Accepted Manuscript Differences in human gingival and dermal fibroblasts may contribute to oral-induced tolerance against nickel Lina Gölz, MD, Elisa Vestewig, doctoral student, Moritz Blankart, MR, Dominik Kraus, MD, Thorsten Appel, MD, Stilla Frede, PhD, Andreas Jäger, MD PII:
S0091-6749(16)30252-4
DOI:
10.1016/j.jaci.2016.03.036
Reference:
YMAI 12093
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 5 June 2015 Revised Date:
4 March 2016
Accepted Date: 22 March 2016
Please cite this article as: Gölz L, Vestewig E, Blankart M, Kraus D, Appel T, Frede S, Jäger A, Differences in human gingival and dermal fibroblasts may contribute to oral-induced tolerance against nickel, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.03.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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TITLE PAGE
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Differences in human gingival and dermal fibroblasts may contribute to oral-induced
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tolerance against nickel Lina Gölz1, Elisa Vestewig1,2, Moritz Blankart1, Dominik Kraus 3, Thorsten Appel4, Stilla
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Frede2*, Andreas Jäger1*
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Department of Orthodontics, Dental Clinic, University Hospital of Bonn, Bonn, Germany
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Clinic of Anesthesiology and Intensive Care Medicine, University Hospital of Bonn, Bonn,
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Germany
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Department of Prosthodontics, University of Bonn, Bonn Germany
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Center of Dento-Maxillo-Facial Medicine, University of Bonn, Bonn Germany
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contributed equally
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Lina Gölz, MD1
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Elisa Vestewig, doctoral student1,2
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Moritz Blankart, MR1
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Dominik Kraus, MD3
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Thorsten Appel, MD 4
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Stilla Frede, PhD 2*
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Andreas Jäger, MD 1*
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Funding: LG received financial support from the German Society of Orthodontics (Deutsche
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Gesellschaft für Kieferorthopädie (DGKFO)).
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Correspondance to: Dr. Lina Gölz, Welschnonnenstraße 17, 53111 Bonn, Department of Orthodontics, University of Bonn, Bonn, Germany; e-mail:
[email protected]; phone: +49 (0)228-28722433; Fax: +49(0)228-28722558
ACCEPTED MANUSCRIPT Key words: nickel hypersensitivity, allergic contact dermatitis, oral tolerance, dermal
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fibroblasts, gingival fibroblasts, dendritic cells, NF-κB, HIF-1α, CCL20, IL-10
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ACCEPTED MANUSCRIPT CAPSULE SUMMARY
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In contrast to dermal fibroblasts, gingival (oral mucosal) cells display anti-inflammatory pro-
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tolerogenic capacities which may predispose for oral tolerance against nickel via repression of
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NF-κB and HIF-1α signaling opening the door for new therapeutic approaches.
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ACCEPTED MANUSCRIPT LETTER TO THE EDITOR
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To the Editor:
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The prevalence of allergic contact dermatitis (ACD) has increased steadily with nickel (Ni)
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still as the main sensitizer.1-2 In this context, it seems contradictory that orthodontic materials
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contain high amounts of Ni which lead to inevitable release of Ni ions (Ni2+) into the oral
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cavity.3 However, intraoral allergic reactions are rare and current evidence indicates that
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orthodontic treatment prior to piercings may even prevent Ni hypersensitivity probably via
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oral-induced tolerance against Ni.4 The detailed mechanisms still remain unclear. Previous
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studies imply that immunological differences between mucosal and dermal dendritic cells
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(DCs) contribute to oral-induced immune tolerance.5 Thereby, mucosal DCs display a pro-
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tolerogenic character, whereas dermal DCs have a pro-allergenic phenotype. Interestingly,
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DCs are strongly influenced by their microenvironment determining the subsequent
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phenotype of DCs.5-7 Mediators like transforming growth factor (TGF)-β, interleukin (IL)-10
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and certain innate immune receptors such as Toll-like receptor (TLR)4 seem to play a pivotal
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role in these events.5-8
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As fibroblasts primary constitute tissue structure and are in direct contact with immune
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cells like DCs, we determined whether dermal fibroblasts promote a pro-allergenic
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inflammatory microenvironment in contrast to oral gingival (mucosal) fibroblasts which may
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favor a pro-tolerogenic anti-inflammatory surrounding. Therefore, primary human
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oral/gingival (HGFs) and dermal fibroblasts (HDFs) were challenged with Ni ions (Ni2+) and
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subsequent cell signaling and the expression of certain mediators responsible for immune cell
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activation and recruitment were evaluated (for details, see this article`s Online Repository at
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www.jacionline.org).
