Background: Chronic Fatigue Syndrome, also known as Myalgic Encephalomyelitis (CFS/ME), is a debilitating condition that presents with a range of symptoms, including fatigue, cognitive dysfunction, muscular and joint pain, and may be immune-mediated. In particular, patients exhibit abnormal cytokine expression. Similarly, in Multiple Sclerosis (MS), patients display neuroimmunological symptoms, and abnormal cytokine expression, with some overlap in symptomology with CFS/ME. The purpose of this study was to compare Th1, Th2, Th17 cytokines, inflammatory cytokines and chemokines, in healthy controls, CFS/ME and MS patients. Methods: Serum samples were collected from healthy controls (n = 16, mean age = 50 ± 11.85 years), CFS/ME patients (n = 16, mean age = 49.88 ± 9.54 years) and MS patients (n = 11, mean age = 52.75 ± 12.81 years). The concentrations of 27 cytokines (IFN- γ, TNF- α, IL-12, IL-2, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-5, IL-17, IL-1ra, IL-7, IL-8, IL-9, eotaxin, IP-10, MCP-1, MIP1 α, MIP1 β, PDGF-bb, RANTES, basic FGF, GCSF, GMCSF, VEGF and IL-15) were measured using a Bio-Plex Pro™ kit. Results: IFN- γ, IL-10 and IL-5 were significantly higher in the serum of both CFS/ME and MS patients compared to the healthy controls (p ≤ 0.041). However, only the MS patients had significantly elevated levels of IL-12, IL-1 β, IL-4, IL-13, IL-6, IL-17, IL-1ra, IL-7, IL-9, eotaxin, IL-10, MIP1 α, basic FGF, GCSF and VEGF compared to the CFS/ME patients and controls (p ≤ 0.04). There were no significant differences between groups for IL-8, MCP-1, MIP1 β, RANTES, GMCSF, TNF- α, and IL-2. Conclusion: CFS/ME and MS patients both displayed abnormal cytokine levels, with dual expression of Th1 and Th2 cytokines. Further research into cytokines such as IFN- γ, IL-10 and IL-5, with the use of a specific CFS/ME case definition and sensitive cytokine assays, is required to improve the understanding of the pathophysiology of CFS/ME.
Chronic Fatigue Syndrome (CFS/ME) is a debilitating illness described as fatigue lasting more than 6 months, accompanied by various symptoms including cognitive difficulties, sore throat, muscle or joint pain, and headaches [
Multiple Sclerosis (MS) is a chronic disease targeting the central nervous system and believed to be immunological in nature, resulting in demyelination in the brain and spinal cord, particularly in the white matter [
Cytokine expression abnormalities are exhibited in both CFS/ME and MS, with dual expression of Th1/Th2 cytokines. However, previous studies of cytokines in CFS/ME have been inconclusive. The objective of this study therefore is to investigate and compare the cytokine profiles of healthy controls, CFS/ME and MS patients.
A cross-sectional case-control study design was used, and samples were obtained from a previous large prospective cohort study. Ethics approval was granted by the Griffith University Human Research Ethics Committee (EC00162), reference number MSC 22/12/HREC. A participant information sheet was provided and written consent was obtained from all participants. CFS/ME patients were classified using the ICC definition [
A Bio-Plex Pro™ kit (Bio-Plex Pro Human 27-plex Assay, Bio-Rad, Hercules, CA, USA) was used to measure serum cytokine concentrations of 27 cytokines, as per manufacturer’s instructions. Cytokines IFN-γ, TNF-α, IL-12, IL-2, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-5, IL-17, IL-1ra, IL-7, IL-8, IL-9, eotaxin, IP-10, MCP-1, MIP1α, MIP1β, PDGF-bb, RANTES, basic FGF, GCSF, GMCSF, VEGF and IL-15 were assessed in duplicates. Serum was acquired from peripheral whole blood, diluted by a factor of 1:4, and then incubated in a 96-well plate for 30 minutes with capture antibody-coupled magnetic beads. During the incubation the plate was sealed and protected from light with aluminum foil, and placed on a shaker at 850 ± 50 rpm. The plate was then washed with buffer using a Bio-Plex Pro wash station. The plate was then incubated with biotinylated detection antibodies for 27 cytokines as previously mentioned for 30 minutes. After washing, 1× SA-PE florescence was added and the plate was incubated for 10 minutes. The plate was then washed and assay buffer was added to the wells to re-suspend the magnetic beads. The plate was incubated for 30 seconds prior to analysis. Bio-Plex MAGPIX software was used to assess the concentration of the 27 cytokines.
