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POSSIBLE ETIOLOGICAL ROLE OF IL-10, IL-17 LEVELS, CD4+ AND CD68 IN THE PATHOGENESIS OF HERNIATED DISC IN IRAQI PATIENTS
D.M. Al-Muathen1, S.A.H. Al-Sharqil, S.A. Brakhas2, A.A. Taha3
1 Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq;
2 Department of Immunology, Allergy Specialized Center, Ministry of Health, Baghdad, Iraq;
3 Ghazy Al-Hariri Hospital for surgical specialties, Medical City Complex, Baghdad, Iraq.
* Corresponding author: dhilal.almuthen@gmail.com
Abstract. Herniated (prolapsed) discs HD is a multifactor disease that afflicts around 9% of the global population and occurs when the nucleus pulposus NP bulges and pushes on the ruptured annulus fibrosus AF, releasing substances that may irritate the surrounding nerves and cause inflammation that leads to extensive histopathological and immunological changes. The aim of this study is to demonstrate the etiological role of IL-10, IL-17 levels, CD4+, and CD68 in the pathogenesis of herniated disc in Iraqi patients. The immunohistochemical analysis was performed on 30 excised HD specimens taken during endoscopic discectomy, and stored in the fixative solution (formalin 10%). Cytokines (IL-10 and IL-17) were measured by using their Enzyme-Linked Immunosorbent Assay ELISA kits, in sera of 40 patients who were diagnosed with HDs as a case group, in addition to 20 healthy as a control group. The results revealed that the CD4+ expression was negative in 70% of the HD samples, while only 8 HD samples were negative for CD68 expression. On the other hand, results of ELISA analysis recorded high levels of IL-10 with low levels of IL-17 in HD patients sera compared with control sera. According to the above it can concluded that immunohistochemical analysis of HD tissues showed infiltration of CD4+ and CD68. Also compared to controls high levels of IL-10 in HD samples combined with low levels of IL-17.
Keywords: herniated disc, cytokines, immunohistochemical analysis, CD4+, CD68.
List of Abbreviations
AF - Annulus fibrosus AIC - Anti-inflammatory cytokines CD - Cluster of differentiation ELISA - Enzyme linked immunosorbent assay
HD - Herniated Disc IDD - Intervertebral disc degeneration IFN-y - Interferon-gamma IL - Interleukin IVD - Intervertebral Disc NKs - Natural killer cells NP - Nucleus pulposus PIC - Pro-inflammatory cytokines SD - Standard deviation SPSS - Statistical package for the social sciences
T h cell - T helper cell
TNF-alfa - Tumor necrosis factor-alfa
Introduction
The inflammatory reaction is triggered by Herniated disc HD when the annulus fibrosus
AF of the intervertebral disc IVD ruptures, causing the nucleus polposus NP to herniate and breaking down the physical barrier between the (IVD) and the immune system (Rodrigues et al., 2016). The first immune reaction (primary response) is triggered when the body's immune system identifies the «immune-privileged» NP as a «foreign antigen». Next, granulation tissue forms as part of the healing process for injured (NP) and (AF), and blood vessels infiltrate the area, exposing the NP tissues to more immune cells (Sollmann et al., 2019). The invasion of immune cells including neutrophils, macrophages, and CD lymphocytes is facilitated by mediators released by satellite glial cells and macrophagein the dorsal root ganglion, such as histamine, prostaglandins, cytokines, and chemokines (Virri et al., 2001). Activation of T cells is a telltale indicator of a cellmediated autoimmune response, by transforming into two distinct phenotypes - Th1 and Th2 - in response to an antigen, they can secrete the cytokines required for the differentiation
of B-cells into antibody-producing plasma cells (Geiss et al., 2009). However, CD68 macrophages are present in the NP of all people with morphologic evidence of intervertebral disc degeneration IDD, despite their absence in human disc tissues of fetuses, infants, and adolescents, both the extent of the hernia and the amount of cell death inside the tissue were correlated with the presence of CD68 macrophages. It has been suggested that in HD, only a certain number of macrophages would be recruited and activated per region of hernia to maintain tissue homeo-stasis (Cunha et al., 2018). Pro-inflammatory cytokines PIC, such as interferon-gamma IFN-Y and IL-12 are secreted by Th1 cells to organize the inflammatory center and boost cellular immunity. Anti-inflammatory cytokines AIC, such as IL-6 and IL-10, are secreted by Th2 cells, which are responsible for the humoral immune response. The immune response to