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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, sensitiv- ity = 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 Small fiber neuropathy, Cytokines, White blood cells, Gene expression Background Small fiber neuropathy (SFN) affects the small caliber *Correspondence: A-delta and C-fibers . SFN patients typically report Nurcan Üçeyler acral or generalized burning pain and par- and dys- email@example.com esthesias . While the underlying pathomechanism Department of Neurology, University of Würzburg, Josef-Schneider-Str. 11, 97080 Würzburg, Germany remains unclear, inflammatory processes and processes of nociceptor degeneration and sensitization may be of © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kreß et al. BMC Neuroscience (2023) 24:1 Page 2 of 12 relevance . There is growing evidence for the induc - microscopy (CCM), quantitative sudomotor axon reflex tion of nociceptor degeneration by local inflammation, test (QSART), and skin punch biopsy were performed oxidative stress following impaired calcium homeosta- as part of a larger study; hence, detailed data on meth- sis, and alteration of the energy metabolism via activa- ods, results, and discussion have been published else- tion of the axonal caspases . Nociceptor sensitization where . In brief: QST (Somedic, Hörby, Sweden) was may result directly from inflammatory processes, which performed at the dorsum of the right foot following the may arise by cytokine or chemokine activation . Basic standardized protocol of the German Research Network mechanisms of the inflammatory response and potential on Neuropathic Pain. PREP were recorded after electri- underlying pathways are well characterized in neurode- cal A-delta fiber stimulation via concentric electrodes generative and metabolic disorders [4, 5], while hardly (Inomed Medizintechnik GmbH, Lübeck, Germany). known in peripheral painful and painless neuropathies. CCM was performed using a retinal tomograph (Heidel- There is evidence for pro- and anti-inflammatory berg Retina Tomograph Rostock Cornea Module, Heidel- cytokines to act as key contributors to SFN pain by berg, Germany). QSART was done on the lateral dorsum directly targeting cutaneous nociceptors . Locally, we of the foot using Q-Sweat (WR Medical Electronics, found elevated gene expression levels of the pro-inflam - Maplewood, MA, USA) and following the manufac- matory cytokines interleukin (IL)-6 and IL-8 in whole turer’s protocol. Six-millimeter skin biopsies were taken skin biopsy samples . Analysis of distinct skin cells from the right lateral lower leg and upper thigh to deter- showed higher expression levels of IL-6 and IL-8 in fibro - mine the intraepidermal nerve fiber density following a blasts and of transforming growth factor beta-1 (TGF) standard protocol . Study participants reporting or in keratinocytes of SFN patients compared to healthy diagnosed with polyneuropathy, diabetes mellitus, renal controls . In addition to a pathophysiological role of insufficiency, uncontrolled thyroid dysfunction, acute or cytokines in neuropathic pain and SFN, there is grow- chronic infection, malignancy within the last five years, ing evidence for their usage as diagnostic and prognostic substance dependence, regular alcohol consumption, or parameters [8–10]. While reports exist for inflammatory severe psychiatric disorder were excluded. Due to the conditions [11, 12], data in neuropathies are rare . prerequisites of individual small fiber tests, the following In our previous study, we analyzed the pro-inflamma - exclusion criteria were additionally applied: epilepsy, car- tory cytokines IL-1ß, IL-2, IL-8, tumor necrosis factor- diac pacemaker, eye disease or surgery, or usage of hard alpha (TNF) and the anti-inflammatory cytokines IL-4, contact lenses. Healthy controls were further free of any IL-10, and TGF, which are examples of frequently inves- neurological disorder. tigated cytokines in painful neuropathies . We found systemically higher expression levels of IL-2, IL-10, and Laboratory blood tests TGF in whole blood samples of SFN patients compared All patients and controls underwent venous blood draw- to healthy controls. Here, we followed-up these cytokines ing under standardized conditions. Blood samples were in a larger study cohort, provide cellular allocation by collected between 8:00 and 9:00 AM after overnight fast- investigation on white blood cells (WBC) and investigate ing, avoiding heavy physical activity or meals, and alco- data on protein level. hol consumption on the previous day. To determine the etiology of SFN and not miss potential exclusion criteria, Methods venous blood was drawn in serum (14 ml) and in ethyl- Patients and healthy controls enediaminetetraacetic acid (EDTA) monovettes (27 ml) From 2015 to 2019, 52 patients with a medical history to perform routine and extended laboratory tests, and and clinical phenotype indicative of SFN (32 women, 20 next-generation