J Neurol DOI 10.1007/s00415-014-7556-8

ORIGINAL COMMUNICATION

Differential gene expression of cytokines and neurotrophic factors in nerve and skin of patients with peripheral neuropathies ¨ c¸eyler • Nadja Riediger • Waldemar Kafke Nurcan U Claudia Sommer



Received: 14 August 2014 / Revised: 17 October 2014 / Accepted: 21 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Pathophysiologically relevant alterations in cytokine and neurotrophic factor levels have been reported in neuropathy subtypes. We characterized gene expression profiles of pro- and anti-inflammatory cytokines and neurotrophic factors in nerve and skin samples of patients with neuropathies of different etiologies. We prospectively studied 133 patients with neuropathies and compared data between subtypes and with healthy controls. All patients underwent sural nerve and/or skin punch biopsy at the lateral thigh and lower leg; controls received skin punch biopsies. Gene expression of pro- and anti-inflammatory cytokines (IL-1b, IL-2, IL-6, TNF, IL-10), neurotrophic factors (BDNF, NGF, NT3, TrkA), and erythropoietin with the erythropoietin receptor (Epo, EpoR) was analyzed. Sural nerve gene expression of the investigated cytokines and neurotrophic factors did not differ between neuropathies of different etiologies; however, IL-6 (p \ 0.01) and IL-10 (p \ 0.05) expression was higher in painful compared to painless neuropathies. Skin IL-6 and IL-10 gene expression was increased in patients compared to controls (p \ 0.05), and IL-10 expression was higher in lower leg skin of patients with non-inflammatory neuropathies compared to inflammatory neuropathies (p \ 0.05). Proximal and distal skin neurotrophic factor and Epo gene expression of patients with neuropathies was reduced compared to controls (NGF, NT3, Epo; p \ 0.05). Neuropathies are associated with an increase in cytokine expression and a decrease in neurotrophic factor expression including nerve and skin.

¨ c¸eyler (&)  N. Riediger  W. Kafke  C. Sommer N. U Department of Neurology, University of Wu¨rzburg, Josef-Schneider-Str. 11, 97080 Wu¨rzburg, Germany e-mail: [email protected]

Keywords Peripheral neuropathy  Sural nerve biopsy  Skin biopsy  Cytokine  Neurotrophic factor  Gene expression

Introduction The pathophysiology of many types of peripheral neuropathies is incompletely understood. The potential role of the immune system and of pro- and anti-inflammatory cytokines and neurotrophic factors in particular have been investigated mainly in the immune-mediated neuropathies chronic inflammatory demyelinating neuropathy (CIDP) and Guillain–Barre´ syndrome (GBS) [8, 49] and in diabetic neuropathy, the most frequent metabolic one. The major finding was an increase of the investigated proinflammatory cytokines in blood, cerebrospinal fluid, and sural nerve biopsy specimens in patients with these neuropathies [12, 18, 21, 23, 35, 37, 39, 46, 48, 50]. In two studies on CIDP, a major role of CD8-positive T cells was found, involving clonal cellular expansion in blood and nerve samples of these patients, linking systemic and local immune alterations [38, 51]. Elevated interleukin-17 levels in different body fluids have also been described [19, 30–32]. Neurotrophic factors are necessary for nerve integrity. Information on their expression in patients with peripheral neuropathy is scarce and conflicting. For example, ciliary neurotrophic factor was found in cerebrospinal fluid (CSF) of patients with GBS and CIDP while it was absent in controls [36]. Also, increased neurotrophic factor expression was reported in sural nerve specimens of CIDP patients compared to autopsy nerves [63]. In skin samples of patients with diabetic neuropathy neurotrophic factor expression was reduced [1].

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In all these studies subject groups were small and restricted to distinct diagnoses, using one biosample, for example either skin, or nerve, or cerebrospinal fluid. Others used a microarray-based approach, where genes involved in immune regulation and repair were found to be regulated [44, 54]. We set out to specifically characterize gene expression profiles of pro- and anti-inflammatory cytokines and neurotrophic factors in sural nerve and skin punch biopsies of a large group of patients with neuropathies of different etiologies. We aimed to identify (a) specific profiles of these factors in nerve and skin characterizing neuropathies in comparison to controls; (b) potential biomarkers that would aid in the differential diagnosis of the neuropathies. Given the widely discussed association between pro-inflammatory cytokines, nerve growth factor (NGF), and neuropathic pain, we (c) hypothesized that painful and painless neuropathies would differ in their respective expression profiles.

Patients and methods Patient assessment and diagnostic classification Between 2007 and 2009 patients were recruited at the Department of Neurology, University of Wu¨rzburg, where they underwent diagnostic work-up including sural nerve and/or skin punch biopsy. The study was approved by the Wu¨rzburg Medical Faculty Ethics Committee, and written informed consent was obtained from every study participant before inclusion. Patients were excluded if they were on immunosuppressive or immunomodulatory treatment or had an ongoing infection. The diagnosis of neuropathy was based on the typical symptoms and signs, questionnaire assessment for pain (Neuropathic Pain Symptom Inventory; Graded Chronic Pain Scale), and neurological examination. For differential diagnosis all patients underwent detailed laboratory studies including the following parameters: glucose metabolism (including HbA1c, oral glucose tolerance test), whole blood and differential cell counts, erythrocyte sedimentation rate, C-reactive protein, serum electrolytes, monoclonal immunoglobulins, vitamin levels (B6, B12), folic acid, renal and liver function tests, thyroid function tests, anti-nuclear antibodies (ANA), antibodies to extractable nuclear antigen (ENA), anti-neutrophil cytoplasmic autoantibody (ANCA), rheumatoid factor, serology of borreliosis, immunofixation, and serum electrophoresis. Urine analysis was performed for BenceJones proteins, if the serum was positive for a monoclonal immunoglobulin. All patients underwent diagnostic lumbar puncture, either at our Department or had undergone lumbar puncture in a hospital prior to admission. The cerebrospinal fluid was examined with regard to cell count

