Histamine H4 Receptor: A Potential Novel Therapeutic Target in Oral Lichen Planus and Oral Tongue Cancer

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Title: Histamine H4 Receptor: A Potential Novel Therapeutic Target in Oral Lichen Planus and Oral Tongue Cancer
Author: Salem, Abdelhakim
Contributor: University of Helsinki, Faculty of Medicine, Clinicum
Doctoral Programme in Oral Sciences
Translational Immunology Research Program (TRIMM), University of Helsinki
Thesis level: Doctoral dissertation (article-based)
Belongs to series: DSHealth Dissertation Series
Abstract: Background and objective: Oral lichen planus (OLP) is a common immune disorder of the oral mucosa, which is categorized as an oral potentially malignant disorder (OPMD) to highlight its potential progression to oral cancer. Oral cancer commonly affects the mobile tongue as oral tongue squamous cell carcinoma (OTSCC), which has dismal prognosis. Histamine signals via four G protein-coupled histamine receptors (H1R-H4R). Classical H1R and H2R medications are ineffective in treating OLP or OTSCC patients. The discovery of H4R has paved the way for novel perspectives in histamine research by modulating immune responses. We therefore hypothesized that H4R is involved in the pathogenesis of OLP and may contribute to oral carcinogenesis. Materials and methods: Tissue samples from OLP, oral epithelial dysplasia (OED) and OTSCC patients, and from healthy control participants were utilized. The in vitro experiments were performed on normal human oral keratinocytes (HOKs), two OTSCC-derived cell lines (HSC-3 and SCC-25), normal salivary gland cells, in addition to supernatants from activated human mast cells (MCs). For in vitro internalization and functional assays, two specific H4R ligands (agonist HST-10, and inverse agonist ST-1007) were used. Protein expression of histamine receptors, transporters and metabolizing enzymes, and other antigens were assessed in tissue samples and cell lines by immunohistochemistry and immunofluorescence staining. The expression levels of mRNA were quantified by qRT-PCR and the highly-sensitive droplet-digital PCR technology. Western blotting assays were performed to assess apoptotic markers following H4R stimulation, while flow cytometry was used to study Annexin-V and PI labelling of dead cells. Histamine levels were analysed using high-performance liquid chromatography. Results: Briefly, H4R is expressed in healthy oral epithelial cells on mRNA and protein levels, and they were able to fully internalize H4R-ligands in a time-dependent manner. In contrast, samples from OLP, OED and OTSCC patients exhibited lower H4R level, which was negatively correlated with MC-count and OTSCC-grade. We also reported that normal HOKs are histamine-producing cells—fully equipped with histamine synthesizing, transporting and degrading molecules. Interestingly, OLP samples exhibit high levels of the histamine synthesizing and transporting molecules, whereas histamine degrading enzyme was strongly inhibited. HOKs showed a dose-dependent Lipopolysaccharides (LPS)-driven release of histamine, while high histamine levels inhibited epithelial adhesion molecules. We next showed that toll-like receptors (TLRs) are essential players in OLP. TLRs were upregulated in OLP lesions, particularly for TLR4, which is necessary for LPS signalling. Importantly, LPS and MC-mediators regulated several oral oncogenes, while H4R-stimulated cells revealed a marked resistance to apoptosis. Furthermore, LPS and histamine influenced human beta defensin 2 (hBD-2) expression, which was highly induced in OLP. Unexpectedly, hBD-2 protein was subsided in OTSCC tissues with a marked downregulation of its transcript in cancer cells. Histamine synergistically induced TNF-α- and IFN-γ-mediated hBD-2 production in HOKs. Interestingly, targeting H4R seems to regulate TNFα- and LPS-mediated expression of hBD-2. Conclusions: Briefly, human oral epithelial cells are “non-professional” histamine producing cells—capable to synthesize, release, and degrade low levels of endogenic histamine. High levels of histamine may downregulate H4R as well as key integrity molecules in HOKs and may enhance subsequent bacterial invasion in OLP. In this regard, our findings suggest a potential role of TLRs in OLP pathogenesis, by mediating LPS signalling and enhancing further immune response and histamine production. In addition, our results indicate that histamine/H4R crosstalk signalling with LPS and MCs could in part be involved in OLP and the potential inflammation-driven tumorigenesis. This was further supported by the ability of H4R to regulate cell apoptosis and modulate antibacterial response in HOKs. Further functional and preclinical studies are therefore warranted.Punajäkälä on tavallinen suun limakalvolla esiintyvä tulehdustauti, joka aiheutuu elimistön immuunijärjestelmän häiriöstä. Perinteiset antihistamiinit ovat osoittautuneet tehottomiksi punajäkälän hoidossa, mutta histamiini 4 -reseptorin (H4R) löytyminen on avannut uusia tutkimusmahdollisuuksia aiheesta. Tämän työn hypoteesina oli, että H4R saattaisi vaikuttaa sekä punajäkälän että kielisyövän syntymiseen. Työn ensimmäisessä vaiheessa osoitimme, että terveet suun limakalvon epiteelisolut ilmensivät H4-reseptoria voimakkaammin verrattuna punajäkälän vaurioittamiin kudosnäytteisiin. Näistä näytteistä löysimme sen sijaan suuria määriä histamiinin tuottamiseen ja kuljettamiseen liittyviä molekyylejä ja vain vähän histamiinia hajottavaa entsyymiä. Tässä yhteydessä pystyimme todentamaan limakalvon keratinosyyttien itsessään toimivan histamiinia tuottavina soluina. Lipopolysakkaridit (LPS) kiihdyttivät histamiinin vapautumista näistä soluista, mutta korkeat histamiinipitoisuudet heikensivät epiteelisolujen kiinnittymisrakenteiden toimintaa. Tutkimuksen seuraavassa vaiheessa havaitsimme Tollin kaltaisten reseptoreiden (TLR) yhdistyvän keskeisesti punajäkälän taudinkuvaan, sillä näiden esiintyminen oli korostunut punajäkälälle altistuneella limakalvolla. Pystyimme myös todentamaan, että LPS ja syöttösolujen tulehdusvälittäjät säätelivät useita epiteelisolujen syöpägeenejä H4R:n osallistuessa näiden apoptoosin säätelyyn. Tämän lisäksi LPS ja histamiini vaikuttivat defensiinien ilmentymiseen limakalvon pintasolukossa. Epiteelisolut pystyivät siis tuottamaan ja hajottamaan histamiinia, joka suurina pitoisuuksina laski histamiini 4-resetorin ja lisäsi bakteerirakenteiden esiintymistä suun punajäkälässä. Tutkimuksemme mukaan H4R vaikuttaa lupaavalta kohteelta, jonka toimintaan vaikuttamalla voitaisiin auttaa punajäkälän ja suusyövän hoidossa. Tarkentavat jatkotutkimukset aiheesta ovat näin ollen perusteltuja.
URI: URN:ISBN:978-951-51-5119-3
Date: 2019-04-17
Subject: Dentistry; Oral Pathology; Oncology
Rights: This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.

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