gene-panel sequencing. Detailed data men; median age: 54 years; range: 19–73) and 21 age and on these laboratory tests were published previously . sex-matched healthy controls (13 women, 8 men; median Venous blood was used to perform an oral glucose toler- age: 48 years; range: 22–66) were prospectively recruited ance test and for WBC and serum isolation. as part of a larger study . Detailed medical and pain history, including pain character, intensity (determined Gene expression analysis in white blood cells on a numeric rating scale [NRS]: range 0 = no pain; White blood cell isolation 10 = maximum pain imaginable), and localization were Eighteen ml venous blood was drawn in EDTA mon- recorded. All study participants underwent complete ovettes and incubated (30 min, room temperature (RT)). neurological examination and nerve conduction studies During incubation, nine 15-ml falcon tubes (Greiner bio- to exclude polyneuropathy. Five small fiber-specific tests, one, Kremsmünster, Austria) were loaded with 7.5 ml namely quantitative sensory testing (QST), recording of erythrocyte lysis (EL)-buffer on ice (Quiagen, Hilden, pain-related evoked potentials (PREP), corneal-confocal Germany) and assembled with 3 ml of EDTA blood each. K reß et al. BMC Neuroscience (2023) 24:1 Page 3 of 12 Throughout an incubation period of 25 min, falcon tubes ensured. To guarantee high quality, cDNA concentra- were vortexed twice after 7 min and 15 min. After cen- tion was measured directly after RT-PCR and before trifugation (400 g, 10 min, 4 °C), discarding the superna- qRT-PCR. tant fluid, and resuspending cell pellets in 3 ml EL-buffer, samples were centrifuged again (400 g, 10 min, 4 °C). Quantitative real‑time PCR Subsequently, supernatant was removed, cell pellets We investigated the following gene targets: pro- were re-suspended in 1.5 ml ribonucleic acid (RNA) pro- inflammatory cytokines IL-1ß (Hs00174097_m1), IL-2 tect cell reagent (Quiagen, Hilden, Germany), aliquoted (Hs00174114_m1), IL-8 (Hs00174103_m1), and TNF 500 µl each, and frozen at -80 °C before RNA extraction. (Hs00174128_m1) and anti-inflammatory cytokines IL-4 (Hs00174122_m1), IL-10 (Hs00174086_m1), and RNA extraction TGF (Hs99999918_m1). Primers were commercially For RNA isolation, the miRNeasy Mini Kit (Quiagen, designed and validated primers (TaqMan, Thermo Fisher Hilden, Germany) was applied and frozen samples con- Scientific, Waltham, MA, USA). A Micro Amp Opti - taining WBC RNA and RNA protect cell reagent were cal 96-Well Reaction Plate (Thermo Fisher Scientific, used. Samples were suspended in 700 µl of Qiazol Lysis Waltham, MA, USA), containing a negative control with- reagent (guanidine thiocyanate and phenol mixture) out cDNA, and a calibrator sample was used to meas- and incubated for 5 min at RT. Then, 140 µl chloroform ure the samples. The calibrator sample (determined as (Carl Roth, Karlsruhe, Germany) was added and the the sample with threshold cycle (Ct-) values next to the samples were shaken vigorously for 15 s. Samples were respective control groups’ mean Ct values), was indi- then incubated for up to 3 min at RT and centrifuged vidually assigned to each target gene. The reaction con - (12.000g, 15 min, 4 °C). Afterwards, the upper aqueous tained: 5 µl cDNA, 2 µl TaqMan Universal Master Mix phase was discarded and 525 µl 100% ethanol was added. (Thermo Fisher Scientific, Waltham, MA, USA), 1.75 µl 700 µl of the suspension was then transferred into a sil- sterile distilled water (Braun, Melsungen, Germany), and ica-membrane RNeasy spin column (supplied with the 0.25 µl of the target primer. Endogenous control eukary- kit) and centrifuged twice (8.000g, 20 s, RT). Three fur - otic, 18 s RNA (Hs99999901_s1) was used as housekeep- ther centrifugation steps (each 8.000g, 20 s, RT) using ing gene as it was validated on human biomaterial in our silica-membrane RNeasy spin columns were performed. previous studies [7, 14, 16, 17]. 18 s reaction mixture con- mRNA was eluted in 30 µl RNAse-free-water. RNA con- tained 2.5 µl cDNA, 2 µl TaqMan Universal Master Mix centration was measured via Nanodrop spectrophotom- (Thermo Fisher Scientific, Waltham, MA, USA), 4.25 µl eter (Peqlab, Erlangen, Germany). To assess RNA purity, sterile, distilled water (Braun, Melsungen, Germany), and the ratio of the absorbance (A) at different wavelengths 0.25 µl 18sRNA. Target genes were measured as tripli- was calculated. A median ratio of 2.0 for A cates, 18 s RNA as duplicates. the following three steps: 260 nm/280 nm (range 1.85–2.05) was defined as pure RNA. Samples first incubation (50 °C, 2 min), second incubation (95 °C, were stored at − 80 °C before further processing. 10 min), and 40 cycles (95 °C, 15 s and 60 °C, 1 min). The analysis was done by using StepOnePlus Cycler −deltadel- Reverse transcription PCR (Thermo Fisher Scientific, Waltham, MA, USA). 