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and protein levels. A complete electrophysiological assessment with standard nerve conduction studies in motor and sensory nerves of the upper and lower limbs and needle electromyography in affected muscles was performed in all patients as needed [24]. The following diagnostic subgroups were distinguished: Chronic inflammatory demyelinating neuropathy (CIDP): patients were diagnosed as CIDP when the INCAT criteria were fulfilled (inflammatory neuropathy cause and treatment) [22]. ‘‘CIDPclin’’: these patients had the typical clinical presentation and laboratory findings, and showed a demyelinating neuropathy in neurophysiological and histological assessment as is characteristic for CIDP, but did not fulfill the neurophysiological INCAT criteria. ‘‘CIDPsens’’: these patients had purely sensory symptoms with a duration of C2 months, signs of demyelination in neurophysiological assessment, signs of demyelination and inflammation in the sural nerve biopsy, elevated CSF protein, a positive response on steroid treatment, and normal to slightly reduced intraepidermal nerve fiber density (IENFD) in the skin punch biopsy taken from the distal lateral thigh and stained with the pan-axonal marker protein gene product 9.5 (PGP9.5, see below) [4, 16]. Multifocal motor neuropathy (MMN), multifocal acquired demyelinating sensory and motor neuropathy (MADSAM), and Guillain–Barre´ syndrome (GBS): MMN, MADSAM, and GBS were diagnosed according to published criteria [3, 14, 29]. Paraproteinemic neuropathies: neuropathies with an IgM paraprotein with and without anti-MAG antibodies were summarized in this group [15]. Vasculitic neuropathy: these patients were divided in those with systemic vasculitis and those with non-systemic vasculitic neuropathy (NSVN) [7]. Chronic idiopathic axonal polyneuropathy (CIAP): these patients reported slow disease onset with slow progression. Clinical presentation was sensory-motor and neurophysiology revealed axonal neuropathy. Histology was axonal but without signs of inflammation. CSF was normal and distal IENFD was reduced. Steroid treatment had no effect [61]. Progressive idiopathic axonal neuropathy (PIAN): patients had a neuropathy with an acute or subacute beginning with slow progression; symptoms were sensory– motor, with axonal neurophysiology and histology; histology also showed inflammation. CSF protein was increased and patients had a positive response upon steroid treatment. Skin biopsy revealed distal IENFD reduction or loss [61]. Diabetic neuropathy: diabetic neuropathy was diagnosed if the patient had diabetes mellitus type I or II and if typical clinical, laboratory, and electrophysiological findings were present.

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Hereditary neuropathy: hereditary neuropathy was diagnosed if genetic testing was positive, or if clinical presentation, neurophysiological data, and family history were indicative. Idiopathic small fiber neuropathy (SFN): SFN diagnosis was made according to published criteria [10, 25]. ‘‘Other origin’’: cases of definite other etiology were summarized in this category such as neuropathy due to vitamin deficiency, amyloidosis, or paraneoplastic neuropathy. ‘‘Unknown etiology’’: in this group we summarized all cases in which a definitive diagnosis as detailed above was not possible at the time point of examination. In addition, we recruited a control group of 20 healthy volunteers for skin punch biopsies. Of these ten (5 men, 5 women; median age 56 years, range 26–84) were suitable for further analysis; ten were excluded due to reduced IENFD as a sign of small fiber impairment.

isopropanol (500 ll) and incubated (25 °C, 10 min). After another spin down step (12,000g, 10 min, 4 °C) the pellet was washed using ethanol 75 % and spun down again (7,500g, 5 min, 4 °C). Then the samples were air-dried and the pellet was dissolved in diethylpyrocarbonate treated water before incubation in a water bath (55 °C, 10 min). Reverse transcription PCR All PCR reagents and cyclers were used from Life Technologies (Carlsbad, CA, USA). Five hundred nanograms of RNA was reverse transcribed to cDNA using TaqMan Reverse Transcription ReagentsÒ in a total volume of 100 ll including the following reagents: 10 ll 109 PCRbuffer, 6.25 ll Multiscribe reverse transcriptase, 2 ll RNase inhibitor, 22 ll MgCl2 and 20 ll dNTPs. The PCR cycler conditions were as follows: annealing (25 °C, 10 min), reverse transcription (48 °C, 60 min), enzyme inactivation (95 °C, 5 min).

Skin punch biopsy Quantitative real-time PCR Two skin punch biopsies (5 mm; device by Stiefel GmbH, Offenbach, Germany) were taken in local anesthesia as previously described [57]: one from the distal lateral calf 10 cm above the malleolus and one from the proximal lateral thigh. One half of each sample was processed for the assessment of intraepidermal nerve fiber density (IENFD) [57] following internationally accepted quantification rules [26]. One half was flash-frozen in liquid nitrogen and was stored at -80 °C before further processing for gene expression analysis.

