Immune Aging Marker Associated with Periodontitis in
Systemic Lupus Erythematosus Patients
Nanda Rachmad Putra Gofur1, Nurdiana2, Kusworini Handono3, Handono Kalim4
1Dept of Biomedical Science, 2Dept of Pharmacology, 3Dept of Clinical Pathology, 4Dept of Internal Medicine Faculty of Medicine – Universitas Brawijaya, Malang Indonesia
Abstract:-
Background
Periodontitis was reported more often found in SLE patients than healthy controls, 54.3% higher than healthy 28.2%, and was estimated related to autoimmune condition. Recently, SLE associated with the immune aging, SLE patients had similarities with immune system of elderly. Expression of IL-2 and IL10 associated with immune aging as T cell, were associated with cytotoxic activity, signaling and low number of naïve T cells were known as biomarker of immune aging.
Objectives
To analyze correlation between periodontitis severity with disease activity, IL-2, IL-10expression in SLE patients.
Methods
Subjects were 61 patients with SLE ( age 18-55 years; SLEDAI score 0-42) collected from Dr. Saiful Anwar General Hospital, Malang Indonesia. Periodontitis severity was measured using Periodontal Index (PI) criteria. Expression of IL-2 and IL-10 using ELISA .
Result
Clinical manifestations of periodontitis were bleeding gum 88.3%, high calculus index 44.9%, found periodontal pocket 73.8% and loose teeth 13.2% among patients. PI score patients was 2.45 ± 0.82. There were significantly positive correlation between PI score and SLEDAI score (r:0.930; p = 0.000), with IL-2 (r: -0927.; p = <0.0001), with IL-10 (r: 0.886; p = <0.0001).
Conclusion
Our study showed that periodontitis were associated with SLE disease activity, and biomarker of immune aging. Furthermore, biomarker could be predictor for periodontal condition, prognosis of periodontitis and best treatment for periodontitis on SLE patient.
Keywords :- SLE, Periodontitis, IL-2, IL-10.
I. INTRODUCTION
Periodontal disease including gingivitis and periodontitis is one of the most common chronic diseases. Worldwide, periodontitis disease suggest approximately 22.9% of the adult population, and in Indonesia a prevalence of 38%1,2. Recently, a prevalence of periodontitis is increasing due to age and systemic disease. Periodontitis is characterized by chronic inflammation and infection of periodontal tissues and causing bone resorption. Mostly, the further stage is tooth loss. Due to tooth loss condition can lead to decreasing intake and nutrients to the body, resulting in higher mortality3.
Main causes of periodontitis is gram-negative bacteria, mostly found on dental plaque. Bacteria could have potent mechanisms to attack and damage human defenses. It produces bacteria strain making PMN and macrophages damaged. As example, collagenases could damage collagen tissue directly4. Moreover, the abnormalities of human immune response may also contribute tissue damaged and severity of the disease5.
Recent studies reported an correlation between periodontitis with Systemic Lupus Erythematosus (SLE) disease. In SLE patients, a prevalence of periodontitis was reported 54.3% higher than healthy patients with no systemic abnormality of 28.2%6. This condition affect stability of oral cavity. But, the association between SLE and periodontitis never been explained clearly. The connection of SLE with periodontitis is assuming that in SLE patients had similarities of immune response with elderly, associated with immune aging7.
Immune response changing in SLE coming from interaction between genetic and environmental factors. It results in hyperactivity of immune cells, including T and B lymphocytes, and production of autoantibodies such as anti-dsDNA antibodies. Correlation of autoantibodies with target antigens results in formation of immune complexes deposited in various places and triggers tissue through organ damaged. Impact of immune responses on SLE patients, triggered immune activation and lead to various diseases, due to immune aging8.
cross-reactions or autoantigens new reactivity lead to tissue damage. It is believed that SLE immune response of hyperactivity, triggering immune aging and causing secretion of inflammatory cytokines, mostly IL-2 and IL-10 which leads to increased infection and destroy periodontal tissue8. Furthermore, no study prove that if there is a correlation between the severity of periodontitis and SLE biomarkers. This study was comparing the periodontal findings in SLE patients and systemically healthy controls, and to determine if there is a correlation between periodontal condition and SLE biomarkers.
II. AIM OF THE STUDY
Determine the association of periodontitis in SLE patients with immune aging.
Knowing correlation between SLEDAI, IL-2, and IL-10 with the severity of periodontitis.
Provide new biomarker both in blood serum for SLE and Periodontitis diagnostic.