2 taCt For reverse transcription polymerase chain reaction method was performed for data evaluation. (PCR), 250 ng RNA was used and sterile distilled water (Braun, Melsungen, Germany) was added to reach a total Protein expression analysis of cytokines volume of 32.8 µl each. Samples were supplemented with Serum collection 5 µl Random Hexamer (TaqMan Reverse Transcrip- Nine ml venous blood was drawn in serum monovettes tion Reagents, Thermo Fisher Scientific, Waltham, MA, and incubated (30 min, RT). Monovettes were centri- USA) and incubated (3 min, 85 °C). Two µl Oligo-DT and fuged (400g, 10 min, 4 °C), serum was aliquoted 500 µl 60.2 µl Master Mix (produced of 10 µl 10 × PCR buffer, each, and frozen at − 80 °C before further use. 6.25 µl multiscribe reverse transcriptase, 2 µl RNase inhibitor, 22 µl M gCl , and 20 µl dNTPs) were added. Enzyme‑linked immunosorbent assay PCR was run in a PCR-Cycler Advanced Primus 96-PCR To determine the protein levels of IL-2, IL-8, and TNF (Peqlab Biotechnology, Erlangen, Germany) under the in serum of patients and healthy controls, the following following conditions: annealing (10 min, 25 °C), reverse enzyme-linked immunosorbent assay (ELISA) kits were transcription (60 min, 48 °C), and enzyme inactivation used: Invitrogen human IL-2, human IL-8 and human (5 min, 95 °C). cDNA was stored at − 20 °C before fur- TNF-alpha ELISA kit (each Thermo Fisher Scientific, ther processing. RT-PCR was done shortly before run- Waltham, MA, USA). ELISA was performed accord- ning qRT-PCR. Using cDNA samples ≤ 2 months was ing to the manufacture’s protocol. The provided analytic Kreß et al. BMC Neuroscience (2023) 24:1 Page 4 of 12 Table 1 Basic clinical and laboratory data of study population Patients (n = 52) Controls (n = 21) a a Age [years] (range) 54 (19–73) 48 (22–66) Gender (F/M) 32/20 13/8 Time since diagnosis [years] (range) 0.25 (< 1 month-12) NA Pain duration [years] 3.25 (< 1 month-24) NA Assumed etiology of SFN NA Determined (In some patients ≥ 1 pathological finding was present, thus, the sum exceeds 100%) 22/52 (42%) Diabetes or impaired glucose tolerance 22/52 (42%) Vitamine B12 deficiency 2/52 (4%) Hereditary 5/52 (10%) Thyroid dysfunction 6/52 (12%) Idiopathic 30/52 (58%) Abnormal results in routine laboratory tests b (In some patients ≥ 1 pathological finding was present) 9/52 (17%) Not investigated Leucocytosis (Ref.: 5–10*10 /µl) 2/52 (4%) Leucopenia (Ref.: 5–10*10 /µl) 1/52 (2%) Creatinine (Ref.: 0–0.95 mg/dl) ↑ 2/52 (4%) CRP (Ref.: 0–0.5 mg/dl) ↑ 2/52 (4%) Gamma GT (Ref.: < 40 U/l) ↑ 1/52 (2%) Abnormal results in extended laboratory tests c (In some patients ≥ 1 pathological finding was present) 28/52 (51%) NA HbA1c (Ref.: ≤ 6.1%) ↑ 7/52 (13%) Vitamin B12 (Ref.: ≥ 197 pg/ml) ↓ 2/52 (4%) TSH (Ref.: 0.3–4.0 mlU/l) ↑ 1/52 (2%) TSH (Ref.: 0.3–4.0 mlU/l) ↓ 1/52 (2%) Detected autoantibodies (antinuclear antibodies, extractable nuclear antigen antibodies, anti-neutrophil 2/52 (4%) cytoplasmic antibodies) Pathological oGTT (2 h glucose level ≤ 140 mg/dl) 15/52 (29%) NA Pain distribution NA Acral 15/52 (29%) Generalized 22/52 (42%) Both 15/52 (29%) Pain intensity [NRS] (range) NA Current pain intensity 4 (0–8) Maximum pain intensity 8 (3–10) Mean pain intensity 5 (0–8) Female 5 (0–8) Male 4 (0–8) Signs of small fiber impairment in neurological examination 30/52 (58%) None Thermal hypoesthesia 13/52 (25%) Hypo-/hyperalgesia 15/52 (29%) Allodynia 4/52 (8%) Dysesthesia/paresthesia 7/52 (13%) Additional symptoms NA Gastrointestinal symptoms 3/52 (5%) Obstipation 1/52 (2%) Diarrhea 2/52 (4%) Autonomic symptoms 29/52 (56%) Hypo-/hyperhidrosis 27/52 (52%) Sexual dysfunction 7/52 (13%) K reß et al. BMC Neuroscience (2023) 24:1 Page 5 of 12 Table 1 (continued) Patients (n = 52) Controls (n = 21) Impairment of micturition 6/52 (12%) Repetitive syncope 0/52 (0%) CRP C-reactive protein, F female, HbA1c hemoglobin A1c, IENFD intraepidermal nerve fiber density, M male, NA not applicable, NRS numeric rating scale, oGTT oral glucose tolerance test, Ref. reference, SFN small fiber neuropathy, TSH thyroid stimulating hormone, WBC white blood cells Data are given as median b 3 3 3 Individual data: leucocytosis: 12.0*10 /µl; 15.6*10 /µl; leucopenia: 3.6*10 /µl; creatinine ↑: 0.97 mg/dl; 1.0 mg/dl; CRP ↑: 0.84 mg/dl; 2.47 mg/dl; gamma GT ↑: 86.2 U/l Individual data: HbA1c ↑: 6.2% (3x); 6.3%; 6.6%; 6.9%; 7.7%; vitamin B12 ↓: 137 pg/ml; 195 pg/ml; TSH ↑: 9.2 mlU/l; TSH ↓: 0.1 mlU/l; detected autoantibodies: antinuclear antibodies 1:80; antinuclear antibodies 1:160 distributed, thus the non-parametric Mann–Whitney- U-test and the Spearmann’s test for correlation analysis were applied. Receiver operating characteristic (ROC)- curve analysis was used to calculate the area under the curve (AUC), specificity, sensitivity, and the optimal cut- off value of IL-2, IL-8, TNF and their combination to evaluate the accuracy in differentiating SFN patients and healthy controls. ROC curve plotting and analysis was achieved by using the web-based tool easy ROC (version 1.3.1), which is based on R Langue Environment . Optimal cut-off values were defined by Youden