For each sample 5 ll cDNA was used in quantitative realtime PCR (qRT-PCR) which was performed in a StepOnePlusTM Cycler. Table 1 summarizes the investigated targets and provides the respective Assay-IDs. As an endogenous control 18sRNA was used. The qRT-PCR reactions contained 12.5 ll TaqMan Master Mix and 1.25 ll of the specific primer in a total volume of 25 ll. The cycler conditions were as follows: incubation (2 min,

Sural nerve biopsy

Table 1 Target genes and assays investigated in skin and sural nerve specimens

Sural nerve biopsy was performed in all patients following a standard procedure [13]. The major part of the sural nerve specimen was used for stains of routine diagnostics; 2–3 mm were dissected, immediately shock-frozen in liquid nitrogen, and stored at -80 °C for further gene expression analysis. Gene expression analysis from nerve and skin biopsies All reagents and cyclers used for quantitative real-time PCR (qRT-PCR) were purchased from Life Technologies (Carlsbad, CA, USA). Nerve and skin biopsy specimens were processed following earlier published protocols [57]. In brief, tissue samples were thawed on ice and homogenized in TRIzol reagentÒ (Invitrogen, Karlsruhe, Germany) using an Ultraturrax homogenizer (Polytron PT 1600EÒ, Kinematica, Luzern, Switzerland). Chloroform (200 ll) was added and incubated (25 °C, 3 min). After spin down (12,000g, 15 min, 4 °C) the supernatant was mixed with

Marker

Assay-ID

Nerve Nerve growth factor (NGF)

Hs00171458_m1

Neurotrophic factor-3 (NT3)

Hs00267375_s1

Tyrosine-kinase A (TrkA)

Hs00176787_m1

Tumor necrosis factor-alpha (TNF)

Hs00174128_m1

Interleukin-1b (IL-1b)

Hs00174097_m1

Interleukin-6 (IL-6) Interleukin-10 (IL-10)

Hs00174131_m1 Hs00174086_m1

Skin Brain derived neurotrophic factor (BDNF)

Hs00380947_m1

Nerve growth factor (NGF)

Hs00171458_m1

Neurotrophic factor-3 (NT3)

Hs00267375_s1

Interleukin-2 (IL-2)

Hs001741141_m1

Interleukin-6 (IL-6)

Hs00174131_m1

Interleukin-10 (IL-10)

Hs00174086_m1

Erythropoietin (Epo)

Hs00171267_m1

Epo receptor (Epo-R)

Hs00181092_m1

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50 °C), second incubation (95 °C, 10 min), 40 cycles (15 s, 95 °C and 1 min, 60 °C).

Table 2 Patients’ clinical characteristics and diagnostic subgroups Item

Number (% of entire group)

M, F (N)

93, 40

Median age (range)

64 years (29–81)

Median disease duration (range in years)

3 years (0.08–25)

Evaluation of qRT-PCR results We applied the comparative deltaCT method (i.e., relating target gene expression with individual 18sRNA expression) for nerve and skin samples. Lower deltaCT values (i.e., sample detection at earlier PCR cycles) indicate higher gene expression. For intuitive data presentation we chose to illustrate the results as 1/deltaCT, since these reciprocal values result in higher values for higher gene expression. In addition, we compared gene expression in the patient and control groups. To guarantee inter-plate comparability one standard sample was run on each PCR plate. Also, each plate contained a negative control sample without cDNA template to exclude genomic contamination. All target genes were measured as triplicates; the 18sRNA values were very stable and were measured as duplicates. Statistical analysis IBM SPSS 21 Software (Munich, Germany) was used for data analysis. Data analysis gave non-normally distributed values for qRT-PCR results; we therefore applied the nonparametric Mann–Whitney U test for independent groups. Results are illustrated as boxplots giving the median values and the upper and lower 25 % percentile. Since the delta CT method was used for gene expression analysis (i.e., difference between CT value of the respective target gene and the value of the housekeeping gene 18sRNA, see below) lower values indicate higher gene expression. For intuitive illustration, graphs present reciprocal 1/deltaCT values, which allows illustration of low deltaCT values (i.e., high gene expression) as higher boxplots. Statistical significance was assumed at p \ 0.05.

Diagnostic subgroups (N and % of entire group): CIDP

28 (21 %)

Unknown etiology

23 (17 %)

Vasculitic neuropathy NSVN

16 (12 %) 11 (69 %)

CIDPclin

11 (8 %)

CIDPsens

11 (8 %)

PIAN

7 (5 %)

Diabetic neuropathy

6 (4.5 %)

Other non-inflammatory (e.g., toxic, druginduced) Other inflammatory (sarcoidosis, Sjo¨gren’s syndrome, neuroborreliosis) CIAP

6 (4.5 %) 5 (4 %) 4 (3 %)

Hereditary neuropathy

4 (3 %)

Paraproteinemic neuropathy (IgM)

4 (3 %)

Anti-MAG

3 (2 %)

MMN

3 (2 %)

GBS

2 (2 %)

MADSAM

2 (2 %)

SFN

1 (1 %)

Painful, painless (N)

86, 47

Median IENFD Proximal (range)

8 fibers/mm (2–38)

Distal (range)

3 fibers/mm (0–12)

Basic description of the cohort

CIAP chronic idiopathic axonal polyneuropathy, CIDP chronic inflammatory demyelinating polyneuropathy, CIDPclin patients with a clinical presentation typical of CIDP, but not fulfilling electrophysiological INCAT criteria; CIDPsens patients with pure sensory clinical presentation and otherwise like CIDP, but not fulfilling electrophysiological INCAT criteria; F female, GBS Guillain–Barre´ syndrome, IENFD intraepidermal nerve fiber density, INCAT Inflammatory Neuropathy Cause and Treatment Group, MADSAM multifocal acquired demyelinating sensory and motor neuropathy, M male, MMN multifocal motor neuropathy, N number, NSVN nonsystemic vasculitic neuropathy, PIAN progressive idiopathic axonal neuropathy, SFN small fiber neuropathy

We included 133 patients with neuropathies of different etiologies with a median age of 64 years (range 29–81 years). The group consisted of 93 men (median age: 64 years, 37–81) and 40 women (median age: 67 years, 29–78). The median duration of neuropathy until presentation at our department was 3 years (range 0.08–25 years). Clinical characteristics of the study group and neuropathy subgroups are summarized in Table 2. In 89/133 (67 %) patients an inflammatory neuropathy was diagnosed (CIDP, CIDPclin, CIDPsens, vasculitic neuropathy, PIAN,