III. MATERIAL AND METHOD
The design of this study was an observational analytic study with cross sectional approach. The research received an ethical approval from the UB Medical Ethics Committee from Faculty of Medical, Brawijaya University Malang, East Java. All patients included in this study were required to sign an informed consent.
The study was conducted on 61 SLE patients and 61 healthy subject. Study held from September 2017 until June 2018 on Rheumatology Department Saiful Anwar Hospital Malang, Indonesia. In all SLE patients clinical examination of the oral cavity to assess the presence of periodontal abnormalities using periodontal index (PI), gingival index (GI), plaque index, pocket depth and numbers of loose teeth. Clinical examination and laboratory tests are conducted to assess the activity of the disease. Severity of SLE measured using SLEDAI criteria and biomarker using elisa to measured IL-2, and IL-10 expression. Inclusion criteria was female subjects with a confirmed diagnoses of SLE, willing to become the subject of study, could read and write and had full consciousness. Exclusion criteria were smoking, pregnancy, diabetes, and another systemic disease. For the healthy subject had similar inclusion, and exclusion criteria.
Severity of Periodontitis Assessment and SLE Patients
Periodontal assessments were collected from all subjects. Periodontal assessments consisted of the following: periodontal index (PI), gingival index (GI), plaque index, pocket depth and numbers of loose teeth. Severity of SLE using SLEDAI index with clinical examination and laboratory test.
Biomarker of SLE Using ELISA
Sample 10-15 mL subject vein blood was performed at Poly Rheumatic / Internal Diseases RSSA. We used serum for cytokine examination9. Measurement of Cytokine Levels of IL-2, and IL-10. Samples in EDTA were centrifuged for 10 min at 1,000 x g. plasma at <-200C and then measured the levels of IL-2 using enzyme-linked immunosorbent assay Human IL-2 ELISA (Human IL-2 ELISA MAXTM Biolegend Catalog No. 431803). Added 100 uLnologiHuman IL-2 Capture Antibody that has been diluted, and incubated for 1 day at a temperature of 2-8 ° C. Then add assay buffer at room temperature (18-25 º C) for 1 hour, and washed 4 times. Microplate received with calibrator and plasma. After as much as 60 μl then added buffer 11.94 μl, incubation 2 hours while in shaker at room temperature (18-25 º C). Washed 4 times with a wash and add a 100 μl IL-2 human antibody detection solution each strip after it was incubated for 1 hour at room temperature (18-25ºC). 4 days ago added 100 ml of Avidin-HRP solution, incubated at room temperature for 30 minutes. washing 5 times with washing buffer. Added TM 100 μl substrate, Incubation 15 min at room temperature outside dark without dishaker and plus stop solution 100 μl, spectrophotometric print result at 450 nm wave. Doing same step but different using 100 uL Lemism IL-10 Capture Antibody diluted using Elisa Max ™ Deluxe Set (Biolegend Catalog no.430106) for IL-10 in Biomedical laboratory Faculty of Medicine Brawijaya University Malang.
Data Processing and Analysis
The collected data will be analyzed using of SPSS version 20 program. The difference of Immune aging markers on LES patients with and without periodontitis was analyzed by Kolmogorov Smirnof for normality test, Spearman/Pearson for correlation test and Mann Whitney for comparison test11.
IV. RESULTS
Characteristics of Research Subjects
A total 122 subjects (61 with SLE and 61 control) were included in this study. We found that 54/61 (88,53%) subjects with SLE had periodontal disease, based on Periodontal Index assessed. As shown in table 1, the mean age for SLE subject was 29. The mean score of Periodontal index, gingival index, plaque index, a large number of teeth periodontal pockets and loose teeth was 22,66±1,2, 1,85±1,02, 0,75±0,59 mm, and 0,26±0,65, respectively. The mean SLEDAI score was 17.70 ± 12.70.
Differences in the Occurrence of Periodontitis in SLE Subjects and Control
Dental status examination was performed in both groups, and it was obtained that in SLE group, periodontal disorders tend to shown more severe and advanced clinical manifestation compared to those in control group. The clinical manifestations assessed were shown in table 3. There was a significant difference in periodontal index, gingival index, plaque index and periodontal pocket between two groups. SLE subject had higher periodontal index, gingival index, and periodontal pocket but lower plaque index than control. There is no significant different the number of loose teeth between two groups.
Periodontitis Severity and SLE Characteristics
When the periodontitis severity status divided into two groups based on Periodontal Index, SLE subject with severe periodontitis tend to had higher SLEDAI score, anti-dsDNA level, and inflammatory cytokines level compared to mild periodontitis group (table 4 and table 5). There was a significant difference in several SLE characteristics between two groups.
