 17 males (34%) and 33 females (66%) who had an average age of 38.30 ± 13.386 years, ranging from 20 to 64 years old. Table 1 presents more detailed sociodemographic characteristics of both the RA patients and healthy control participants.

Clinical and laboratory characteristics Rheumatoid arthritis patients

Out of the 50 patients with rheumatoid arthritis, 16 individuals (equivalent to 32% of the sample) had a family history of the disease, while 40 (80%) tested positive for RF. 26 (52%) of RA patients have five and less than five years of disease duration. While 24 (48% ) are RA patients with more than 5 years of disease duration. DAS28 score of 3.33. The RA patients were categorized into two groups based on their DAS-ESR score. Based on this 11 (22%) RA patients were in remission while 39 (78%) were active RA patients Table 2 presents the clinical characteristics of the RA patients

Plasma sPD-1 level in RA patients and healthy control

The median sPD-1 plasma concentration in RA patients was (median; 1023 pg/mL, IQR; 850.5 - 1304, p<0.001, Figure 1), which is higher as compared to healthy controls (Median; 531.9 pg/mL, IQR, 364 - 809). In addition, the median sPD-1 plasma concentration in female RA patients; 1105 pg/mL IQR; 940.6 - 1323, p=0.0025, Figure 2) was higher than that in male RA patients (median; 810.3 pg/mL, IQR; 510.3 - 1098).

Comparison of plasma sPD-1 concentration of RA patients with rheumatoid factor

We measured the median difference in plasma sPD-1 concentrations between seropositive and seronegative RA patients. We found concentrations of sPD-1 to be (median; 1103 pg/mL, IQR; 922.2 - 2346) for RF-positive and (median; 759.9 pg/mL, IQR; 678.5 - 1146) for RF-negative RA patients. The results showed a statistically significant difference in sPD-1 concentrations between seropositive and seronegative RA patients (p = 0.0015, Figure 3).

Correlation analysis of plasma sPD1 levels with clinical parameters of RA

Based on the Spearman correlation coefficient analysis, it was observed that the levels of plasma sPD-1 exhibited a significant association with the DAS28score (r=0.7157, p<0.001, Figure 4A) as well as the number of swollen and tender joints (r=0.5533, p<0.001, Figure 4B). However, no significant correlation was found between plasma sPD-1 levels and disease duration (r =0.0420, p=0.8193, Figure 4C).

Comparison of plasma sPD-1 levels with different disease activities of RA patients

Median sPD-1 plasma concentrations in RA patients with different disease activities were (765.5 pg/ml; IQR; 734.6-818.9) for RA patients in remission and (1105; IQR; 956.4-1323) for RA patients with Active disease stage.

We used Kruskal-Walli’s test to analyze the difference in the median sPD-1 plasma concentration among the RA patients and healthy controls. As shown in Figure 5, we found that RA patients in active stage had significantly higher plasma sPD1 levels than RA patients in remission (p=0.0083). in addition as we see this with healthy control RA patients in active stage had significantly higher plasma sPD1 levels than healthy control p< 0.0001. Regardless, sPD1 levels were not statistically significant between RA patients in remission versus healthy control p = 0.7145.

Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease of unknown etiology. According to the 2010 ACR/EULAR, in addition to clinical diagnosis, the seropositive markers RF and ACPA, and the inflammatory markers ESR and CRP are used to diagnose RA(22). However, this biomarker has not shown a strong correlation with RA disease activity (23, 24). For this reason, the best descriptive biomarkers with strong correlations with clinical parameters of RA disease activity are critical for better disease assessment. In the present study, we determined the plasma levels of sPD-1 in RA patients to assess their correlation with disease activity score and used them as a biomarkers for RA disease progression.

In this study, we measured plasma soluble PD-1 levels in RA patients and healthy controls. Expression of membrane-bound and soluble co-stimulatory immune checkpoint molecules such as PD-1 and sPD-1 increases during persistent immune cell activation and inflammatory conditions such as RA (16, 25). In this regard, we found that plasma sPD-1 levels were significantly higher in RA patients than in controls. A Chinese study by Wan, Bing et al. provides consistent evidence. They found higher expression of PD-1 and sPD-1 in RA patients than in healthy controls (26). A study in Denmark by SR Greisen et al. also supports our finding, that the mean concentrations of sPD1 were increased in RA patients compared to healthy controls (20). In China and Egypt, Liu et al. (27) and W.A. Hassan et al. (12) respectively forwarded consistence evidence. One study has shown a decrease in sPD-1 in RA compared to healthy control (28). In addition, patients with Pemphigus vulgaris, (18) and immune thrombocytopenia, (29) have a reduced expression of sPD-1 as compared to healthy control. This depicted a diverse function of sPD-1 in autoimmune diseases.