MMN, GBS, MADSAM, paraproteinemia, neuropathy in sarcoidosis or Sjo¨gren’s syndrome, neuroborreliosis), while in 21/133 (16 %) cases neuropathy was of non-inflammatory origin (diabetic, hereditary, CIAP, toxic, SFN; see Table 2). In 23/133 (17 %) cases the etiology of the neuropathy remained obscure. At the time of admission to our department 7/133 (5 %) patients were on oral steroids (methylprednisolone or prednisolone), 1/133 (0.1 %) patient each was on azathioprine, had received intravenous

Results

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immunoglobulins, or intravenous cyclophosphamide, i.e., 10/133 (8 %) patients had received immunosuppressive or immunomodulatory treatment before sural and skin punch biopsy. Eighty-six (65 %) patients had a painful neuropathy (i.e., current pain intensity C3 on a numeric rating scale ranging from 0 to 10 with 0 ‘‘no pain’’ and 10 ‘‘worst pain imaginable’’). Forty-six (53 %) patients with painful neuropathy were on analgesic medication: pregabalin (n = 30), gabapentin (n = 12), amitriptyline (n = 5), opioids (n = 4), carbamazepine (n = 3); eight of these patients were on a combination of analgesics.

associated with higher IL-6 and IL-10 gene expression in inflammatory and non-inflammatory neuropathies (shown for IL-6 in Fig. 1c). IL-6 did not correlate with the patients’ current pain intensity (data not shown). Gene expression did not differ between inflammatory and non-inflammatory neuropathies as classified by clinical diagnosis. Also, no intergroup difference was found when comparing the diagnostic subgroups for any of the assessed targets. Disease duration had no influence on the expression of the investigated markers in the sural nerve (cases with \3 versus C3 years).

Painful neuropathies are associated with increased IL-6 and IL-10 gene expression in the sural nerve

Peripheral neuropathies are associated with increased IL-6 and IL-10 expression in skin and reduced cutaneous neurotrophic factors

In sural nerve samples we analyzed the gene expression of IL-1b, IL-6, IL-10, TNF, TrkA, NGF, and NT3 normalized to the individual 18sRNA gene expression (housekeeping gene). We found that painful neuropathies were associated with higher IL-6 (p \ 0.01, Fig. 1a) and IL-10 (p \ 0.05, Fig. 1b) gene expression in the sural nerve when compared with painless neuropathies. Interestingly, pain was

In skin biopsy samples we analyzed the gene expression of IL-2, IL-6, IL-10, TNF, BDNF, Trk, NT3, Epo, and Epo-R normalized to the individual 18sRNA gene expression (housekeeping gene) and compared to healthy controls. We first assessed the group of patients with peripheral neuropathies as a whole. In skin from the distal and proximal leg patients with neuropathies had a higher expression of

Fig. 1 Gene expression of IL-6 and IL-10 in sural nerve specimens Boxplots show 1/deltaCT values, i.e., reciprocal relation of the CT value of the target normalized to the housekeeping gene 18sRNA, of interleukin-6 (IL-6; a **p \ 0.01) and IL-10 (b *p \ 0.05). The reciprocal value allows illustration of low deltaCT values (i.e., high gene expression) as higher boxplots. Gene expression was higher in patients with painful neuropathies compared to patients with painless neuropathies. This distinction was independent of inflammation as shown for IL-6 in c n.s. not significant. N of investigated sural nerve specimens: painful neuropathy, 23; painless neuropathy, 10; inflammatory painful, 22; inflammatory painless, 11; noninflammatory painful, 15; noninflammatory painless, 7

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J Neurol Fig. 2 Gene expression of cytokines, neurotrophic factors, and Epo in distal and proximal skin specimens Boxplots show 1/deltaCT values, i.e., reciprocal relation of the CT value of the target normalized to the housekeeping gene 18sRNA, of the pro-inflammatory cytokine interleukin-6 (IL-6; a, b), the anti-inflammatory cytokine IL10 (c, d), the neurotrophins nerve growth factor (NGF; e, f) and neurotrophin-3 (NT3; g, h), and erythropoietin (Epo; i, j). The reciprocal value allows illustration of low deltaCT values (i.e., high gene expression) as higher boxplots. Gene expression of IL-6 (distal, p [ 0.01; proximal, p \ 0.05; a, b) and of IL-10 (distal and proximal p \ 0.05; c, d) was higher in patients with neuropathies compared to healthy controls. Gene expression of NGF (p \ 0.01) and NT3 (p \ 0.001) was reduced only in proximal skin of patients with neuropathies compared to healthy controls (e–h); NT3 was not measurable in distal skin of healthy controls. Epo gene expression was lower in distal (p \ 0.05) and proximal skin (p \ 0.01) of patients with neuropathies compared to controls (i, j). PNP peripheral neuropathy. N of investigated skin biopsies: PNP, 31; controls, 9

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IL-10 is increased in distal skin of non-inflammatory neuropathies In skin from the distal leg of patients with clinically noninflammatory neuropathies IL-10 gene expression was higher compared to patients with inflammatory neuropathies (p \ 0.05) and compared to control skin (p \ 0.01; Fig. 3a). In proximal skin IL-10 gene expression was higher in patients with inflammatory neuropathies only (p \ 0.05; Fig. 3b). No correlation could be found with regard to prior-to-biopsy treatment with immunosuppressive or immunomodulatory drugs in the respective 10/133 (8 %) patients. Also, further diagnostic subgroups did not differ (data not shown).