HD tissue is regulated by these two classes of cytokines (Yao et al., 2017). Clinically, there are some local studies related to ILs related to different tpics of medicine such as IL-38 that was appeared new player in Graves' ophthalmopathy (Jawad et al., 2023), while, IL-10 was consider as biomarker in patients of rheumatoid arthritis (Al-Jumaily et al., 2023). However, IL-10 prevents the catabolic effects of PIC by decreasing matrix-degrading enzymes and matrix metalloproteinases. On the other hand, numerous studies have shown that IL-17A has a direct effect on NP cells in vitro by adding IL-17A to NP cell cultures (Liu et al., 2019), the IDD and immune-induced inflammation in HD have both been linked to Th17 lymphocytes, a novel subtype of T-cells that mediate IL-17 production (He et al., 2020). Therefor, the aim of this study is to display the etiological roles of IL-10, IL-17 levels, CD4+, and CD68 via their im-munohistochemical estimation in the pathogenesis of herniated disc in Iraqi patients.
Materials and Methods
Thirty HD tissue samples were taken during endoscopic discectomy procedures performed at Ghazy Al-Hariri Hospital for surgical specialties / Medical City complex and Al-Zaytoon Hospital for general surgery (Private), from
February 2022 to December 2022, and im-munohistochemical analysis was performed on the tissue samples while they were stored in the fixative solution (formalin 10%), at the Iraqi specialized laboratory (private). Paraffin-embedded tissue blocks were cut at 5p,m thick section. And all sections were deparaffinized in xylene, then decreasing grades of ethanol and incubated with phosphate-buffered saline. Antigen retrieval as required by the primary antibody. The steps of the staining protocol with monoclonal antibodies toward CD4+ lymphocytes and CD68 macrophages were followed from (Genemed, Biotechnologies, Inc.). According to Kawaguchi et al., (2000), the positive staining intensity of CD4+ was given the grade of: Absent (-), A few cells (+/-), Common (+) and Abundant (++). The intensity of positive staining with the CD68 was given according to Ford et al. (2020) and was graded as: No cells (0), a few cells (1), moderate cells (2), and abundant cells (3). Furthermore, fourty Patients with (HD) and twenty other healthy controls each had 5ml of venous blood drawn. Before being kept at -20°C for further analysis, the blood samples were centrifuged at 3000 r.p.m. for 5 minutes at room temperature to separate the sera. The Allergy Specialized Center/Al-ris-safa served as the site for the immunological investigation and the cytokines IL-10 and IL-17 were measured by using their (ELISA) kits in the serum.
Ethical approval
All participants agreed to provide the investigator with the specimens. The ethics committee of the College of Science, Mustansiriyah University approved this work. Informed consent according to the Declaration of Helsinki was obtained from all participants.
Statistical analysis
SPSS 26 for Windows, Excel data visualization. Continuous variables were represented as means with SD where applicable. The frequencies of categorical variables were represented as percentages. Box plots were used to determine whether numerical data followed a normal distribution and to highlight extreme outliers for
further study. We compared normally distributed data with equal variance using a two-sample t-test. The statistical significance criterion was set at P < 0.05.
Results
To confirm that, the HD tissues were analyzed utilizing Immunohistochemical methods for CD4+, the staining intensity of CD4+ lymphocyte expression was negative in 21 samples (70%) which had scored (-), while only 9 samples (30%) showed a few cells expression that scored (+/-). The intensity of CD68 macrophages expression was negative in eight biopsies (26.67%) which had scored (0), while, thirteen biopsies (43.33%) showed
a few cells that scored (1). Meanwhile,five biopsies (16.67%) showed moderate cells expression that scored (2), and finally four biopsies (13.33%) showed abundant cells expression that scored (3). All these result were shown in Table 1, Figures 1, 2, 3. The results in this study showed that the HD Patients group recorded a high level of IL-10 according to the mean values estimator compared with the control group, while the control group recorded a high level of IL-17 according to the mean values estimator compared with the HD Patients group which indicates that there was a significant difference in the levels of the readings of each marker compared between the two groups (Table 2).