Correlation between Periodontitis Status and SLE
Manifestations Severity
The correlation between periodontitis status (based on Periodontal Index) with SLE manifestation severity were assessed using Pearson correlation test and the results were shown in table 5. It can be seen that there was a significant and strong correlation between the periodontitis status and severity of SLE manifestations in all five characteristics of SLE (figure 1).
V. DISCUSSION
Periodontitis is chronic inflammation disease on periodontal tissue. Periodontitis began from complex interactions between host and bacteria causing destruction of the gingival tissue, ligament periodontal, cementum and alveolar bone. Recently, periodontitis associated several systemic disease. There has been an increasing interest in the relationship between periodontitis and autoimmune disease, Systemic Lupus Erythematosus (SLE). SLE patients have abnormalities of immune response called immune aging. SLE patients have similarities systemic condition with elderly. It caused increasing prevalence and severity of periodontitis12. Increasing occurring of periodontitis also proven in vivo study with non-human primates and rodentsa13.
These abnormalities have been described for adaptive immune B and T cells. Immune aging effect on periodontal tissues have been suggested based on molecular changes the cells array and inflammation condition of the periodontium. It affected differentiation and bone process (osteoblasts, osteoclasts), changing microbial condition, environment and systemic condition related to host because of cytokines14,15.
The regulation of the expression of enzymes and proteins could affect on periodontal inflammatory response, such as stimulating, migration and stoppaging of immune cells, exacerbation and resolution. This response is functioning by complement, cytokines and other biomarker molecules. Cytokine proteins may have important roles during different human physiological and pathological processes. For example, much Interleukin concentration changes in GCF, suggest these cytokines as a predictable marker of gingival inflammation in chronic periodontitis patients. It results proven from studies with periodontal disease is related to other general disease such cardiovascolar and autoimmune. There are others fact that an association between periodontitis and atherosclerotic vascular disease, including stroke, myocardial infarction, peripheral vascular disease, abdominal aortic aneurysm, coronary heart disease, cardiovascular death and in our case systemic lupus erythematosus16.
There are many cytokine that playing role on systemic lupus erythematosus. TCD4+ cells were initially subdivided into two subsets, designated Th1 and Th2, based on their cytokine production patterns. Th1 cells secrete IL-2 and IFN-γ, whereas Th2 cells produce 5, 6, 4, 10 and IL-13. Th1 is an important standard in the response against intracellular microorganisms and is responsible for inducing cell-mediated inflammation4,17. Cytockine that have an important role on periodontitis is IL-2, and IL-10 among others.
Recent study, IL-2 was decreasing related to pocket, which is related to advanced periodontitis, resulting in a greater severity of the disease. Interleukine-2 is a multifunctional cytokine, considered a central regulator of host resistance against a variety of pathogens and has been recently demonstrated an active role in the pathogenesis of periodontal diseases. P. gingivalis can influence responses of T cell lineages to evade or suppress their adaptive responses. This is achieved by inhibiting the expression and accumulation of IL-2, which attenuates T cell proliferation and communication. IL-2 is affected by P. gingivalis at the protein level and partially through suppression of activator protein 1 (AP-1). AP-1 is a transcription factor.T cells were not able to maintain a stable IL-2 accumulation. On other hand decreasing of IL2 could be affecting P.gingivalis at the protein level and partially through suppression of AP-1 protein and inducing bone resorption18.
periodontitis. Lower levels of IL-2 indicated higher incidence of periodontitis and bad prognosis.
The result with our study proven that systemic IL-10 had positive correlation with severity of periodontitis. Higher levels of IL-10 indicated increasing of periodontitis and poor prognosis. Literature suggest that there is a positive correlation with anti-inflammatory cytokines IL-10, discovered on periodontal disease. Interleukin-10 is known controlling and production many mediators. Myeloid cells are key target cells of IL-10 in infection stimulated alveolar bone loss. Significant of CD40L T cell responses in infection stimulated alveolar bone destruction. IL-10 is essential as a modulator of the response to infection at the Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling axis of host responses. IL-10 was first identified by its ability to incresing the immune response by inhibiting the production of a number of cytokines resulting mortality. IL-10 has been recognized to have potent and broad-spectrum inflammatory activity, which has been proven in various models of infection, inflammation, and even in cancer20.