method. Discriminatory efficacy of single cytokines was deter - mined by AUC comparison. DeLong’s test procedure was used to non-parametrically test the hypothesis of the equality of the AUCs of combined cytokines. To perform DeLong’s test R package pROC (version 4.2.1) was used. Scatter and box plots were created with GraphPad Prism 18.104.22.168 software. G*Power version 22.214.171.124 (http:// www. p s yc ho. uni- due ss e ldor f. de/ a bt ei lungen/ aa p/ g p owe r3/) was used for post-hoc sample size calculation. P < 0.05 was considered significant. Post-hoc sample size calcu - lation revealed that n = 84 patients and n = 34 controls Fig. 1 HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0" Gene should be included in our study assuming a large effect expression of cytokines in WBC of SFN patients compared to controls. size. With n = 52 SFN patients and n = 21 controls, we The scatter plots show the gene expression of pro- (IL-1ß, IL-2, performed an exploratory study. 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. Results Anti-inflammatory cytokine expression did not differ between groups Clinical and laboratory data (b). Number of samples investigated: SFN patients = 52; controls = 21. Baseline clinical and laboratory data of the study cohort IL− interleukin, SFN small fiber neuropathy, TGF transforming growth is summarized in Table 1. In 22/52 (42%) SFN patients, factor beta-1, TNF tumor necrosis factor-alpha. *p < 0.05 a potential underlying reason was found and 30/52 (58%) SFN patients were classified as having an idi - opathic SFN. Laboratory tests were normal in 43/52 sensitivities of the assays were given as follows: 9.1 pg/ml (83%) of the patients except for the following abnormali- for IL-2, < 5.0 pg/ml for IL-8, and 1.7 pg/ml for TNF. ties: cell count (n = 3), serum (n = 5), elevated HbA1c (n = 7), vitamin B12 deficiency (n = 2), thyroid dysfunc- Statistical analysis tion (n = 6), and detection of autoantibodies (n = 2). An We used SPSS 26 (IBM Deutschland GmbH, Ethningen, impaired glucose tolerance was detected in 15/52 (29%) Germany) for statistical analysis. Data were not normally of the patients. 15/52 (29%) patients reported acral, 22/52 Kreß et al. BMC Neuroscience (2023) 24:1 Page 6 of 12 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 ar ea under the curve, IL− interleukin, ROC receiver operating characteristic, TNF tumor necrosis factor-alpha K reß et al. BMC Neuroscience (2023) 24:1 Page 7 of 12 Table 2 Diagnostic value of cytokines in SFN Biomarker AUC (95% CI) Sensitivity % Specificity % Optimal cut-off IL-2 0.68 70 62 0.69 IL-8 0.69 76 62 0.65 TNF 0.67 52 86 0.73 IL-2 + IL-8 0.78 64 91 0.76 IL-2 + TNF 0.78 54 91 0.81 IL-8 + TNF 0.74 90 48 0.54 IL-2 + IL-8 + TNF 0.81 70 86 0.67 AUC area under the curve, CI confidence interval, IL− interleukin, SFN small fiber neuropathy, TNF tumor necrosis factor-alpha Table 3 Comparison of different AUCs in the gene expression levels of the pro-inflammatory cytokines IL-1ß and IL-6 (Fig. 1a) and the anti-inflamma - Cytokine combination p-value 95% CI tory cytokines IL-4, IL-10, and TGF (Fig. 1b). IL-8 vs. IL-2 0.93 − 0.21–0.22 IL-8 vs. TNF 0.75 − 0.13–0.18 Highest accuracy to distinguish SFN patients and healthy IL-2 vs. TNF 0.88 − 0.19–0.22 controls combining IL‑2, IL‑8, and TNF IL-8 vs. IL-8 + IL-2 0.14 − 0.22–0.03 The total AUC of the cytokines IL-2, IL-8, and TNF IL-8 vs. IL-8 + TNF 0.39 − 0.10–0.04 to distinguish between patients and healthy controls IL-2 vs. IL-2 + IL-8 0.07 0.68–0.78 was > 0.5, but remained < 0.7 when investigating sin- IL-2 vs. IL-2 + TNF 0.08 0.68–0.79 gle cytokines separately (Fig. 2a, Table 2). A combina- TNF vs. TNF + IL-8 0.27 0.67–0.72 tion of two cytokines resulted in an AUC = 0.74 (for TNF vs. TNF + IL-2 0.04 − 0.24–0.00 IL-8 and TNF) and an AUC = 0.78 for IL-2 and IL-8 or TNF + IL-2 + IL-8 vs. IL-2 + IL-8 0.46 − 0.39–0.09 IL-2 and TNF (Fig. 2b, Table 2). We achieved the high- TNF + IL-2 + IL-8 vs. TNF + IL-2 0.40 − 0.03–0.07 est AUC = 0.81 by combining IL-2, IL-8, and TNF and TNF + IL-2 + IL-8 vs. TNF + IL-8 0.14 − 0.03–0.20 reached a sensitivity of 70% together with 86% specific - TNF + IL-2 + IL-8 vs. IL-8 0.08 − 0.01–0.25 ity (Fig. 2c, Table 2). When comparing AUC, individual TNF + IL-2 + IL-8 vs. IL-2 0.05 0.81–0.68 cytokines and the majority of combinations of two or TNF + IL-2 + IL-8 vs. TNF 0.02 0.02–0.26 three cytokines did not differ in their discriminative efficacy between SFN and healthy controls (Table 3). AUC area under the curve, CI confidence interval, IL− interleukin, TNF tumor necrosis factor-alpha The combinations of TNF + IL-2 and TNF + IL-2 + IL-8 showed best discrimination between the two groups (Table 3). (42%) generalized, and 15/52 (29%) simultaneously acral and generalized pain. The median current pain intensity Higher expression of TNF and IL‑8 in WBC of female SFN was 4/10 NRS with a range from 0–8. Female patients patients compared to female controls reported a mean pain intensity of 5/10 NRS (range 0–8), We found higher gene expression levels of IL-8 and TNF male patients of 4/10 NRS (range 0–8). In 30/52 (58%) of in female SFN patients (n = 32) compared to female con- SFN patients, signs of small fiber impairment were found trols (n = 13) (IL-8: p = 0.01; TNF: p = 0.03) (Fig. 3a), on neurological examination, namely: thermal hypoes- while we did not detect intergroup differences in male thesia (n = 13), hypo-/hyperalgesia (n = 15), allodynia patients (n = 20) and male controls (n = 8) (Fig. 3b). (n = 4), dys-/paresthesia (n = 7). 