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ISBN 978-952-62-1469-6 (Paperback)ISBN 978-952-62-1470-2 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1403

ACTA

Maria Siponen

OULU 2017

D 1403

Maria Siponen

ORAL LICHEN PLANUS – ETIOPATHOGENESISAND MANAGEMENT

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;FINNISH DOCTORAL PROGRAM IN ORAL SCIENCES

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 4 0 3

MARIA SIPONEN

ORAL LICHEN PLANUS – ETIOPATHOGENESIS AND MANAGEMENT

Academic dissertation to be presented with the assentof the Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein the Leena Palotie auditorium (101A) of the Faculty ofMedicine (Aapistie 5 A), on 28 January 2017, at 12 noon

UNIVERSITY OF OULU, OULU 2017

Copyright © 2017Acta Univ. Oul. D 1403, 2017

Supervised byProfessor Tuula SaloProfessor Ylermi SoiniDocent Pentti Nieminen

Reviewed byProfessor Kristiina HeikinheimoProfessor Karin Nylander

ISBN 978-952-62-1469-6 (Paperback)ISBN 978-952-62-1470-2 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2017

OpponentDocent Bengt Hasséus

Siponen, Maria, Oral lichen planus – etiopathogenesis and management. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Finnish DoctoralProgram in Oral SciencesActa Univ. Oul. D 1403, 2017University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Oral lichen planus (OLP) is a chronic immune-mediated mucosal disease with unknown etiology.According to the current view, the pathogenesis of OLP involves activation of T-cell mediatedimmunity against the epithelial keratinocytes. A proportion of OLP patients are affected by painfulsymptoms, and the risk of oral cancer is increased in OLP. There is no curative treatment for OLP.Topical corticosteroids are used most commonly in the management of OLP. However, theevidence base for the effectiveness of any therapy is weak.

The objective of this thesis was to study novel aspects of OLP etiopathogenesis andmanagement. An epidemiologic, retrospective case-control study was conducted to determinewhether systemic diseases, in particular thyroid diseases, are associated with OLP. In addition, arandomized controlled trial comparing the effectiveness of topical tacrolimus, triamcinoloneacetonide and placebo in symptomatic OLP was carried out. Furthermore, immunohistochemicalexpression of toll-like receptors 4 and 9, hyaluronan and its principal receptor CD44 antigen,hyaluronan synthases 1-3, hyaluronidases 1-2 and cathepsin K was studied in OLP tissue samplesand in healthy oral mucosa. The effect of topical tacrolimus on the expression of these moleculesin OLP was also studied.

The results of the present study showed that a history of hypothyroidism was associated withan approximately twofold risk of having OLP. Furthermore, both tacrolimus and triamcinoloneacetonide were more effective than placebo in reducing the signs and symptoms of OLP. Nostatistically significant differences were noted in the efficacy between tacrolimus andtriamcinolone acetonide. In addition, the expression of the studied molecules was altered in theepithelium or stroma in OLP compared to healthy oral mucosa. Tacrolimus treatment decreasedthe expression of CD44 antigen in the stroma and the expression of cathepsin K in the epitheliumin OLP.

In conclusion, the present study extends our knowledge about systemic associated factors andmanagement of OLP. In addition, the results improve our understanding of molecular levelchanges that occur in OLP.

Keywords: case-control study, cathepsin K, CD44 antigen, hyaluronan, hyaluronan synthase 1,hyaluronan synthase 2, hyaluronan synthase 3, hyaluronidase 1, hyaluronidase 2, hypothyroidism,immunohistochemistry, oral lichen planus, placebo, randomized controlled trial, tacrolimus, thyroiddisease, toll-like receptor 4, toll-like receptor 9, triamcinolone acetonide

Siponen, Maria, Suun punajäkälä – etiopatogeneesi ja hoito. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Kansallinen suunterveystieteiden tohtoriohjelmaActa Univ. Oul. D 1403, 2017Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Suun punajäkälä on krooninen immuunivälitteinen limakalvotauti, jonka etiologia on tuntema-ton. Taudin syntymekanismiin liittyy tämän hetkisen näkemyksen mukaan T-soluvälitteisenimmuniteetin aktivoituminen epiteelin keratinosyyttejä vastaan. Suun punajäkälä aiheuttaa osallepotilaista kivuliaita oireita ja lisää suusyövän riskiä. Parantavaa hoitoa tautiin ei ole. Yleisim-min suun punajäkälän oireiden hoidossa käytetään paikallisia kortikosteroidivalmisteita. Kuiten-kin eri hoitomuotojen tehosta on vain heikkoa näyttöä.