Discussion

Fig. 3 Gene expression of IL-10 in distal and proximal skin biopsies Boxplots show 1/deltaCT values, i.e., reciprocal relation of the CT value of the target normalized to the housekeeping gene 18sRNA. The reciprocal value allows illustration of low deltaCT values (i.e., high gene expression) as higher boxplots. Gene expression of interleukin10 (IL-10) in the distal skin of patients with non-inflammatory neuropathies was higher than in patients with inflammatory neuropathies (*p \ 0.05) and in controls (**p \ 0.01; a). In proximal skin IL-10 gene expression was higher in patients with inflammatory cytokines compared to controls. N of investigated skin biopsies: inflammatory PNP, 11; non-inflammatory PNP, 8; controls, 9

IL-6 (distal, p \ 0.01; proximal, p \ 0.05; Fig. 2a, b) and of IL-10 (distal and proximal p \ 0.05, Fig. 2c, d) compared to healthy controls. In contrast, gene expression of the neurotrophins NGF (p \ 0.01) and NT3 (p \ 0.001) was reduced only in proximal skin of patients with neuropathies compared to healthy controls (Fig. 2e–h); NT3 was not measurable in distal skin of healthy controls. Also, Epo gene expression was lower in distal (p \ 0.05) and proximal skin (p \ 0.01) of patients with neuropathies compared to controls (Fig. 2i, j). No correlation was found when comparing skin and nerve cytokine, neurotrophic factor, and Epo gene expression (data not shown). Also, no intergroup differences were found for the other investigated targets.

We here report the results of our study investigating cytokine and neurotrophic factor gene expression profiles in a large cohort of patients with neuropathies of different etiologies seen at one neuromuscular center, and including skin and sural nerve biopsy specimens. We show that painful peripheral neuropathies are associated with an increase in sural nerve IL-6 and IL-10 gene expression and that neuropathy per se is associated with an increase in skin IL-6 and IL-10 and a decrease in neurotrophic factor gene expression. These findings may suggest that neuropathies not only affect peripheral nerves, but also involve skin homeostasis. Studies on cytokine expression in peripheral neuropathies have so far mainly been restricted to patients with CIDP and GBS focusing either on body fluids or on sural nerve biopsy specimens. Assessing serum and plasma, researchers found increased levels of TNF in patients with CIDP and GBS compared to controls [18, 39], as well as increased levels of IL-6 in CSF and in serum [35]. Immunohistochemistry revealed increased TNF in sural nerve biopsies from patients with CIDP [37] and of TNF and IL-1b in GBS [46]. Investigating a large number of nerve samples we could not find cytokines whose expression differed between neuropathy subgroups. In skin, where we were able to compare to controls, we found an increase in distal and proximal IL-6 and IL-10 gene expression in neuropathy patients. This finding might indicate that peripheral neuropathies not only involve the nerves, but also skin and skin homeostasis. One mechanism might be that diseased cutaneous small-caliber nerve fibers induce cytokine gene expression in keratinocytes and fibroblasts [11]; however, with the lack of knowledge about the precise source cells expressing the investigated targets (e.g., epidermal or dermal cells, cutaneous immune cells) and also investigating gene expression only, this remains speculative.

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Pro- and anti-inflammatory cytokines play a critical role in neuropathic pain induction and maintenance [6, 60]. Preclinical and clinical studies indicate that an imbalance of algesic pro-inflammatory and analgesic anti-inflammatory cytokines may be a major factor in the development of neuropathic pain [59]. Painful neuropathies are associated with increased systemic (serum, plasma, CSF) [5, 34, 58] and local (sural nerve biopsy) [17, 33] levels of pro-inflammatory, i.e., algesic cytokines. Here we found that gene expression of both the algesic IL-6 [40, 43] and the analgesic IL-10 cytokine [52, 55] was increased in sural nerve specimens of patients with painful compared to painless neuropathies. IL-6 and IL-10 are both produced in Schwann cells, macrophages and T-lymphocytes in peripheral nerve. Elevated IL-6 levels have been detected in body fluids and tissue of patients with different pain states [28, 33, 56, 57]. Also, antibodies against IL-6 receptors have been reported effective in human neuropathic pain [42]. In this context, our finding of increased IL-6 gene expression in the sural nerve independent of nerve inflammation (Fig. 1) fits well in the current pathophysiological concept of neuropathic pain and is in according with previous findings [33]. The parallel increase of IL-10 expression is as yet unexplained and may indicate a physiologic process toward the resolution of inflammation. The reason for the lack of correlation between skin and nerve target gene expression is unclear. One reason may be the low number of cases in the respective subgroups being compared with each other. Neurotrophic factors are key players for nerve integrity. While increased neurotrophic factor expression has been reported in sural nerve specimens of CIDP patients compared to autopsy nerves [62], NGF expression in skin samples of patients with diabetic neuropathy was reduced [2]. In our cohort of mixed neuropathies, neurotrophic factor expression was also reduced in skin. This reduction was seen for NGF, NT3 and also for Epo in samples from the thigh, but only for Epo in samples from the lower leg. Our findings are in line with recent reports on successful treatment of experimental SFN with glial derived neurotrophic factor (GDFN) via overexpression in keratinocytes or via local application of a small molecule enhancing GDNF signaling [20]. There is a need for objectively measurable diagnostic markers to distinguish between different subtypes of neuropathies. This distinction is mostly a challenge in clinical practice due to insufficient criteria and atypical clinical presentations [4, 41]. Recent studies have investigated skin or sural nerve biopsy specimens of small CIDP patient groups mostly using microarray technology and different potential biomarkers were described in each of these studies [9, 27, 45, 47]. However, so far assessments as for their sensitivity and specificity in comparison to a

