Elevated interleukin-8 expression by skin fibroblasts as a potential contributor to pain in women with Fabry disease

Abstract

Fabry disease (FD) is a lysosomal storage disorder of X-linked inheritance. Mutations in the α-galactosidase A gene lead to cellular globotriaosylceramide (Gb3) depositions and triggerable acral burning pain in both sexes as an early FD symptom of unknown pathophysiology. We aimed at elucidating the link between skin cells and nociceptor sensitization contributing to FD pain in a sex-associated manner. We used cultured keratinocytes and fibroblasts of 27 adult FD patients and 20 healthy controls. Epidermal keratinocytes and dermal fibroblasts were cultured and immunoreacted to evaluate Gb3 load. Gene expression analysis of pain-related ion channels and pro-inflammatory cytokines was performed in dermal fibroblasts. We further investigated electrophysiological properties of induced pluripotent stem cell (iPSC) derived sensory-like neurons of a man with FD and a healthy man and incubated the cells with interleukin 8 (IL-8) or fibroblast supernatant as an in vitro model system. Keratinocytes displayed no intracellular, but membrane-bound Gb3 deposits. In contrast, fibroblasts showed intracellular Gb3 and revealed higher gene expression of potassium intermediate/small conductance calcium-activated potassium channel 3.1 (KCa 3.1, KCNN4) in both, men and women with FD compared to controls. Additionally, cytokine expression analysis showed increased IL-8 RNA levels only in female FD fibroblasts. Patch-clamp studies revealed reduced rheobase currents for both iPSC neuron cell lines incubated with IL-8 or fibroblast supernatant of women with FD. We conclude that Gb3 deposition in female FD patient skin fibroblasts may lead to increased KCa3.1 activity and IL-8 secretion. This may result in cutaneous nociceptor sensitization as a potential mechanism contributing to a sex-associated FD pain phenotype.

Fig 1. Gb3 accumulations in FD skin cells and gene expression analysis of FD keratinocytes.

Keratinocytes of FD patients were incubated with DAPI, Stx 647, and antibodies against CytK 10 and α-tubulin to visualize Gb3 deposits within cells. A) There were no Gb3 accumulations detectable within the cytoplasm of FD hEK. Investigation of membrane-bound deposits (B) revealed no Gb3 deposits in Ctrl hEK (B`), but Gb3 positive membranes of FD hEK (B”). C) For detection of Gb3 deposits in hDF, incubation with Stx 555 was performed. High levels of Gb3 accumulations were found within FD hDF (arrows, C”), while control fibroblasts also displayed few positive Stx signals (arrows, C`). Abbreviations: Ctrl = control; CytK 10 = cytokeratin 10; DAPI = 4’,6-diamidino-2-phenylindol; FD = Fabry disease; Gb3 = globotriaosylceramide; hDF = human dermal fibroblasts; hEK = human epidermal keratinocytes; qRT-PCR = quantitative real-time PCR; Stx = Shiga toxin subunit B; TRPV1 = transient receptor potential vanilloid 1. *p<0.05. Scale bars = 25 μm.

Fig 2. Ion channel gene expression analysis of FD fibroblasts.

Pain related ion channel gene expression was investigated in hDF using qRT-PCR. Expression of TRPV1 (p<0.001, A`) and SCN9A (p < .01, A”) was reduced compared to Ctl hDF, while there was no difference for HCN2 (A”`), and KCNMA1 (A”“) expression. B) Whole patient cohort hDF KCNN4 expression was higher compared to Ctrl (p<0.001). C) Sex stratification of the patient cohort revealed a higher KCNN4 gene expression in male (p<0.05), and female FD hDF (p<0.001). Please see supplementary file for raw data. Abbreviations: Ctrl = control; FD = Fabry disease; HCN2 = hyperpolarization-activated cyclic nucleotide-gated ion channel 2; hDF = human dermal fibroblasts; qRT-PCR = quantitative real-time PCR; TRPV1 = transient receptor potential vanilloid 1; KCNMA1 = potassium calcium-activated channel subfamily M alpha 1; KCNN4 = calcium-activated potassium channel 3.1; SCN9A = voltage-gated sodium channel 1.7. *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Cytokine gene expression analysis of FD fibroblasts.

To investigate cytokine gene expression in hDF, qRT-PCR was used. Gene expression of IL-8 was higher in whole FD patient fibroblasts, compared to Ctrl (p<0.01, A). Analysis of data stratifying patient groups for sex revealed higher IL-8 gene expression only for female hDF compared to Ctrl (p<0.001) and male FD hDF (p<0.05, B). Gene expression of TNF (C), IL-1β (D), and IL-6 (E) was not different between FD patients and Ctrl. Please see supplementary file for raw data. Abbreviations: Ctrl = control; FD = Fabry disease; hDF = human dermal fibroblasts; IL-1β = interleukin 1β; IL-6 = interleukin 6; IL-8 = interleukin 8; qRT-PCR = quantitative real-time PCR; TNF = tumor necrosis factor-alpha. *p<0.05, **p<0.01, ***p<0.001.

Fig 4. Electrophysiological analysis of iPSC-derived neurons upon cytokine exposure.

AP parameters (A) were investigated under naïve conditions and after incubation of iPSC-derived sensory-like neurons with IL-8 or hDF supernatant. No difference was found in RMP (B) or any other AP parameter, except for hyperpolarization amplitudes. Hyperpolarization (C) was higher in Ctrl neurons incubated with Ctrl, PatM hDF supernatant, and IL-8 compared to naïve Ctrl neurons (p<0.001; p<0.01; p<0.001). FD neurons revealed increased hyperpolarization amplitudes incubated with Ctrl or PatF supernatant compared to naïve Ctrl neurons (p<0.001; p<0.001). Rheobase currents (D) to elicit first AP were lower in Ctrl neurons after incubation with PatF and PatM supernatant compared to naïve neurons (p<0.01; p<0.01). In FD neurons, incubation with PatF hDF supernatant or IL-8 lead to reduced rheobase currents compared to naïve neurons (p<0.05; p<0.05). Abbreviations: AP = action potential; Ctrl = control; FD = Fabry disease; hDF = human dermal fibroblasts; IL-8 = interleukin 8; iPSC = induced pluripotent stem cell; PatF = female Fabry disease patient; PatM = male Fabry disease patient; RMP = resting membrane potential. *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Potential mechanism underlying sex-associated pain phenotype.

Mutation of the GLA gene leads to a non- or dys-functional enzyme, which results in Gb3 accumulation in FD fibroblasts. These deposits may be involved in increased gene expression of the cytokine-linked ion channel KCa3.1. Upregulated ion channel gene expression then may lead to an elevated expression of IL-8 only in female FD patients with a pain phenotype. Potentially increased IL-8 secretion close to nociceptor endings in the skin may then lead to sensitization via reduction of AP induction thresholds contributing to FD-associated pain. Abbreviations: GLA = α-galactosidase A; AP = action potential; FD = Fabry disease; Gb3 = globotriaosylceramide; IL-8 = interleukin 8; KCa3.1 = potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4.