Tämän väitöskirjatyön tarkoituksena oli tutkia uusia näkökohtia liittyen suun punajäkälänetiopatogeneesiin ja hoitoon. Epidemiologisessa tapaus-verrokkitutkimuksessa selvitettiin, liitty-vätkö yleissairaudet, erityisesti kilpirauhassairaudet, suun punajäkälään. Lisäksi satunnaistetus-sa kontrolloidussa tutkimuksessa verrattiin paikallisen takrolimuusin, triamsinoloniasetonidin jalumelääkkeen tehoa oireisesta suun punajäkälästä kärsivillä potilailla. Tutkimuksessa selvitettiinmyös tollin kaltaisten reseptorien 4 ja 9, hyaluronaanin ja sen pääasiallisen reseptorin CD44-antigeenin, hyaluronaanisyntaasien 1–3, hyaluronidaasien 1–2 sekä katepsiini K:n immunohisto-kemiallista ilmentymistä suun punajäkälänäytteissä ja terveessä suun limakalvossa. Lisäksi tut-kittiin takrolimuusihoidon vaikutusta näiden molekyylien ilmentymiseen suun punajäkälässä.

Tämän tutkimuksen tulokset osoittivat, että kilpirauhasen vajaatoimintaan liittyi noin kaksin-kertainen riski sairastaa suun punajäkälää. Lisäksi havaittiin, että suun punajäkälässä sekä takro-limuusi että triamsinoloniasetonidi ovat tehokkaampia kuin lumelääke oireiden ja kliinisen tau-dinkuvan lievittämisessä. Takrolimuusin ja triamsinoloniasetonidin tehossa ei todettu tilastolli-sesti merkitseviä eroja. Lisäksi suun punajäkälänäytteissä tutkittujen molekyylien ilmentyminenoli muuttunut joko epiteelissä tai stroomassa verrattuna terveeseen limakalvoon. Takrolimuusi-hoito vähensi CD44-antigeenin ilmentymistä stroomassa ja katepsiini K:n ilmentymistä epitee-lissä suun punajäkälässä.

Yhteenvetona voidaan todeta, että tämä tutkimus lisää tietoa suun punajäkälään liittyvistäsysteemisistä tekijöistä ja suun punajäkälän hoidosta. Lisäksi löydökset lisäävät ymmärtämystäsuun punajäkälässä tapahtuvista molekyylitason muutoksista.

Asiasanat: CD44-antigeeni, hyaluronaani, hyaluronaanisyntaasi 1, hyaluronaanisyntaasi 2,hyaluronaanisyntaasi 3, hyaluronidaasi 1, hyaluronidaasi 2, immunohistokemia, katepsiini K,kilpirauhasen vajaatoiminta, kilpirauhassairaus, lumelääke, satunnaistettu kontrolloitu tutkimus,suun punajäkälä, takrolimuusi, tapaus-verrokkitutkimus, tollin kaltainen reseptori 4, tollin kaltainenreseptori 9, triamsinoloniasetonidi

To my parents

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9

Acknowledgements

The studies in this thesis were conducted at the Institute of Dentistry and the Cancer

and Translational Medicine Research Unit, University of Oulu, the Department of

Oral and Maxillofacial Diseases and Department of Pathology, Oulu University

Hospital, the Department of Oral and Maxillofacial Diseases, Kuopio University

Hospital and the Institute of Biomedicine/Anatomy, School of Medicine,

University of Eastern Finland.

First of all, I wish to express my deepest gratitude to my principal supervisor

Professor Tuula Salo for providing me with interesting and relevant research topics

and for her invaluable “round-the-clock” advice and support during the thesis work.

In addition to professional supervision and co-operation, Professor Salo’s support

has extended to personal life as well. After I moved to Kuopio, many visits to Oulu

were much nicer thanks to Tuula’s B&B.

I am also forever grateful for the expert opinions, kind guidance and

encouragement of my co-supervisors Docent Pentti Nieminen and Professor Ylermi

Soini.

I am greatly indebted to the pre-examiners of this thesis, Professor Kristiina

Heikinheimo from the University of Eastern Finland and University of Turku and

Professor Karin Nylander from the University of Umeå for their expertise and

excellent contributions.

The work leading to this thesis began in Oulu in 2002 when a novel drug,

tacrolimus, had shown promise in the management of oral lichen planus.