Table 1
Intensity of CD4+ T lymphocyte and CD68 macrophage expression in HD tissue
Grade / (Intensity of CD4+ T lymphocyte) No. Cases of HD No. Cases of HD (%)
-= Absent 21 70
+/- = A few cells 9 30
+ = Common 0 0
++ = Abundant 0 0
Total 30 100
Grade / (Intensity of CD68 macrophages) No. Cases of HD No. Cases of HD (%)
0 = No cells 8 26.67
1 = A few cells 13 43.33
2 = Moderate cells 5 16.67
3 = Abundant cells 4 13.33
Total 30 100
Note: Results are expressed as percentage
Table 2
The levels of Interleukin-10 and Interleukin-17 in sera of the herniated disc groups and control groups
Interleukins Groups No. Mean ± SD P value
IL-10 Control 20 74.20 ± 17.25 0.000*
Patients 40 116.9 ± 32.25 0.000*
IL-17 Control 20 594.2 ± 254.5 0.000*
Patients 40 268.7 ± 67.06 0.000*
* significant at the P<0.05 level (2-tailed).
Fig. 1. Immunohistochemical staining method detection of CD4+ T lymphocyte using in HD tissue showing: (A) + (few) expression (X10), (B) - (absent) expression (X10)
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Fig. 2. Immunohistochemical staining method detection of CD68 macrophages using HD tissue showing 0 (No cells) expression ([A and B] X40)
Fig. 3. Immunohistochemical staining method detection of CD68 macrophages using in HD tissue showing: (A) A few cells expression (B) Moderate cells expression (C) Abundant cells expression (A, B and C X40)
Discussion
In the present study, the immunohistochemical analysis for CD4+ revealed that the staining intensity of CD4+ lymphocyte expression was negative in 21 samples (70%) which had
scored (-), while only 9 samples (30%) showed a few cells expression that scored (+/-). Histological examinations of human HD have previously shown that T lymphocytes penetrate the tissue. Kawaguchi et al. (2000) examined
38 lumbar HD specimens and found a high percentage (42%) of CD68 cells (monocytes, macrophages, and dendritic cells) infiltrating the tissue, but the percentage of T cells was much lower. Similarly, in a different research while macrophages were more prevalent (37% of cases) in a sample of 205 cases, numerous T-cells and activated T-cells were found in the same percentage of cases (17%) (Park et al, 2002). Even fewer CD4+ lymphocytes were seen in HD tissue samples in recent findings, it will be shown later in this presentation that this pattern of cell expression agrees with the decreased presence of the lymphocyte products IL-17 in these tissues. Alternatively, the intensity of CD68 macrophages expression was negative in eight biopsies (26.67%) which had scored (0), while, thirteen biopsies (43.33%) showed a few cells that scored (1). Meanwhile, five biopsies (16.67%) showed moderate cells expression that scored (2), and finally four biopsies (13.33%) showed abundant cells expression that scored (3). Iqbal et al. (2014) noted that CD68 was a useful cytochemical marker for immunostaining monocytes/macrophages in the histochemical investigation of inflammatory and malignant tissues. An increased risk of neovascularization and, perhaps, macrophage infiltration was associated with extruding HDs as opposed to protruding ones (Djuric et al, 2020). Exposure of the disc tissue to the host's immune system causes macrophage infiltration due to the leuko-attractant nature of IL-6 (Jas-sim et al, 2019), and tumor necrosis factor TNF-alfa, or as a result of an autoimmune response. In a study including 10 lumbar HD patients, Shamji et al. (2010) found that the proportion of CD68 macrophages was considerably greater in the NP of lumbar HD compared with healthy IVD, and the current results corroborated these findings. The presence of a specific cellular population (CD68 macrophages). Also the present results indicated that the HD Patients group recorded a high level of IL-10 according to the mean values estimator compared with the control group, while the control group recorded a high level of IL-17 according to the mean values estimator compared with the HD Patients group. In addition to that results
