Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Abstract

Background: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is characterised by severe prolonged fatigue, and decreases in cognition and other physiological functions, resulting in severe loss of quality of life, difficult clinical management and high costs to the health care system. To date there is no proven pathomechanism to satisfactorily explain this disorder. Studies have identified abnormalities in immune function but these data are inconsistent. We investigated the profile of markers of immune function (including novel markers) in CFS/ME patients.

Methods: We included 95 CFS/ME patients and 50 healthy controls. All participants were assessed on natural killer (NK) and CD8(+) T cell cytotoxic activities, Th1 and Th2 cytokine profile of CD4(+) T cells, expression of vasoactive intestinal peptide receptor 2 (VPACR2), levels of NK phenotypes (CD56(bright) and CD56(dim)) and regulatory T cells expressing FoxP3 transcription factor.

Results: Compared to healthy individuals, CFS/ME patients displayed significant increases in IL-10, IFN-γ, TNF-α, CD4(+)CD25(+) T cells, FoxP3 and VPACR2 expression. Cytotoxic activity of NK and CD8(+) T cells and NK phenotypes, in particular the CD56(bright) NK cells were significantly decreased in CFS/ME patients. Additionally granzyme A and granzyme K expression were reduced while expression levels of perforin were significantly increased in the CFS/ME population relative to the control population. These data suggest significant dysregulation of the immune system in CFS/ME patients.

Conclusions: Our study found immunological abnormalities which may serve as biomarkers in CFS/ME patients with potential for an application as a diagnostic tool.

Figure 1

Selection Process for Experimental Groups. Participants for the present project were grouped into CFS/ME, or non-fatigued control groups based on the CDC 1994 case definition symptom criteria. Participants, that is, both CFS/ME and non-fatigued controls, were comprised of both male and females selected using advertisements and through the CFS/ME support groups. Non-fatigued controls were randomly selected from the general population using newspaper and email advertisements. The above flow chart illustrates the process used to generate the final research population.

Figure 2

Reduced lytic function of cytotoxic cells in CFS/ME. In vivo assessment of NK and CD8⁺T cell lysis (cytotoxic activity) of tumour cell lines K562 and P815 respectively in CFS/ME (black bars) in comparison to controls (white bars). Lytic activity is represented as percent (%) lysis on the y-axis. *Denotes statistical significant results. Data presented as mean ± SEM.

Figure 3

mRNA Expression of cytotoxic molecules in NK and CD8⁺T cells. Quantitative reverse transcriptase (RT)-PCR demonstrated the relative expression of granzyme A, granzyme K, perforin and IFN-γ in NK (A) and CD8⁺T cells (B). In NK and CD8+T cells expression levels of GZMA, GZMK and IFN-G were decreased in CFS/ME (black bars) compared to the controls (white bars). PRF1 was however increased in CFS group. *Denotes statistical significant results (P ≤ 0.05). Data presented as mean ± SEM.

Figure 4

Distribution of NK phenotypes. NK phenotypes that were examined are denoted as either NK Bright (CD56^brightCD16^-) or NK Dim (CD56^dimCD16⁺). (A). The box plots represent the raw data of NK Bright cells in the two groups. CFS/ME patients were more decreased in the cell numbers for this particular NK phenotype. (B). However raw data of CD56^dimCD16⁺NK cells were examined in the control and CFS/ME groups, these were found to be similar. (C). Using the raw data from the flow cytometry results, total counts of NK cells were deduced. These measurements are plotted using bar graphs, CD56^brightCD16^-NK cells are more reduced in the CFS/ME (black bars) group in comparison to the controls (white bars). *Denotes statistical significant results (P ≤ 0.05). Data presented as mean ± SEM.

Figure 5

Examination of the expression levels of CD4+T cell Related Cytokiness in CFS/ME following mitogenic stimulation. CD4⁺T cells, Th1, Th2 and Th17 cytokine levels in CFS/ME (black bars) and control participants (white bars) measured after mitogenic stimulation with PHA. The concentrations of cytokines were measured in pg/mL. Both anti-inflammatory (IL-10) and pro-inflammatory (IFN-γ, TNF-α) cytokines were increased in the CFS/ME group following mitogenic stimulation. *Statistically significant results at p < 0.05. Data presented as mean ± SEM.

Figure 6

FoxP3 expression and CD4+CD25+T cells in CFS/ME. The percentage of CD4+T cells expressing CD4+CD25+FoxP3+ markers are represented in the bar graph. Tregs of interest in this study were those positive for FoxP3 and CD4+CD25+ in CFS/ME (black bars) and control (white bars) participants. *Represent statistically significant results at p < 0.05. Data presented as mean ± SEM.

Figure 7

VPAC2R immune cells in CFS/ME. VPAC2R expression on CD4⁺T cells was assessed in CFS/ME (black bars) and controls (white bars). The data presented here are based on percentage of cells positive for CD4 and VPACR2. *Represent statistically significant results at p < 0.05. Data presented as mean ± SEM.