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Since Ni2+ exerts some effects via TLR4-binding and NF-κB is a key regulator of
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inflammation,8 downstream activation of the transcription factor nuclear factor (NF-)κB and
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the expression of its target gene IL-1β were investigated. Nuclear accumulation of NF-κB was
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markedly induced in HDFs after Ni2+ stimulation compared to HGFs (Fig. 1, A). In line with
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these findings, IL-1β mRNA expression was significantly elevated in HDFs (Fig. 1, B). In
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contrast, reduced nuclear NF-κB levels were found in HGFs. Accordingly, Ni2+ provoked
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significantly higher expression levels of IL-1β mRNA in dermal compared to gingival cells
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(45-fold, Fig. 1, B). In good agreement with the mRNA results, Ni2+ challenge induced an
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increase of IL-1β protein release by HDFs. Instead, in HGFs IL-1β protein was only slightly
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elevated and significantly less compared to HDFs (22-fold, Fig. 1, C).
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ACCEPTED MANUSCRIPT As divalent cations like Ni2+ are known for their hypoxia-mimicking capacities,E4,E5
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we determined the activation of the transcription factor hypoxia inducible factor (HIF)-1α and
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its target gene vascular endothelial growth factor (VEGF) which is liable for vascularization,
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endothelial permeability and immune cell invasion.E6, E7 Notably, HIF-1α mRNA expression
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was not altered by Ni2+ stimulation in HDFs nor in HGFs (data not shown). However, HIF-1α
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activation measured as nuclear accumulation was increased in HDFs. Similar results were
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detectable in the cytoplasm of HDFs after Ni2+ challenge (data not shown). Notably, nuclear
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HIF-1α accumulation in HGFs was less intense compared to HDFs (Fig. 1, D). Accordingly,
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VEGF mRNA levels were more than twice as high in HDFs compared to HGFs which was
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statistical significant (Fig. 1, E). Regarding VEGF protein, results were in line with mRNA
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data, as significantly higher protein levels were detected in HDFs compared to HGFs (4-fold,
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Fig. 1, F).
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To antagonize inflammatory processes, anti-inflammatory mediators are secreted
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which are linked to the induction of immune tolerance.E8,E9 Therefore, we investigated IL-10
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and TGF-β mRNA and protein expression after Ni2+ challenge. A low constitutive expression
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of IL-10 mRNA could be demonstrated in HDFs which was not affected by Ni2+ incubation
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(Fig 2, A). Instead, in HGFs Ni2+ incubation increased the expression levels of IL-10
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significantly (10- to 30-fold). Accordingly, we found very low IL-10 protein levels in HDFs
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and significantly higher amounts in HGFs supernatants (30-fold; Fig 2, B). Notably, TGF-β
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was not altered in HDFs or HGFs (data not shown).
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Next, we determined CCL20 (MIP-1α) because of its chemoattractant and
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inflammatory capacities leading to DC recruitment.E10 We could demonstrate that CCL20
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mRNA was induced 200-fold by Ni2+ in HDFs (Fig 2, C). In contrast, Ni2+ provoked only
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minor changes in HGF CCL20 expression. Regarding CCL20 protein release, a time-
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dependent increase in HDFs supernatants was detectable (35-fold). Instead, CCL20 protein
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concentrations in HGFs supernatants were below the detection limit (Fig 2, D).
