Table 1.
RT-PCR Assay Characteristics and Primer Sequences.
Figure 1.
Concentrations of serum cytokines, determined by using a Human Cytokine multiplex immunoassay.
No differences are found analyzing serum protein levels of IL-6, IL-10 and IL-17 between patients and healthy controls (Fig. 1A, 1B, 1C). Protein levels of pro-inflammatory cytokine IL-23 are significantly higher in the peripheral blood of patients with CLBP compared to healthy controls. Values are expressed as mean ± standard deviation. (IL-23: 0.94±0.29 pg/ml in healthy controls vs. 1.21±0.43 pg/ml in CLBP patients; p = 0.009; Fig. 1D).
Figure 2.
Expression levels of T cell related cytokine mRNA measured by qPCR.
TNF-α, IL-4 and IL-10 were neither detectable in CD4+ T cells of CLBP patients nor in healthy controls. The expression of IFN-γ did not exhibit significant differences in CLBP patients as compared to healthy controls (IFN-γ: 4.19±3.54 in CLBP patients vs. 3.60±2.20 in healthy controls, n.s.; Fig. 2A) whereas IL-23 expression of in CD4+ T cell samples of CLBP patients was found to be significantly decreased (4.88±2.44 in CLBP patients vs. 7.73±3.77 in healthy controls, p = 0.006; Fig. 2B).
Figure 3.
Expression levels of T cell subset related mRNA measured by qPCR.
TGF-β and FoxP3 mRNA expression, specific for Tregs, was significantly higher in patients with CLBP than in healthy controls (TGF-β: 0.21±0.07 in CLBP patients vs. 0.14±0.05 in healthy controls, p = 0.014; Fig. 3A), (FoxP3: 0.21±0.14 in CLBP patients vs. 0.14±0.06 in healthy controls, p = 0.009; Fig. 3B). TH17 specific expression of IL-17 was neither detectable in CD4+ samples of CLBP patients nor in healthy controls. Expression levels of RORγT did not differ in CLBP patients and healthy controls (RORγT: 0.028±0.02 in CLBP patients vs. 0.025±0.01 in healthy controls, p = n.s.; Fig. 3C).
Figure 4.
Gating strategy for the detection of Tregs.
PBMCs extracellular stained with PerCP labeled anti-human CD4-antibody, PE labeled anti CD25-antibody, Brilliant Violet (BV570) labeled anti CD127-antibody and intracellular stained with Alexa Fluor (AF488) labeled anti-human FoxP3-antibody. Lymphocyte population was gated from PBMCs according to forward scatter (FSC) characteristics and side scatter (SSC) characteristics (left). Gated lymphocytes were then separated in CD4+CD25high cells/T cells (middle) and CD4+CD25highCD127lowFOXP3+ cells/CD4+T cells (right, named Treg). Upper row represents the result of a healthy control with less CD4+CD25high T cells (3.28%) and less CD4+CD25highCD127lowFoxP3+ T cells (1.94%) compared to a patient with CLBP (lower row, 5.74% and 3.11%).
Figure 5.
Gating strategy for the detection of TH1 and TH17 cells.
PBMCs stimulated with cell stimulation cocktail for 5 h followed by intracellular staining with Brilliant Violet (BV421) labeled anti-human IL-17 antibody and FITC labeled anti-human IFN-γ antibody.
Figure 6.
Flow cytometric quantification of Tregs and TH17 cells.
Results show significantly higher percentage of anti-inflammatory Tregs in patients with CLBP in both staining protocols (CD4+CD25high cells: 4.45±0.88% in CLBP patients vs. 3.49±0.53% in healthy controls, p<0.001; Fig. 6A), (CD4+CD25highCD127lowFoxP3+ cells: 2.89±1.07% in CLBP patients vs. 1.93±0.66% in healthy controls, p = 0.001; Fig. 6B). Number of TH17 cells as percentage of T cells in peripheral blood show significantly lower percentage of pro-inflammatory TH17 cells in patients with CLBP (TH17 cells: 0.46±0.24% in CLBP patients vs. 1.14±0.73% in healthy controls, p<0.001; Fig. 6C).
Figure 7.
Ratios of TH17/CD4+CD25high, TH17/Tregs and TH1/TH2 cells.
Ratios of Th17/CD4+CD25high and Th17/CD4+CD25highCD127lowFoxP3+ were significantly decreased in CLBP patients as compared to healthy controls (Th17/CD4+CD25high: 0.12±0.08 in CLBP patients vs. 0.33±0.23% in healthy controls, p<0.001; Fig. 7A), (Th17/CD4+CD25highCD127lowFoxP3+: 0.23±0.17 in CLBP patients vs. 0.64±0.79 in healthy controls, p<0.001; Fig. 7B). Ratio of TH1/TH2 cells in peripheral blood of patients with CLBP and healthy controls were tendencially decreased in patients with CLBP, but did not reach significance (9.76±7.27 in CLBP patients vs. 14.72±12.81 in healthy controls, p = 0.19, Fig. 7C).
Figure 8.
NRS pain scores, KAB stress scores and T cell subsets before and after treatment.
35% [n = 13] of all patients benefited by the 4 weeks intensive multimodal therapy with long lasting pain- and stress reduction (Fig. 8A). Even all responders showed a significant pain- and stress reduction of ≥50%, no transformation were observed regarding T cell subsets. None of our analyzed T cell subsets (TH1, TH2, TH17, Tregs) normalized after successful therapy (Fig. 8B). (NRS at rest before/after: p = 0.025, NRS at rest before/follow up: p = 0.003, NRS during movement before/after: p = 0.046, NRS during movement before/follow up: p = 0.012, KAB before/after: p = 0.024, KAB before/follow up: p = 0.019).