Results were statistically analysed using SPSS. Duplicated measurements were averaged, and outliers were excluded [
The mean (±standard deviation) ages for the healthy controls, CFS/ME patients, and MS patients were 50 ± 11.846, 50 ± 9.542, and 58 ± 11.990 years respectively (
MS patients were further categorized by disease subtype and current treatments (see
Characteristic | Control (n = 16) mean (SD) | CFS/ME patients (n = 16) mean (SD) | MS patients (n = 11) mean (SD) |
---|---|---|---|
Age | 50.06 (11.846) | 49.88 (9.542) | 57.82 (11.990) |
WBC | 5.56 × 106 (1.602) | 5.17 × 106 (1.552) | 7.53 × 106 (1.161) |
Gender | Control (n = 16) N (%) | CFS/ME patients (n = 16) N (%) | MS patients (n = 11) N (%) |
Female | 8 (50.0%) | 11 (68.8%) | 8 (72.7%) |
Male | 7 (43.8%) | 5 (31.3%) | 3 (27.3%) |
Characteristic | MS patients N (%) |
---|---|
Type of disease Clinically isolated syndrome Relapsing remitting Primary progressive Secondary progressive | 1 (9.1%) 7 (63.6%) 1 (9.1%) 2 (18.2%) |
Treatment None IFN-β Glatiramer acetate | 4 (36.4%) 6 (54.5%) 1 (9.1%) |
CFS/ME patients are often undergoing therapeutic treatment to manage their symptoms. Medications taken by CFS/ME patients were divided into categories (see
T helper (Th) 1 cytokines assessed included IFN-γ, TNF-α, IL-12, IL-1β and IL-2 (see
Medications | No N (%) | Yes N (%) | ≥2 drugs of that type N (%) |
---|---|---|---|
Anti-inflammatory | 10 (62.5%) | 4 (25%) | 2 (12.5%) |
Anti-microbial | 15 (93.8%) | 1 (6.3%) | - |
Psychoactive drugs (anti-depression/anxiety etc.) | 7 (43.8%) | 4 (25%) | 5 (31.3%) |
Analgesic | 11 (68.8%) | 4 (25%) | 1 (6.3%) |
Natural/supplements/vitamins | 13 (81.3%) | 1 (6.3%) | 2 (12.5%) |
Other (proton inhibitors, BP medication, digestive, anticoagulants) | 10 (62.5%) | 4 (25%) | 2 (12.5%) |
CFS/ME patients had significantly higher serum levels than controls (p = 0.041), and MS also had significantly higher serum levels compared with control (p = 0.002). There was a significant difference between CFS/ME and MS (p = 0.006). There was no significant difference in level of TNF-α between groups. IL-12 and IL-1β were both significantly higher in MS (p ≤ 0.021) than in CFS/ME and control. IL-2 was not significantly different between groups.
Th2 cytokines that were assessed included IL-4, IL-6, IL-10, IL-13, and IL-5 (see
IL-13 was significantly elevated in MS compared with CFS/ME and control (p ≤ 0.001), and CFS/ME and control were not significantly different. IL-6 was also significantly higher in MS patients compared with control (p = 0.049), with no significant difference between MS and CFS/ME patients, and CFS/ME and control groups. However for IL-10, CFS/ME and MS patients were both significantly elevated compared to the control (p ≤ 0.001), with no significant difference between the two groups. IL-5 was also significantly higher in both MS and CFS/ME compared with controls (p ≤ 0.001), although there was no significant difference between these two groups. IL-17 was significantly different in MS patients compared to CFS/ME (p = 0.001) and control (p = 0.033) groups.
MS samples also had significantly higher levels of IL-1ra, IL-7, IL-9, eotaxin, IP-10, MIP1α, basic FGF, and GCSF (p ≤ 0.035), while CFS/ME and control samples were not significantly different (see
was no significant difference between the three groups for IL-8, MCP-1, MIP-1β, PDGF-bb, RANTES, VEGF and GMCSF serum levels.
In order to test the effect of MS therapeutic treatment on serum cytokine levels, ANOVA and Bonferroni post hoc tests were performed between drug groups for each cytokine. There was no significant difference between MS patients undergoing treatment compared with no treatment for any of the cytokines tested. Similarly, the medications for CFS/ME patients were analysed, with no significant effect found.
This study simultaneously compared the cytokine profiles of healthy controls, CFS/ME patients and MS patients, and found significant differences in IFN-γ, IL-10, Il-5, IL-12, IL-1β, IL-4, IL-13, IL-6, IL-17, IL-1rα, IL-7, IL-9, eotaxin, IL-10, MIP-1α, basic FGF, GCSF and VEGF. CFS/ME and MS patients both exhibited significantly elevated levels of IFN-γ, IL-10 and IL-5, while MS alone exhibited significantly elevated levels of IL-12, IL-1β, IL-4, IL-13, IL-6, IL-17, IL-1rα, IL-7, IL-9, eotaxin, IL-10, MIP-1α, basic FGF, and GCSF. This comparison with MS may assist in the investigation of the pathophysiology of CFS/ME, as well as contribute to the determination of potential biomarkers for CFS/ME.