In the present study, we also observed gender differences in the expression of soluble PD-1. Plasma sPD-1 levels were significantly higher in female RA patients than in male RA patients. This finding supports existing evidence for the role of sex hormones in the development of autoimmune diseases and its role in the expression of PD-1 on immune cells. Sex hormones are associated with various autoimmune diseases, such as SLE, RA, and multiple sclerosis. For example, in SLE, estrogen is an immunomodulate that allows autoreactive B cells to bypass tolerance mechanisms and proliferate, while treatments with estrogen receptor antagonists such as testosterone have been shown in certain SLE patients and mice models to reduce the disease from SLE (30). In addition, sex hormones play a role in PD-1 expression on various immune cells. Experiments showed that PD-1 expression in FoxP3 + cells was significantly reduced in estrogen receptor α knockout mice (27).

Rheumatoid arthritis is characterized by the production of autoantibodies such as ACPA and RF. In this study, we assessed the existence of an association between RF seropositivity and sPD-1 concentrations. We found that sPD-1 concentrations were statistically significantly higher in RF seropositive RA patients than in RF seronegative RA patients. This is supported by different researchers by showing an association between sPD-1 levels and autoantibody production in RA patients (12, 31). In addition, a study in China by Wan, Bing et al. showed that increased levels of soluble costimulatory molecules, especially sPD-1, were strongly positively correlated with rheumatoid factor (26). Furthermore, a study in Egypt found that patients with seropositive RA had significantly higher mean plasma concentrations of sPD-1 than patients with seronegative RA (12) suggesting the role of sPD-1 in RA disease progression. In addition, sPD-1 has a role in autoantibody production which is explained by the fact that follicular helper T cells in RA are known to express more PD-1, and these cells are important for B cell activation and the formation of antigen-specific plasma cells and memory B cells (32, 33). The production of autoantibodies is one of the key factors in RA disease progression. Thus, the presence of the regulatory molecule PD-1 on TFH suggests that these cells are an important regulatory point controlling the activation of autoantibody-producing memory B cells (34). However, soluble PD-1 is also upregulated in chronic diseases such as RA. Therefore, antagonism of soluble PD-1 may exacerbate autoantibody production.

In this study, we identified the correlation between sPD-1 levels and DAS28 score as an indicator of disease activity in RA patients. An increase in sPD-1 may help predict the severity of RA. A high DAS28 score indicates high disease activity and uncontrolled inflammation. In this regard, we found a statistically significant positive correlation between plasma sPD-1 levels and DAS28 scores. Our results are consistent with previous studies (12, 34). In addition, a study in Denmark found that sPD-1 levels before treatment were not significantly associated with clinical parameters such as DAS28, but 9 months after treatment initiation, sPD-1 was significantly associated with DAS28, as this finding showed the effect of treatment on the level of sPD-1 (20). In this study, sPD-1 concentration is significantly higher among RA patients with moderate disease activity and high disease activity as compared to RA patients with low disease activity and those who are in remission. Our study is consistent with a previous study done by Liu et al (31). This finding suggested the role of sPD-1 in the RA inflammatory process which is supported by the idea that sPD-1 enhances pro-inflammatory cytokine expression and aggravates joint pathology in RA (27). A characteristic feature of highly active RA patients is an increased secretion of inflammatory markers and the production of autoantibody.

In this study, plasma sPD-1 is significantly higher in RA patients than in healthy controls. In addition, plasma sPD-1 level is higher in female RA patients than in males. Plasma sPD-1 level is higher in RA patients with RF positive than RF negative. This considerable association of sPD-1 with autoantibody production gives us insight into its role in the pathogenesis of RA.

An increase in plasma sPD-1 concentration is statistically associated with clinical disease indicators of RA patients such as DAS28 score, and inflammatory marker ESR level. In our study, we revealed that plasma sPD-1 concentration is significantly higher in RA patients who have high DAS28 scores. This result indicates that sPD-1 is associated with the disease activity of RA.