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large control group and to other neuropathy subtypes are missing [53]. In our study, we found IL-6 and IL-10 expression increased in nerve and skin of patients with peripheral neuropathies, and NGF, NT-3, and Epo expression reduced in skin. Whether these markers might be useful to facilitate neuropathy diagnostics needs to be tested in future studies. The major limitation of our study is the low number of healthy controls that does not match with the patient group with regard to age and gender distribution. This was mainly due to the fact that we accepted only those volunteers as healthy controls that had a normal IENFD in the skin punch biopsy assessment. Another limitation is that we only assessed gene expression, but did not analyze protein levels. This is due to the limited amount of biosamples that did not allow quantitative measurements using, e.g., ELISA. Additionally, the limited amount of material hampered studying all targets we investigated in nerve also in skin and vice versa. Our study can also not answer the question if cytokine alterations are the cause or the consequence of neuropathies; for this longitudinal studies would be needed. Another aspect is that we cannot be sure if the sural nerve specimens investigated really contained the area with the most intensive pathology. Particularly in inflammatory neuropathies with discontinuous inflammation all over the sural nerve it is at least possible that a non- or less-inflamed nerve area was biopsied, which may have influenced the gene expression results. Neuropathies are not restricted to the peripheral nerves but involve skin gene expression presumably via the interaction between cutaneous cells and nerves. These markers could be useful to differentiate peripheral neuropathies from other differential diagnoses via a simple skin punch biopsy. Acknowledgments Expert technical help by B. Broll, B. Dekant, and S. Mildner is gratefully acknowledged. The study was supported by the Bundesministerium fu¨r Bildung und Forschung (BMBF), Deutscher Forschungsverbund Neuropathischer Schmerz (DFNS), and by intramural funds of the University of Wu¨rzburg. This work is part of the doctoral thesis of N. Riediger. Conflicts of interest of interest.

The authors declare that they have no conflicts

References 1. Anand P (1996) Neurotrophins and peripheral neuropathy. Philos Trans R Soc Lond B Biol Sci 351:449–454 2. Anand P, Terenghi G, Warner G, Kopelman P, Williams-Chestnut RE, Sinicropi DV (1996) The role of endogenous nerve growth factor in human diabetic neuropathy. Nat Med 2:703–707 3. Asbury AK, Cornblath DR (1990) Assessment of current diagnostic criteria for Guillain–Barre syndrome. Ann Neurol 27(Suppl):S21–S24

J Neurol 4. Ayrignac X, Viala K, Koutlidis RM, Taieb G, Stojkovic T, Musset L, Leger JM, Fournier E, Maisonobe T, Bouche P (2013) Sensory chronic inflammatory demyelinating polyneuropathy: an under-recognized entity? Muscle Nerve 48:727–732 5. Backonja MM, Coe CL, Muller DA, Schell K (2008) Altered cytokine levels in the blood and cerebrospinal fluid of chronic pain patients. J Neuroimmunol 195:157–163 6. Calvo M, Dawes JM, Bennett DL (2012) The role of the immune system in the generation of neuropathic pain. Lancet Neurol 11:629–642 7. Collins MP, Mendell JR, Periquet MI, Sahenk Z, Amato AA, Gronseth GS, Barohn RJ, Jackson CE, Kissel JT (2000) Superficial peroneal nerve/peroneus brevis muscle biopsy in vasculitic neuropathy. Neurology 55:636–643 8. Dalakas MC (2014) Pathogenesis of immune-mediated neuropathies. Biochim Biophys Acta. doi:10.1016/j.bbadis.2014.06.013 9. Dalakas MC (2011) Potential biomarkers for monitoring therapeutic response in patients with CIDP. J Peripher Nerv Syst 16(Suppl 1):63–67 10. Devigili G, Tugnoli V, Penza P, Camozzi F, Lombardi R, Melli G, Broglio L, Granieri E, Lauria G (2008) The diagnostic criteria for small fibre neuropathy: from symptoms to neuropathology. Brain 131:1912–1925 11. Diamond J (1994) Nerve-skin interactions in adult and aged animals. Prog Clin Biol Res 390:21–44 12. Doupis J, Lyons TE, Wu S, Gnardellis C, Dinh T, Veves A (2009) Microvascular reactivity and inflammatory cytokines in painful and painless peripheral diabetic neuropathy. J Clin Endocrinol Metab 94:2157–2163 13. Dyck PJ, Dyck PJB, Engelstad J (2005) Pathological alterations of nerves. In: Dyck PJ, Thomas PK (eds) Peripheral Neuropathy. Elsevier Saunders, Philadelphia, pp 733–829 14. EFNS, PNS JTFot (2010) European Federation of Neurological Societies/Peripheral Nerve Society guideline on management of multifocal motor neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society–first revision. J Peripher Nerv Syst 15:295–301 15. EFNS, PNS JTFot (2010) European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of paraproteinemic demyelinating neuropathies. Report of a Joint Task Force of the European Federation of Neurological Societies and the Peripheral Nerve Society–first revision. J Peripher Nerv Syst 15:185–195 16. Eftimov F, van Schaik I (2013) Chronic inflammatory demyelinating polyradiculoneuropathy: update on clinical features, phenotypes and treatment options. Curr Opin Neurol 26:496–502 17. Empl M, Renaud S, Erne B, Fuhr P, Straube A, Schaeren-Wiemers N, Steck AJ (2001) TNF-alpha expression in painful and nonpainful neuropathies. Neurology 56:1371–1377 18. Exley AR, Smith N, Winer JB (1994) Tumour necrosis factoralpha and other cytokines in Guillain–Barre syndrome. J Neurol Neurosurg Psychiatry 57:1118–1120 19. Han RK, Cheng YF, Zhou SS, Guo H, He RD, Chi LJ, Zhang LM (2014) Increased circulating Th17 cell populations and elevated CSF osteopontin and IL-17 concentrations in patients with Guillain–Barre syndrome. J Clin Immunol 34:94–103 20. Hedstrom KL, Murtie JC, Albers K, Calcutt NA, Corfas G (2014) Treating small fiber neuropathy by topical application of a small molecule modulator of ligand-induced GFRalpha/RET receptor signaling. Proc Natl Acad Sci USA 111:2325–2330 21. Herder C, Lankisch M, Ziegler D, Rathmann W, Koenig W, Illig T, Doring A, Thorand B, Holle R, Giani G, Martin S, Meisinger C (2009) Subclinical inflammation and diabetic polyneuropathy: MONICA/KORA Survey F3 (Augsburg, Germany). Diabetes Care 32:680–682