IFN-γ plays an immunoregulatory function, enhancing antigen-presentation to lymphocytes and natural killer (NK) cell cytotoxicity [
IL-1β, a potent inflammatory cytokine, was significantly elevated in MS patients compared to CFS/ME patients and controls. Previous research has demonstrated elevated levels of IL-1β in cerebrospinal fluid correlated with cortical lesion load in MS patients [
IL-12 was also significantly elevated in MS patients compared to CFS/ME patients and controls. IL-12 is a pro-inflammatory cytokine which affects the differentiation of Th1 cells and plays an influential role in NK cell activity [
MS patients exhibited elevated levels of IFN-γ, IL-1β and IL-12, indicating a state of inflammation. IL-12 may be influencing the release of IFN-γ from NK cells, and IFN-γ is likely contributing to migration of immune cells to the CNS. IL-1β may be further contributing to disease progression in MS patients. CFS/ME patients exhibited abnormal levels of IFN-γ, independent of IL-12 elevation. This may be either contributory or compensatory to the reduced NK activity observed in in CFS/ME patients.
IL-10 was significantly elevated in both CFS/ME and MS patients. Previous studies have also found elevated levels of IL-10 in CFS/ME patients [
The differentiation of naive B cells into antibody secreting plasma cells is profoundly influenced by IL-5, which is produced by Th2 and NK cells [
IL-4 stimulates antibody secretion by B cells and B cell proliferation and is a major regulatory cytokine [
The significant alteration in IL-10 present in both CFS/ME and MS patients indicates a struggle to suppress overactive immune responses. Additionally, elevated levels of IL-5 in both conditions suggest a Th2 cytokine profile, and may serve to compensate for elevated Th1 cytokines levels and inflammation present in both CFS/ME and MS. In CFS/ME patients, IL-5 may be released by NK cells concurrently with IFN-γ. Additionally, IL-5, IL-4, and IL-6 may be contributing to pathogenic B cells in MS, through activation of B cell differentiation and antibody secretion. The elevation of IL-17 is indicative of Th17 involvement in MS pathology.
MS patients also exhibited elevated levels of IL-1rα, IL-7, IL-9, eotaxin, IP-10, MIP1α, basic FGF, and GCSF. IL-1rα polymorphisms are been detected in genetic studies of MS patients [
MS patients have exhibited elevated levels of chemotactic factors eotaxin, IP-10, and MIP-1α suggest high migration of macrophages, T cells, B cells, and NK cells. As MS is a neurodegenerative disease, these immune cells are likely migrating towards the CNS. Hence these results suggest that patients may be in a state of relapse, with further recruitment of immune cells and potential worsening of disease. CFS/ME patients did not exhibit abnormal levels of these chemokines, indicating that chemotactic driven migration of immune cells was not present in these patients. CFS/ME patients also did not exhibit changes in growth factor production, unlike MS patients whose elevated expression may be compensatory to neural damage.
Expression of Th1 and Th2 cytokines were observed in both CFS/ME and MS. Th2 cytokine elevations may be a compensatory response to an overactive immune reaction. The findings of this study implicate widespread immune dysfunction in CFS/ME, through abnormal IFN-γ, IL-10 and IL-5 serum levels. Immune dysfunction may involve NK cells, Tregs, B cells and eosinophils, with potential similarities to MS pathophysiology. These provide potential areas for future research. However, many cytokines have not been sufficiently researched in CFS/ME outside of this study, and so further research should be conducted to investigate the roles of these cytokines in CFS/ME. The cytokines that were abnormally elevated in MS patients were consistent with those associated with disease, and are indicative of high disease severity. However, some of the elevations of cytokines may have been influenced by IFN-β treatment.
In conclusion, this study has reported expression of Th1 and Th2 cytokines in CFS/ME and MS patients. Elevated levels of IFN-γ, IL-10 and IL-5 were found in both CFS/ME and MS. In the MS cohort, the results were consistent with the majority of findings from previous studies, where general elevation of pro-inflammatory, some anti-inflammatory, Th1 and Th2 cytokines has been observed [
We would like to acknowledge the Mason Foundation (Grant Number MA43120) and Queensland Government Department of Science, Information Technology, Innovation and the Arts Smart Futures Fund (Grant Number 216702MRE) for the founding of this project.
Authors would like to disclose there are no competing interests.
NaomiWong,ThaoNguyen,Ekua WebaBrenu,SimonBroadley,DonaldStaines,SonyaMarshall-Gradisnik, (2015) A Comparison of Cytokine Profiles of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and Multiple Sclerosis Patients. International Journal of Clinical Medicine,06,769-783. doi: 10.4236/ijcm.2015.610103