ACPA; anti-citrullinated protein antibodies,

ACR; American college of rheumatology,

BSA; Bovine serum albumin,

CD; cluster of differentiation,

CIA; Collagen-induced arthritis,

CRP; C-reactive protein,

DAS28; Disease activity score using 28 joints count,

DMARDs ; disease-modifying anti-rheumatic drugs

EDTA; ethylene diamine tetra acetic acid,

ELISA; enzyme-linked immunosorbent assay,

ESR; erythrocytes sedimentation rate,

EULAR; European alliance of association for rheumatology,

HC; healthy control

IQR; inter quartile range

PBS; phosphate buffer saline,

PD-1; programmed death -1,

PD-L1; programmed death ligand-1,

RA; rheumatoid arthritis, RE; remission,

RF; rheumatoid factor

SLE; systemic lupus erythematosus,

sPD-1 Soluble form programmed death-1.

Ethical approval and consent to participate

The ethical review board (IRV) of the University of Gondar approved the protocol of this study. With a reference number Ref. No. SBMLS/2754. Written informed consent was obtained for the willingness of study participants to participate.

Competing interests

The authors declare that they have no competing interests

Funding

No specific fund for this study

Availability of data and materials

All required datasets from which the conclusions of the findings concluded are included in the manuscript

Authors' contributions

All authors involved in conceptualization, proposal development, project management, research and monitoring, data analysis, supervision, the data collection process, performed, document preparation, interpretation, writing, andmanuscript preparation. All authors read and approved the final manuscript.

Acknowledgments

I am extremely thankful to my advisors, Fitsumbrhan Tajebe, Tadelo Wondmagegn, and Marko Negash from the Department of Immunology and Molecular Biology, School of Biomedical and Laboratory Sciences, University of Gondar, for their lofty guidance, valuable suggestions, full encouragement, and enthusiasm. Very special thanks to my advisor Dr. Becky Abdissa, Department of Internal Medicine, School of Medicine, Addis Ababa University who helped me in the selection and diagnosis of patients.

I would like to thank Tikur Anbesa Specialized Hospital, Laboratory unit, and Rheumatology Clinic administrative and staff members for allowing me to conduct research. I would like to thank the study participants for their willingness to participate in this study.

Finally, and most importantly, I would like to thank Bule Hora University for funding this research, the Department of Immunology for my education, and the University of Gondar for sponsoring my graduate studies.

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Table 1: Sociodemographic characteristics of study participants at Tikur Anbesa Specialized Referral hospital, from February 2021 to June 2021

Characteristics		Healthy control (n=50) N (%)	RA cases (n=50) N (%)
Sex	Female	33 (66%)	39 (78%)
Sex	Male	17 (34%)	11 (22%)
Residence area	Urban	43(86%)	39 (78%)
Residence area	Rural	7(14%)	11(22%)
Religion	Orthodox	40(80%)	39 (78%)
	Muslim	5 (10%)	7(14%)
	Protestant	5(10%)	4(8%)
Marital status	Single	9 18%)	4 (8%)
	Married	41(18%)	45 (90%)
	Divorced	--	1 (2%)
Educational status	Illiterate	4 (8%)	3 (6%)
	Complete primary school	14(28%)	25 (50%)
	Complete secondary school	10(20%)	13(26%)
	Complete college and above	22(44%)	9 (18%)
Types of occupation	Student	2(10%)	1 (2%)
	Housewife	8 (4%)	27 (54%)
	Government worker	19 (38%)	14 (28%)
	Private worker	11(22%)	5 (10%)
	Farmer	1(2%)	1 (2%)
	Merchant	2 (4%)	2 (4%)

N= number, %= percent

Table 2: Clinical, laboratory characteristics and disease activity of rheumatoid arthritis patients

Clinical and laboratory parameter		RA patients (n=50)
RF positivity	RF Positive	40 (80%)
RF positivity	RF Negative	10(20%)
Family history of the disease	Yes	16(32%)
Family history of the disease	No	34(68%)
Disease stage	Active RA patients	39 (78%)
Disease stage	On remission	11 (22%)
ESR (mm/hr.); median; range		48.14(9 – 180)
Disease duration	≤ 5years	26 (52%)
Disease duration	More than five year	24 (48%)
DAS28-ESR score (median; range)		3.45 (1.61 – 5.52)

DAS 28 = Disease activity score using 28 joint count, ESR = Erythrocyte sedimentation rate, RA = Rheumatoid arthritis, RF = Rheumatoid factor

No competing interests reported.

Upregulation of circulating soluble programmed death-1 molecule and its correlation with the disease activity of rheumatoid arthritis patients in Ethiopia

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Abstract

Figures

Background

Methods