29/52 (56%) patients When comparing cytokine levels of female and male SFN reported autonomic symptoms; dyshidrosis was the most patients, we did not find a difference in the gene expres - common (n = 29). sion levels of the investigated pro- and anti-inflammatory cytokines (Fig. 3c). Gene expression data Higher expression of IL‑2, IL‑8, and TNF in WBC of SFN Blood cytokine expression profiles differ among SFN patients patients compared to controls with moderate and severe pain intensity and among patients Gene expression of the pro-inflammatory cytokines IL-2, with acral and generalized pain IL-8, and TNF was higher in patients (n = 52) compared When stratifying data for pain intensity, we detected to controls (n = 21) (IL-2: p = 0.02; IL-8: p = 0.009; TNF: higher gene expression levels of IL-2 in patients with no p = 0.03) (Fig. 1a). We did not find intergroup differences Kreß et al. BMC Neuroscience (2023) 24:1 Page 8 of 12 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 to moderate pain intensity (NRS < 4) (n = 35) compared Discussion to patients with severe pain (NRS ≥ 4) (n = 17) (p = 0.02) We investigated systemic gene and protein expression (Fig. 4a). Dividing the SFN group into patients with acral levels of selected pro- and anti-inflammatory cytokines (n = 15) and generalized pain (n = 21), we found higher of SFN patients compared to healthy controls. We gene expression levels of IL-10 in patients with acral pain found higher gene expression of the pro-inflammatory (p = 0.004) (Fig. 4b) compared to patients with general- cytokines IL-2, IL-8, and TNF in patients compared to ized pain. Patients with alternating acral and general- controls, which also distinguished well between patients ized pain were excluded in this analysis. Thus, the total and controls when combined. amount of patients does not reach n = 52. Cytokines play a major role in the pathogenesis of neu- ropathic pain [19, 20] and large-fiber neuropathies on a local and systemic level [21–23]. Cytokines are also cru- IL‑2 gene expression negatively correlates with pain intensity cial for activation and recruitment of immune cells and We further found a negative correlation between the rel- are produced by a wide range of blood cells e.g. mono- ative gene expression of IL-2 and the current pain inten- cytes, natural killer cells, peripheral blood mononuclear sity in SFN patients (p = 0.02) (Fig. 5a). Gene expression cells (PBMC), and T-lymphocytes . Their expression of IL-8 and TNF did not correlate with pain intensity patterns vary depending on disease etiology and investi- (Fig. 5b, c). gated biomaterial [14, 25, 26]. Previously, we found higher systemic RNA levels of IL-2, TGF, and IL-10 in whole blood samples of SFN Protein levels of IL-8 and TNF are similar in serum of SFN patients compared to controls . Here, we show higher patients and controls IL-2, IL-8, and TNF gene expressions in patients with Protein levels of the pro-inflammatory cytokines IL-8 and SFN compared to controls when assessing RNA from the TNF did not differ in serum of patients (n = 26; n = 15 WBC fraction of blood samples, whereas the protein lev- female, n = 11 male) compared to controls (n = 8; n = 5 els of IL-8 and TNF did not differ. female, n = 3 male) (Fig. 6). IL-2 serum levels remained below the detection thresholds of the ELISA kits used. K reß et al. BMC Neuroscience (2023) 24:1 Page 9 of 12 Higher serum TNF expression was previously reported in SFN due to sarcoidosis compared to controls [29, 30]. TNF serum expression was also higher in patients with diabetic polyneuropathy compared to diabetes without polyneuropathy . Further, patients with bortezomib therapy-induced neuropathy showed higher serum levels of TNF in contrast to controls . There are also reports on an analgesic effect of anti-TNF therapy in patients with SFN due to sarcoidosis . Previous studies assessing demographic data and data on pain history of women and men with chronic pain conditions reported equivocal results [33–36]. Some studies found major variations in pain sensitivity and intensity between women and men [33–35]. Oth- ers described minor differences with lower thermal and mechanical pain thresholds in women compared to men, while the average pain intensity did not differ between sexes . In line with these data, we found no relevant differences in pain intensity levels between Fig. 4 Relative gene expression of cytokines in SFN patients