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Finally, to investigate the recruitment of DCs, a migration assay was performed using
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supernatants of each fibroblast type (Fig 2, E). Supernatants of HDFs cultured under control
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condition reduced DC migration, whereas control supernatants of HGFs induced an increase.
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Most interesting, supernatants of HDF challenged with Ni2+ significantly stimulated DC
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migration, whereas supernatants of similarly treated HGFs caused a pronounced inhibition of
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their migration (Fig 2, E).
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Taken together, increased IL-1β, CCL20 and VEGF protein levels as well as decreased
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IL-10 expressions predispose for an imbalanced inflammatory microenvironment with
ACCEPTED MANUSCRIPT subsequent immune cell recruitment and invasion in dermal tissue which seem to be inhibited
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in gingival/oral tissue. In line with present data, previous studies demonstrated that in contrast
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to dermal DCs, oral DCs respond with an increase of anti-inflammatory IL-10 secretion
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following TLR4 activation which then stimulate the induction of regulatory T cells (Treg).6,7
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Tregs are key players for the induction of immune tolerance. Usually, activation of TLR4
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initiates a potent pro-inflammatory response accompanied by the release of pro-inflammatory
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cytokines and chemoattractants recruiting predominantly effectory T cells (Teff) which
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aggravate inflammatory processes and allergic reactions.9 DCs as antigen-presenting cells
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regulate the initiation of hypersensitivities as they determine T cell priming and activation.
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The tolerogenic function of different subsets of DCs depends on their maturation stage,
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certain ontogenics and on immunomodulating agents. As the microenvironment of DCs seem
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to play a crucial role in their maturation and fibroblasts primary constitute the intestinal
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lamina propria in which DCs reside, we hypothesize that gingival fibroblasts contribute to
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their pro-tolerogenic phenotype via altered NF-κB and HIF-1α regulation and subsequent
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expression profile.
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To conclude, present results unveil new aspects of oral-induced tolerance and provide
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additional information for current knowledge which indicates that oral Ni2+ challenge prior to
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piercings may prevent Ni hypersensitivity.4 Accordingly, our findings may open the door for
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new therapeutic approaches solving current difficulties in ACD and oral-induced
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hyposensitivity.
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We thank Mario Fox and Frank Splettstoesser for his technical support and the volunteers for participating to the study.
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Lina Gölz, MD1
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Elisa Vestewig, doctoral student1,2
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Moritz Blankart, MR1
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Dominik Kraus, MD3
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Thorsten Appel, MD 4
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Stilla Frede, PhD 2*
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Andreas Jäger, MD 1*
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Department of Orthodontics, Dental Clinic, University Hospital of Bonn, Bonn, Germany; 2Clinic of Anesthesiology and Intensive Care Medicine, University Hospital of Bonn, Bonn, Germany; 3Department of
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Prosthodontics, University of Bonn, Bonn Germany; 4Center of Dento-Maxillo-Facial Medicine, University of
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Bonn, Bonn Germany; Email:
[email protected] *
These authors contributed equally to this work
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LG received financial support from the German Society of Orthodontics (Deutsche
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Gesellschaft für Kieferorthopädie (DGKFO)).
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Disclosure of conflicts of interest: The authors declare that there are no conflicts of interest.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
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FIG 1. Altered NF-κB and HIF-1α activation.
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Activation of the transcription factors NF-κB (A) and HIF-1α (D) were determined followed
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by the analysis of mRNA and protein of their target genes IL-1β (B and C) and VEGF (E and
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F) in dermal (HDFs) and gingival fibroblasts (HGFs) stimulated with 100 µmol/l Ni2+.
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(Means ± SEMs are depicted, *p