22. Hughes R, Bensa S, Willison H, Van den Bergh P, Comi G, Illa I, Nobile-Orazio E, van Doorn P, Dalakas M, Bojar M, Swan A (2001) Randomized controlled trial of intravenous immunoglobulin versus oral prednisolone in chronic inflammatory demyelinating polyradiculoneuropathy. Ann Neurol 50:195–201 23. Hussain G, Rizvi SA, Singhal S, Zubair M, Ahmad J (2013) Serum levels of TNF-alpha in peripheral neuropathy patients and its correlation with nerve conduction velocity in type 2 diabetes mellitus. Diabetes Metab Syndr 7:238–242 24. Kimura J (2001) Electrodiagnosis in diseases of nerve and muscle: principles and practice. Oxford University Press, New York 25. Lacomis D (2002) Small-fiber neuropathy. Muscle Nerve 26:173–188 26. Lauria G, Cornblath DR, Johansson O, McArthur JC, Mellgren SI, Nolano M, Rosenberg N, Sommer C, European Federation of Neurological S (2005) EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy. Eur J Neurol 12:747–758 27. Lee G, Xiang Z, Brannagan TH 3rd, Chin RL, Latov N (2010) Differential gene expression in chronic inflammatory demyelinating polyneuropathy (CIDP) skin biopsies. J Neurol Sci 290:115–122 ¨ c¸eyler N, Frettlo¨h J, Hoffken O, Krumova EK, Lissek 28. Lenz M, U S, Reinersmann A, Sommer C, Stude P, Waaga-Gasser AM, Tegenthoff M, Maier C (2013) Local cytokine changes in complex regional pain syndrome type I (CRPS I) resolve after 6 months. Pain 154:2142–2149 29. Lewis RA, Sumner AJ, Brown MJ, Asbury AK (1982) Multifocal demyelinating neuropathy with persistent conduction block. Neurology 32:958–964 30. Li S, Jin T, Zhang HL, Yu H, Meng F, Concha Quezada H, Zhu J (2014) Circulating Th17, Th22, and Th1 cells are elevated in the Guillain–Barre syndrome and downregulated by IVIg treatments. Mediators Inflamm 2014:740947 31. Li S, Yu M, Li H, Zhang H, Jiang Y (2012) IL-17 and IL-22 in cerebrospinal fluid and plasma are elevated in Guillain–Barre syndrome. Mediators Inflamm 2012:260473 32. Liang SL, Wang WZ, Huang S, Wang XK, Zhang S, Wu Y (2012) Th17 helper cell and T-cell immunoglobulin and mucin domain 3 involvement in Guillain–Barre syndrome. Immunopharmacol Immunotoxicol 34:1039–1046 33. Lindenlaub T, Sommer C (2003) Cytokines in sural nerve biopsies from inflammatory and non-inflammatory neuropathies. Acta Neuropathol (Berl) 105:593–602 34. Ludwig J, Binder A, Steinmann J, Wasner G, Baron R (2008) Cytokine expression in serum and cerebrospinal fluid in noninflammatory polyneuropathies. J Neurol Neurosurg Psychiatry 79:1268–1273 35. Maimone D, Annunziata P, Simone IL, Livrea P, Guazzi GC (1993) Interleukin-6 levels in the cerebrospinal fluid and serum of patients with Guillain–Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy. J Neuroimmunol 47:55–61 36. Massaro AR, Soranzo C, Carnevale A (1997) Cerebrospinal-fluid ciliary neurotrophic factor in neurological patients. Eur Neurol 37:243–246 37. Mathey EK, Pollard JD, Armati PJ (1999) TNF alpha, IFN gamma and IL-2 mRNA expression in CIDP sural nerve biopsies. J Neurol Sci 163:47–52 38. Mausberg AK, Dorok M, Stettner M, Muller M, Hartung HP, Dehmel T, Warnke C, Meyer Zu, Horste G, Kieseier BC (2013) Recovery of the T-cell repertoire in CIDP by IV immunoglobulins. Neurology 80:296–303 39. Misawa S, Kuwabara S, Mori M, Kawaguchi N, Yoshiyama Y, Hattori T (2001) Serum levels of tumor necrosis factor-alpha in chronic inflammatory demyelinating polyneuropathy. Neurology 56:666–669