stratified women and men. for pain phenotype. The scatter plots show the gene expression of One study described a positive correlation between pro- and anti-inflammatory cytokines when comparing patients with the protein expression of 17 different cytokines (meas - moderate (NRS < 4) to severe (NRS ≥ 4) pain a and with acral pain to ured in cerebrospinal fluid, plasma, and salvia) and pain 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 intensity in patients with neuropathic pain syndromes patients with acral pain compared to generalized pain (b). Numbers . In our previous study, we did not find a correlation of samples investigated: moderate pain = 35; severe pain = 17; acral between pain questionnaire data (using the short form pain = 15; generalized pain = 21. IL− interleukin, NRS numeric rating of the Mc Gill pain questionnaire, the Neuropathic scale, SFN small fiber neuropathy, TGF transforming growth factor Pain Symptom Inventory, and the Graded Chronic Pain beta-1, TNF tumor necrosis factor-alpha. *p < 0.05; **p < 0.01 Scale) and cytokine expression in SFN patients . Here, we detected a negative correlation between WBC cytokine gene expression of IL-2 and the current pain While confirming previous data of an elevated systemic intensity, whereas no correlation was found for IL-8 IL-2 gene expression in SFN patients , higher systemic and TNF. With the current study, we provide further IL-2 RNA levels were also found in PBMC of patients evidence for distinct cytokine patterns in patient bio- with painful polyneuropathies of various etiologies com- material, that may be of use in the clinical management pared to controls . Thus, an elevated systemic IL-2 of SFN patients after future mechanistic exploration. gene expression is not specific for SFN but may be of Apart from the pathophysiological role of cytokines, potential importance in the development and mainte- one study provided diagnostic information using serum nance of neuropathic pain in SFN. levels of IL-6, IL-17, and TNF in patients with diabetic In a former study, we found higher IL-8 gene expres- neuropathy . In line with our results, the AUC sion in fibroblasts obtained from skin punch biopsies of remained below 0.7 for single cytokines and increased SFN patients . Here, we provide evidence for elevated after combination of two cytokines to at least 0.7 . IL-8 RNA levels also in the WBC fraction. Elevated IL-8 Although no single cytokine was likely to show suffi - RNA levels in skin and blood cells of SFN patients may cient diagnostic performance in SFN, our findings point indicate a local and systemic inflammatory state. This towards the potential usefulness of cytokine combina- makes IL-8 an interesting target gene for potential ther- tions. To explore clinical utility, further studies includ- apeutic means in SFN pending further validation. It is of ing co-influencing parameters and analyzing a larger note that an enhanced expression level of IL-8 was also study cohort are necessary. found in blood, tissue samples, and cerebrospinal fluid In contrast to the results of the gene expression anal- of patients with various chronic pain conditions based ysis in WBC, we did not find intergroup differences on small fiber pathology such as burning mouth syn - in serum protein levels of the investigated cytokines, drome  or in patients with postherpetic neuralgia which may be due to the small sample size as we con- . firmed by post-hoc sample size calculation. Another Kreß et al. BMC Neuroscience (2023) 24:1 Page 10 of 12 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 limitation of our study was the restricted subgroup in a clinically well-characterized study cohort. Further analysis due to the small study cohort. Still, we pro- studies are needed to determine the underlying mecha- vide evidence for higher WBC IL-2, IL-8, and TNF nisms potentially linking systemic cytokine expression expression in SFN compared to controls investigated with SFN pain. Conclusions We found higher systemic gene expression levels of IL-2, IL-8, and TNF in SFN patients, which may be of poten- tial relevance in the development and maintenance of neuropathic pain in SFN. Our data may have implications for accomplishing SFN diagnostics by objective markers and for patient stratification in clinical management and research, which needs further determination in larger Fig. 6 Protein expression of cytokines in serum of SFN patients patient cohorts. 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 Abbreviations between groups. Number of samples investigated: SFN patients = 26 A Absorbance (n = 15 female, n = 11 male); controls = 8 (n = 5 female, n = 3 male). AUC Ar ea under the curve IL− interleukin, SFN small fiber neuropathy, TNF tumor necrosis CCM Corneal confocal microscopy factor-alphaCI Confidence interval CRP C-reactive protein EDTA Ethylenediaminetetraacetic acid K reß et al. BMC Neuroscience (2023) 24:1 Page 11 of 12 ELISA Enzyme-linked immunosorbent assay Received: 30 April 2022 Accepted: 20 December 2022 F Female HbA1c Hemoglobin A1c IENFD Intraepidermal nerve fiber density IL Interleukin M Male References NA Not applicable 1. Devigili G, Tugnoli V, Penza P, Camozzi F, Lombardi R, Melli G, et al. The NRS Numeric rating scale diagnostic criteria for small fibre neuropathy: from symptoms to neuro - oGTT Oral glucose t olerance test pathology. Brain. 