Cytokine expression profiles in white blood cells of patients with small fiber neuropathy

Abstract

Background: The role of cytokines in the pathophysiology, diagnosis, and prognosis of small fiber neuropathy (SFN) is incompletely understood. We studied expression profiles of selected pro- and anti-inflammatory cytokines in RNA from white blood cells (WBC) of patients with a medical history and a clinical phenotype suggestive for SFN and compared data with healthy controls.

Methods: We prospectively recruited 52 patients and 21 age- and sex-matched healthy controls. Study participants were characterized in detail and underwent complete neurological examination. Venous blood was drawn for routine and extended laboratory tests, and for WBC isolation. Systemic RNA expression profiles of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-2, IL-8, tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4, IL-10, transforming growth factor beta-1 (TGF) were analyzed. Protein levels of IL-2, IL-8, and TNF were measured in serum of patients and controls. Receiver operating characteristic (ROC)-curve analysis was used to determine the accuracy of IL-2, IL-8, and TNF in differentiating patients and controls. To compare the potential discriminatory efficacy of single versus combined cytokines, equality of different AUCs was tested.

Results: WBC gene expression of IL-2, IL-8, and TNF was higher in patients compared to healthy controls (IL-2: p = 0.02; IL-8: p = 0.009; TNF: p = 0.03) and discriminated between the groups (area under the curve (AUC) ≥ 0.68 for each cytokine) with highest diagnostic accuracy reached by combining the three cytokines (AUC = 0.81, sensitivity = 70%, specificity = 86%). Subgroup analysis revealed the following differences: IL-8 and TNF gene expression levels were higher in female patients compared to female controls (IL-8: p = 0.01; TNF: p = 0.03). The combination of TNF with IL-2 and TNF with IL-2 and IL-8 discriminated best between the study groups. IL-2 was higher expressed in patients with moderate pain compared to those with severe pain (p = 0.02). Patients with acral pain showed higher IL-10 gene expression compared to patients with generalized pain (p = 0.004). We further found a negative correlation between the relative gene expression of IL-2 and current pain intensity (p = 0.02). Serum protein levels of IL-2, IL-8, and TNF did not differ between patients and controls.

Conclusions: We identified higher systemic gene expression of IL-2, IL-8, and TNF in SFN patients than in controls, which may be of potential relevance for diagnostics and patient stratification.

Keywords: Cytokines; Gene expression; Small fiber neuropathy; White blood cells.

Fig. 1

HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0" Gene expression of cytokines in WBC of SFN patients compared to controls. The scatter plots show the gene expression of pro- (IL-1ß, IL-2, IL-6, IL-8, and TNF) (a) and anti- (IL-4, IL-10, and TGF) inflammatory cytokines. Gene expression of pro-inflammatory cytokines IL-2, IL-8, and TNF was higher in WBC in patients compared to controls. Anti-inflammatory cytokine expression did not differ between groups (b). Number of samples investigated: SFN patients = 52; controls = 21. IL− interleukin, SFN small fiber neuropathy, TGF transforming growth factor beta-1, TNF tumor necrosis factor-alpha. *p < 0.05

Fig. 2

ROC-curve analysis of cytokine gene expression levels in WBC of SFN patients compared to controls. The ROC-curves show the total AUC for IL-2, IL-8, and TNF separately (a), after combination of two out of three cytokines (b), and after merging IL-2, IL-8, and TNF together (c). AUC was highest for IL-8 (AUC = 0.69) when comparing AUC results for each cytokine separately (a). After merging, combing IL-2 with IL-8 or TNF reached the highest values (AUC = 0.78, each). Combination of three cytokines resulted in an AUC of 0.81. Numbers of samples investigated: SFN patients = 52; controls = 21. AUC area under the curve, IL− interleukin, ROC receiver operating characteristic, TNF tumor necrosis factor-alpha

Fig. 3

Relative gene expression of cytokines in WBC stratified for sex. The scatter plots show the gene expression of pro- and anti-inflammatory cytokines when comparing a female patients and female controls; b male patients and male controls; c female patients and male patients. Gene expression of IL-8 and TNF was higher in female patients compared to female controls (a), no intergroup difference was found comparing male patients b and male controls or female patients and male patients (c). Numbers of samples investigated: female patients = 32; female controls = 13; male patients = 20; male controls = 8. IL− interleukin, SFN small fiber neuropathy, TGF transforming growth factor beta-1, TNF tumor necrosis factor-alpha. *p < 0.05

Fig. 4

Relative gene expression of cytokines in SFN patients stratified for pain phenotype. The scatter plots show the gene expression of pro- and anti-inflammatory cytokines when comparing patients with moderate (NRS < 4) to severe (NRS ≥ 4) pain a and with acral pain to generalized pain (b); Gene expression was higher of IL-2 in patients with moderate pain compared to severe pain (a) and of IL-10 in patients with acral pain compared to generalized pain (b). Numbers of samples investigated: moderate pain = 35; severe pain = 17; acral pain = 15; generalized pain = 21. IL− interleukin, NRS numeric rating scale, SFN small fiber neuropathy, TGF transforming growth factor beta-1, TNF tumor necrosis factor-alpha. *p < 0.05; **p < 0.01

Fig. 5

Correlation analysis between WBC cytokine expression profiles and pain intensity in SFN patients. The scatter plots show the gene expression of a IL-2, b IL-8, and c TNF in correlation to the pain intensity (determined on a NRS: range 0 = no pain; 10 = maximum pain imaginable) of SFN patients. Gene expression of IL-2 and current pain intensity correlated negatively in SFN patients (a), while no correlation was found for IL-8 (b) and TNF (c). Numbers of samples investigated: SFN patients = 52; IL− interleukin, NRS numeric rating scale, TNF tumor necrosis factor-alpha. *p < 0.05

Fig. 6

Protein expression of cytokines in serum of SFN patients compared to controls. The scatter plots show the protein expression of pro-inflammatory cytokines IL-8 and TNF in serum of SFN patients and healthy controls. Protein expression of IL-8 and TNF did not differ between groups. Number of samples investigated: SFN patients = 26 (n = 15 female, n = 11 male); controls = 8 (n = 5 female, n = 3 male). IL− interleukin, SFN small fiber neuropathy, TNF tumor necrosis factor-alpha