123

J Neurol 40. Murakami T, Kanchiku T, Suzuki H, Imajo Y, Yoshida Y, Nomura H, Cui D, Ishikawa T, Ikeda E, Taguchi T (2013) Antiinterleukin-6 receptor antibody reduces neuropathic pain following spinal cord injury in mice. Exp Ther Med 6:1194–1198 41. Nobile-Orazio E (2014) Chronic inflammatory demyelinating polyradiculoneuropathy and variants: where we are and where we should go. J Peripher Nerv Syst 19:2–13 42. Ohtori S, Miyagi M, Eguchi Y, Inoue G, Orita S, Ochiai N, Kishida S, Kuniyoshi K, Nakamura J, Aoki Y, Ishikawa T, Arai G, Kamoda H, Suzuki M, Takaso M, Furuya T, Kubota G, Sakuma Y, Oikawa Y, Toyone T, Takahashi K (2012) Efficacy of epidural administration of anti-interleukin-6 receptor antibody onto spinal nerve for treatment of sciatica. Eur Spine J 21:2079–2084 43. Opree A, Kress M (2000) Involvement of the proinflammatory cytokines tumor necrosis factor-alpha, IL-1 beta, and IL-6 but not IL-8 in the development of heat hyperalgesia: effects on heatevoked calcitonin gene-related peptide release from rat skin. J Neurosci 20:6289–6293 44. Puttini S, Panaite PA, Mermod N, Renaud S, Steck AJ, Kuntzer T (2014) Gene expression changes in chronic inflammatory demyelinating polyneuropathy skin biopsies. J Neuroimmunol 270:61–66 45. Puttini S, Panaite PA, Mermod N, Renaud S, Steck AJ, Kuntzer T (2014) Gene expression changes in chronic inflammatory demyelinating polyneuropathy skin biopsies. J Neuroimmunol 270(1–2):61–66 46. Putzu GA, Figarella-Branger D, Bouvier-Labit C, Liprandi A, Bianco N, Pellissier JF (2000) Immunohistochemical localization of cytokines, C5b-9 and ICAM-1 in peripheral nerve of Guillain– Barre syndrome. J Neurol Sci 174:16–21 47. Renaud S, Hays AP, Brannagan TH 3rd, Sander HW, Edgar M, Weimer LH, Olarte MR, Dalakas MC, Xiang Z, Danon MJ, Latov N (2005) Gene expression profiling in chronic inflammatory demyelinating polyneuropathy. J Neuroimmunol 159:203–214 48. Rentzos M, Angeli AV, Rombos A, Kyrozis A, Nikolaou C, Zouvelou V, Dimitriou A, Zoga M, Evangelopoulos ME, Tsatsi A, Tsoutsou A, Evdokimidis I (2012) Proinflammatory cytokines in serum and cerebrospinal fluid of CIDP patients. Neurol Res 34:842–846 49. Rinaldi S, Bennett DL (2014) Pathogenic mechanisms in inflammatory and paraproteinaemic peripheral neuropathies. Curr Opin Neurol 27:541–551 50. Satoh J, Yagihashi S, Toyota T (2003) The possible role of tumor necrosis factor-alpha in diabetic polyneuropathy. Exp Diabesity Res 4:65–71

123

¨ c¸eyler N, Gobel K, Som51. Schneider-Hohendorf T, Schwab N, U mer C, Wiendl H (2012) CD8? T-cell immunity in chronic inflammatory demyelinating polyradiculoneuropathy. Neurology 78:402–408 52. Soderquist RG, Sloane EM, Loram LC, Harrison JA, Dengler EC, Johnson SM, Amer LD, Young CS, Lewis MT, Poole S, Frank MG, Watkins LR, Milligan ED, Mahoney MJ (2010) Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration. Pharm Res 27:841–854 53. Sommer C, Toyka K (2011) Nerve biopsy in chronic inflammatory neuropathies: in situ biomarkers. J Peripher Nerv Syst 16(Suppl 1):24–29 54. Steck AJ, Kinter J, Renaud S (2011) Differential gene expression in nerve biopsies of inflammatory neuropathies. J Peripher Nerv Syst 16(Suppl 1):30–33 55. Tu H, Juelich T, Smith EM, Tyring SK, Rady PL, Hughes TK Jr (2003) Evidence for endogenous interleukin-10 during nociception. J Neuroimmunol 139:145–149 ¨ c¸eyler N, Ha¨user W, Sommer C (2011) Systematic review with 56. U meta-analysis: cytokines in fibromyalgia syndrome. BMC Musculoskelet Disord 12:245 ¨ c¸eyler N, Kafke W, Riediger N, He L, Necula G, Toyka KV, 57. U Sommer C (2010) Elevated proinflammatory cytokine expression in affected skin in small fiber neuropathy. Neurology 74:1806–1813 ¨ c¸eyler N, Rogausch JP, Toyka KV, Sommer C (2007) Differ58. U ential expression of cytokines in painful and painless neuropathies. Neurology 69:42–49 ¨ c¸eyler N, Scha¨fers M, Sommer C (2009) Mode of action of 59. U cytokines on nociceptive neurons. Exp Brain Res 196:67–78 ¨ c¸eyler N, Sommer C (2008) Status of immune mediators in 60. U painful neuropathies. Curr Pain Headache Rep 12:159–164 61. Vrancken AF, Notermans NC, Jansen GH, Wokke JH, Said G (2004) Progressive idiopathic axonal neuropathy–a comparative clinical and histopathological study with vasculitic neuropathy. J Neurol 251:269–278 62. Yamamoto M, Ito Y, Mitsuma N, Hattori N, Sobue G (2003) Pain-related differential expression of NGF, GDNF, IL-6, and their receptors in human vasculitic neuropathies. Intern Med 42:1100–1103 63. Yamamoto M, Ito Y, Mitsuma N, Li M, Hattori N, Sobue G (2001) Pathology-related differential expression regulation of NGF, GDNF, CNTF, and IL-6 mRNAs in human vasculitic neuropathy. Muscle Nerve 24:830–833

Differential gene expression of cytokines and neurotrophic factors in nerve and skin of patients with peripheral neuropathies.

Pathophysiologically relevant alterations in cytokine and neurotrophic factor levels have been reported in neuropathy subtypes. We characterized gene ...
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