2008;131(Pt 7):1912–25. PBMC Peripheral blood mononuclear cells 2. Gross F, Üçeyler N. Mechanisms of small nerve fiber pathology. Neurosci PCR Polymerase chain reaction Lett. 2020;737: 135316. PREP Pain related evoked potentials 3. Menichella DM, Abdelhak B, Ren D, Shum A, Frietag C, Miller RJ. CXCR4 QST Quantitative sensory testing chemokine receptor signaling mediates pain in diabetic neuropathy. Mol QSART Quantitativ e sensory axon reflex test Pain. 2014;10:42. Ref. Reference 4. Sharma D, Gondaliya P, Tiwari V, Kalia K. Kaempferol attenuates diabetic RNA Ribonucleic acid nephropathy by inhibiting RhoA/Rho-kinase mediated inflammatory ROC Receiver operating characteristic signalling. Biomed Pharmacother. 2019;109:1610–9. RT Room temperature 5. Tiwari V, Chopra K. Resveratrol prevents alcohol-induced cognitive defi- SFN Small fiber neur opathy cits and brain damage by blocking inflammatory signaling and cell death TGF Transforming growth factor beta-1 cascade in neonatal rat brain. J Neurochem. 2011;117(4):678–90. TNF T umor necrosis factor-alpha 6. Kreß L, Hofmann L, Klein T, Klug K, Saffer N, Spitzel M, et al. Differential TSH Thyroid stimulating hormone impact of keratinocytes and fibroblasts on nociceptor degeneration and WBC White blood cells sensitization in small fiber neuropathy. Pain. 2021;162(4):1262–72. 7. Üçeyler N, Kafke W, Riediger N, He L, Necula G, Toyka KV, et al. Elevated Acknowledgements proinflammatory cytokine expression in affected skin in small fiber neu- We thank Danilo Prtvar, Daniela Urlaub, and our undergraduate students ropathy. Neurology. 2010;74(22):1806–13. Philine Dinkel, B.Sc., Helen-Desirée Seibert, and Maria Strunz for expert techni- 8. Bergantini L, Bargagli E, d’Alessandro M, Refini RM, Cameli P, Galasso L, cal support. We also thank Christoph Erbacher, M.Sc. who has helped with et al. Prognostic bioindicators in severe COVID-19 patients. Cytokine. Figure design. 2021;141: 155455. 9. Gille B, De Schaepdryver M, Dedeene L, Goossens J, Claeys KG, Van Den Author contributions Bosch L, et al. Inflammatory markers in cerebrospinal fluid: independent All authors have written and / or edited the manuscript. Recruitment of prognostic biomarkers in amyotrophic lateral sclerosis? J Neurol Neuro- patients and healthy controls was performed by LK, NE, CS, and NÜ. LK and surg Psychiatry. 2019;90(12):1338–46. NÜ did the gene expression analysis and data analysis and the interpretation 10. Bhethanabhotla S, Tiwari A, Sharma MC, Vishnubhatla S, Bakhshi S. of the biomaterial data. NE investigated patients and healthy controls and Prognostic significance of IL-6 in hodgkin lymphoma. Indian J Pediatr. analyzed clinical data. CS contributed to clinical examination of the recruited 2019;86(6):551–4. patients. NÜ designed the study concept, raised funding for the study, con- 11. Song J, Park DW, Moon S, Cho HJ, Park JH, Seok H, et al. Diagnostic and tributed to patient recruitment, clinical examination, data analysis, and data prognostic value of interleukin-6, pentraxin 3, and procalcitonin levels interpretation. All authors read and approved the final manuscript. among sepsis and septic shock patients: a prospective controlled study according to the Sepsis-3 definitions. BMC Infect Dis. 2019;19(1):968. Funding 12. Froeschle GM, Bedke T, Boettcher M, Huber S, Singer D, Ebenebe CU. Open Access funding enabled and organized by Projekt DEAL. This publica- T cell cytokines in the diagnostic of early-onset sepsis. Pediatr Res. tion was supported by the Open Access Publication Fund of the University 2021;90(1):191–6. of Würzburg. The study was founded by the German Research Foundation 13. Zheng YH, Ren CY, Shen Y, Li JB, Chen MW. A cross-sectional study on the (Deutsche Forschungsgemeinschaft, DFG, N.Ü.: UE171/3-1). LK was funded by correlation between inflammatory cytokines, negative emotions, and the Interdisciplinary Center for Clinical Research (Z-2/CSP_22). NÜ was funded onset of peripheral neuropathy in type 2 diabetes. Neuropsychiatr Dis by DFG (UE171/15-1). Treat. 2020;16:2881–90. 14. Langjahr M, Schubert AL, Sommer C, Üçeyler N. Increased pro-inflamma- Availability of data and materials tory cytokine gene expression in peripheral blood mononuclear cells of All data generated or analyzed during this study are included in this published patients with polyneuropathies. J Neurol. 2018;265(3):618–27. article. In case of any queries, please contact the corresponding author. 15. Egenolf N, Zu Altenschildesche CM, Kreß L, Eggermann K, Namer B, Gross F, et al. Diagnosing small fiber neuropathy in clinical practice: a deep phe - notyping study. Ther Adv Neurol Disord. 