IL10 had sometimes function to the pro-inflammatory ones, as higher levels of these mediators were associated with a increase probability of having periodontitis. Among other cytokines, interleukin-10 (IL-10) is an important multifunctional cytokine. It inhibit the synthesis of pro-inflammatory cytokines such as interleukin 1 (IL 1), interleukin 2 (IL 2), interleukin 6 (IL 6), interleukin 8 (IL 8), tumor necrosis factor – α (TNF α) and interferon γ (IFNγ). IL-10 increasing the production of metalloproteinases, and incduce synthesis of tissue metalloproteinases in macrophages. Moreover, it stimulates production of osteoprotegrin, which consequently causing bone resorption by bond RANK-RANKL engagement. IL 10 can be a protective cytokine in periodontal disease but in SLE parient seems as pro- inflammatory cytokines, including those implicated in alveolar bone loss. Individuals who are high producers of IL 10 might be more frequently got chronic periodontitis due to proinflammatory role of IL 1020,21,22.
VI. CONCLUSION AND SUGGESTION
Chronic periodontitis has correlation with severity clinical manifestation of SLE (using SLEDAI. SLE patients with periodontitis show that greater frequency immune aging biomarker than SLE patients without chronic periodontitis. Immune aging biomarker and chronic periodontitis could be predictor for severity of SLE. This result could explain association SLE with cormobidities collateral to aging process. Furthermore, the results could be used as information by doctor to determine patient treatment and education plans. Hopefully there will be further research with markers in the oral cavity.
VII. CONFLICT OF INTERESTS
The authors declare that we have no competing and conflict of interests.
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Variables Mean (± SD) Range (Min-Max)
Age (yrs) 29.47±9.62 34 (17-51)
Periodontal Index 2,66±1.20 4.20 (0.1 to 4.9)
Gingival Index 1.95±1.02 3.00 (0 to 3.00)
Plaque Index 0.34±0.44 1,5 (0 to 2.5)
Periodontal pockets (mm) 0.72±0.62 2,5 (0 to 2.5)
Loose teeth 0.26±0.65 3 (0-3)
SLEDAI score 17.70±12.70 42 (0-42)
[image:5.612.39.584.362.559.2]
Table 1:- SLE patient characteristics
Variables Mean (± SD) Range (Min-Max)
IL-2 (pg / ml) 30,56±17,91 66,70 (10,3- 77,00)
IL-10 (pg / ml) 1,13±0,99 3,90(0,10 – 3,80)
[image:5.612.31.582.596.696.2]
Variables SLE (n = 61) Control (n = 61) p-value
[image:6.612.41.580.76.305.2]Age (yrs) 29,50±9,57 28,57±9,33 0,540
Table 3:- Comparisons of periodontitis clinical manifestations between SLE and controlgroups
Variables Mild (PI 0.7-1.9) Severe (PI 2.0-5.0) p-value
(n = 11) (n = 43)
SLEDAI score 5,09±1,86 23,48±10,42 <0,0001
IL-2 (pg / ml) 41,68±12,53 21,25±5,63 <0,0001
IL-10 (pg / ml) 0,25±0,52 1,52±0,92 <0,0001
[image:6.612.38.596.343.494.2]
Table 4:- Comparisons of SLE characteristics between periodontitis group
Variables Normal Mild (PI 0.7- Severe (PI 2.0- Terminal p-value
Periodontium (PI 1.9) 3.8) (PI 3.9-
0-0.6) n= 7 n = 11 n = 32 8.0) n =
14
SLEDAI 4.57 ± 4.11 6.36 ± 3.44 19.55 ± 12.33 29.35 ± <0.0001
score 6.67
[image:6.612.35.589.531.690.2]
Table 5:- Comparison between All Periodontitis Status and SLE Severity
Periodontal Index 2,66±1,02 0,51±0,81 <0,0001
Gingival index 1,95±1,02 0,83±0,65 <0,0001
Plaque Index 0,34±0,44 0,90±0,62 <0,0001
Periodontal pockets (mm) 0,75±0,59 0,34±0,55 <0,0001
Loose teeth 1,49±1,77 0,14±0,51 <0,0001
Variables p-value r
(Sig, 2 tailed)
SLEDAI score <0.0001 0,948
IL-2 (pg / ml) <0.0001 -0,930
IL-10 (pg / ml) <0.0001 0,886
[image:7.612.28.588.72.194.2]
Table 6:- Correlation between Periodontitis Status and SLE Manifestation Severity
Fig 1:- Correlation between periodontal index score and SLE characteristics. A) Periodontal index and SLEDAI score showed significant (p<0.0001) and strong positive correlation (r=0.948); B) Periodontal index and IL-2 level showed significant (p<0.0001)
[image:7.612.40.575.229.423.2]