showed disjoint interval limits accounted for IL-10 and IL-17 in contrast to the studied groups, which indicates that there was a significant difference in the levels of the readings of each marker compared between the two groups. The impact of an autoimmune response to an IVD was still up for debate. It has been shown in the majority of research (Cunha et al., 2018) that the inflammatory response may be used as a good predictive indication of spontaneous regression. Epstein et al. (2010) found that IL-10 regulates the activity and development of a wide variety of immune cells, including B cells, NKs cells, granulocytes, and certain related cells. Attenuation of PICs, antigen presentation and aberrant immune response, and augmentation of immunological tolerance were only some of the many tasks of immune cells that IL-10 regulates through its transmembrane receptor complex (Song et al., 2013). The findings of the present research were consistent with those of the majority of prior studies that have examined IL-10 levels. The expression of IL-10 and IL-17 was shown to be higher in the peripheral blood sera of IDD patients by Wang et al, (2018). It was also quantified the work of Mon-tesano and Cuellar, (2017), discover that IVDs from patients with degenerative disc disease having surgical therapy with anterior/posterior lumbar fusion had a considerably higher concentration of IL-10 than discs from non-painful controls, utilizing a multiplex panel that measures 27 different cytokines. Small but statistically significant increases in this cytokine were also found in 182 HD patients by Park et al, (2002). In contrast to our current findings, Banimostafavi et al, (2021) found that blood levels of IL-10 appear to be lower in lumbar HD patients than in controls in a case-control research in which 87 patients, including 59 patients with low back pain and 28 healthy participants, were investigated following magnetic resonance imaging MRI approval. In the present results, with a higher number of macrophages identified in HD tissue samples, the pattern of cell expression matched expectations for a higher concentration of IL-10 produced by macrophages. As an additional AIC, IL-10 reduces the production of PIC, decreases the ex-
pression of adhesion molecules, and raises the concentration of certain cytokine inhibitors (Shnayder et al, 2022). This was consistent with the present findings, which showed that HD patients' sera contained much more IL-10 than those of controls. It was found no other studies examining IL-17 concentrations in HD tissue sera that were comparable to our own. High levels of this interleukin have been linked to lumbar HD in earlier researches, and IL-17 has been identified as a key player in IVD pathogenesis (Cheng et al., 2013). Serum inflammatory cytokines levels were unbalanced in lumbar HD patients (Haddadi et al, 2022). In addition, Suyama et al, (2022) found that individuals with low back pain had increased expression of IL-17 in their IDD and lumbar HD, indicating the presence of local immunological responses. Clinical sample analysis from many labs supports this, although the authors noted that the mechanisms by which IL-17 was produced in disease and the connections it has with other inflammatory responses remain poorly understood. When samples from lumbar HD patients were studied by Shamji et al. (2010), it was shown that the NP of lumbar HD had a much larger proportion of CD4+ cells and their products, such as IL-17, compared with healthy
IVD. Low IL-17 levels in the current study corroborated previous results that CD4+ cells were under-represented in HD tissues. Moreover previous studies on the role of PICs and AICs have yielded conflicting results, lending credence to the hypothesis that cytokine imbalance may be an important biomarker for IDD rather than the serum level of one or more cytokines (Shnayder et al, 2023).
Conclusions
When examining HD tissues through im-munohistochemical analysis, infiltration of CD4+ and CD68 was observed. The HD patient's sera exhibited elevated levels of IL-10 and lower levels of IL-17 compared to control samples.
Acknowledgment
The authors would like to introduce their thanks and gratefulness to Al-Mustansiriyah University (https://www.uomustansiriyah.edu.iq/) for the support and help to achieve this article. And I should express my gratitude to Dr. Khalid Muhssin Murad (MSc. Orthopedic Surgery, Al-Zaytoon Hospital, chief of the Iraqi Pain Society), for his assistance and for facilitating my clinical part of the study.
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