2021;14:17562864211004318. Declarations 16. Schubert AL, Held M, Sommer C, Üçeyler N. Reduced gene expression of netrin family members in skin and sural nerve specimens of patients with Ethics approval and consent to participate painful peripheral neuropathies. J Neurol. 2019;266(11):2812–20. Our study was approved by the Ethics Committee of the University of 17. Üçeyler N, Riediger N, Kafke W, Sommer C. Differential gene expression Würzburg (#135/15). The ethics statement included all the above-mentioned of cytokines and neurotrophic factors in nerve and skin of patients with (please refer methods) clinical examinations and collecting blood samples peripheral neuropathies. J Neurol. 2015;262(1):203–12. from each participant. All methods were carried out in accordance with 18. Goksuluk D, Korkmaz S, Zararsiz G, Karaagaoglu AE. easyROC: an interac- relevant guidelines and regulations. Patients and controls gave their written tive web-tool for ROC curve analysis using R language environment. R J. informed consent to all study parts before inclusion. 2016;8(2):213. 19. Cohen SP, Mao J. Neuropathic pain: mechanisms and their clinical impli- Consent for publication cations. BMJ. 2014;348: f7656. Not applicable. 20. Jönsson M, Gerdle B, Ghafouri B, Bäckryd E. The inflammatory profile of cerebrospinal fluid, plasma, and saliva from patients with severe Competing interests neuropathic pain and healthy controls-a pilot study. BMC Neurosci. Authors declare no conflicts of interest. 2021;22(1):6. Kreß et al. BMC Neuroscience (2023) 24:1 Page 12 of 12 21. Beppu M, Sawai S, Misawa S, Sogawa K, Mori M, Ishige T, et al. Serum cytokine and chemokine profiles in patients with chronic inflammatory demyelinating polyneuropathy. J Neuroimmunol. 2015;279:7–10. 22. Furukawa T, Matsui N, Fujita K, Miyashiro A, Nodera H, Izumi Y, et al. Increased proinflammatory cytokines in sera of patients with multifocal motor neuropathy. J Neurol Sci. 2014;346(1–2):75–9. 23. Vincent AM, Callaghan BC, Smith AL, Feldman EL. Diabetic neuropa- thy: cellular mechanisms as therapeutic targets. Nat Rev Neurol. 2011;7(10):573–83. 24. Poznanski SM, Lee AJ, Nham T, Lusty E, Larché MJ, Lee DA, et al. Combined stimulation with interleukin-18 and interleukin-12 potently induces interleukin-8 production by natural killer cells. J Innate Immun. 2017;9(5):511–25. 25. Magrinelli F, Briani C, Romano M, Ruggero S, Toffanin E, Triolo G, et al. The association between serum cytokines and damage to large and small nerve fibers in diabetic peripheral neuropathy. J Diabetes Res. 2015. https:// doi. org/ 10. 1155/ 2015/ 547834. 26. Ziegler D, Strom A, Bönhof GJ, Kannenberg JM, Heier M, Rathmann W, et al. Deficits in systemic biomarkers of neuroinflammation and growth factors promoting nerve regeneration in patients with type 2 diabetes and polyneuropathy. BMJ Open Diabetes Res Care. 2019;7(1): e000752. 27. Barry A, O’Halloran KD, McKenna JP, McCreary C, Downer EJ. Plasma IL-8 signature correlates with pain and depressive symptomatology in patients with burning mouth syndrome: Results from a pilot study. J Oral Pathol Med. 2018;47(2):158–65. 28. Zhao W, Wang Y, Fang Q, Wu J, Gao X, Liu H, et al. Changes in neuro- trophic and inflammatory factors in the cerebrospinal fluid of patients with postherpetic neuralgia. Neurosci Lett. 2017;637:108–13. 29. Heij L, Dahan A, Hoitsma E. Sarcoidosis and pain caused by small-fiber neuropathy. Pain Res Treat. 2012. https:// doi. org/ 10. 1155/ 2012/ 256024. 30. Lu SC, Chang YS, Kan HW, Hsieh YL. Tumor necrosis factor-α mediated pain hypersensitivity through Ret receptor in resiniferatoxin neuropathy. Kaohsiung J Med Sci. 2018;34(9):494–502. 31. Li X, Zhu J, Liu N, Liu J, Zhang Z. TNF-alpha in peripheral neuropathy patients with impaired glucose regulation. J Diabetes Res. 2017. https:// doi. org/ 10. 1155/ 2017/ 70240 24. 32. Zhao W, Wang W, Li X, Liu Y, Gao H, Jiang Y, et al. Peripheral neuropathy following bortezomib therapy in multiple myeloma patients: asso- ciation with cumulative dose, heparanase, and TNF-α. Ann Hematol. 2019;98(12):2793–803. 33. Fillingim RB, King CD, Ribeiro-Dasilva MC, Rahim-Williams B, Riley JL 3rd. Sex, gender, and pain: a review of recent clinical and experimental find- ings. J Pain. 2009;10(5):447–85. 34. Mogil JS. Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon. Nat Rev Neurosci. 2012;13(12):859–66. 35. Bartley EJ, Fillingim RB. Sex differences in pain: a brief review of clinical and experimental findings. Br J Anaesth. 2013;111(1):52–8. 36. Meyer-Frießem CH, Attal N, Baron R, Bouhassira D, Finnerup NB, Freynha- gen R, et al. Pain thresholds and intensities of CRPS type I and neuro- pathic pain in respect to sex. Eur J Pain. 2020;24(6):1058–71. 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BMC Neuroscience – Springer Journals
Published: Jan 5, 2023
Keywords: Small fiber neuropathy; Cytokines; White blood cells; Gene expression
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