Randomized controlled trials of topical tacrolimus in oral lichen planus were

lacking, and therefore we designed and initiated one. The following process

revealed the many different aspects of clinical trials. I would like to thank warmly

the patients as well as many colleagues, dental nurses, nurses, pharmacists,

laboratory technicians and secretaries at the University of Oulu, Oulu University

Hospital and Kuopio University Hospital for their co-operation and effort during

the clinical trial.

I wish to acknowledge the kind help of many skilled laboratory technicians,

especially Mrs. Tanja Kuusisto, Mrs. Eija Rahunen, Mrs. Maritta Harjapää, Mrs.

Sanna Juntunen, Mrs. Merja Tyynismaa, Mrs. Eeva-Maija Kiljander, Mrs. Riitta

Vuento and Mr. Kari Kotikumpu. I am also thankful for the technical assistance

with illustrations provided by Mrs. Seija Leskelä, Mr. Mika Kihlström and Ms.

Marjukka Aronen. Professor emeritus Carl M. Allen is warmly acknowledged for

English language proofreading of some of the articles.

10

I greatly appreciate the co-operation of my co-authors DDS Lasse Huuskonen,

Professor Esa Läärä, Docent Joonas Kauppila, Docent Sanna Pasonen-Seppänen,

Docent Arja Kullaa, DDS, PhD Carolina Cavalcante Bitu, DDS, PhD Ahmed Al-

Samadi and DDS, PhD Soili Kallio-Pulkkinen.

I would like to thank warmly Docent Pirjo-Liisa Lukinmaa from the University

of Helsinki, Professor Kaisa Tasanen-Määttä from the University of Oulu and

Professor Raija Tammi from the University of Eastern Finland for their input to this

thesis as members of the follow-up group.

I wish to thank PhD Deborah D. Kaska for performing the English language

proofreading of this thesis and Delingua Oy for Finnish language checking.

I owe my gratitude to Professor Tuomo Karttunen at the Cancer and

Translational Medicine Research Unit, University of Oulu as well as to Docent Jari

Kellokoski and Dr. Kari Konki at the Department of Oral and Maxillofacial

Diseases, Kuopio University Hospital, for enabling the thesis work. I want to thank

also the chiefs and staff in Clinical Pathology and Forensic Medicine, Institute of

Clinical Medicine, at the Institute of Biomedicine/Anatomy and at the Institute of

Dentistry, School of Medicine, University of Eastern Finland, for letting me use the

facilities there and helping me with technical issues.

This thesis has received funding from the following organizations: Finnish

Dental Society Apollonia, Suomen Naishammaslääkärit r.y., Oulu University

Hospital and Kuopio University Hospital. I want to thank especially research

director Esko Vanninen for the funding of the clinical trial in its final stages.

I am forever thankful for my siblings for giving me the opportunity to

concentrate on the thesis for the last couple of years in taking great care of our

father.

Most of all, I am extremely grateful for the support of my dear father, who

despite his longstanding grave illness has taken an interest in my wellbeing and

supported and encouraged me in my work. Dad, your sophistication and sense of

humor inspires me.

Kuopio, November 2016 Maria Siponen

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Abbreviations

AD atopic dermatitis

AITD autoimmune thyroid disease

APC antigen-presenting cell

BM basement membrane

BMZ basement membrane zone

CTSK cathepsin K

CCL5 C-C motif chemokine 5 (RANTES)

CCR1 C-C chemokine receptor type 1

CD1A T-cell surface glycoprotein CD1a, Langerhans cell marker

CD4 T-cell surface glycoprotein CD4, helper T cell marker

CD8A T-cell surface glycoprotein CD8A alpha chain, cytotoxic T cell

marker

CD44 CD44 antigen, hyaluronan receptor

CD68 macrosialin, macrophage marker

CI confidence interval

CS clinical score

DC dendritic cell

DEFB4A defensin beta 4A (beta defensin 2)

e.g. exempli gratia

et al et alia

FFPE formalin fixed paraffin embedded

HA hyaluronan

HAS hyaluronan synthase

HCV hepatitis C virus

HLA human leukocyte antigen, HLA histocompatibility antigen

(major histocompatibility complex, MHC)

HMW high molecular weight

HSP heat shock protein

HYAL hyaluronidase

ICC intraclass correlation

IF immunofluorescence

IFNG interferon gamma

IHC immunohistochemistry

Ig immunoglobulin

IL interleukin

12

LP lichen planus

LPS lipopolysaccharide

Melan-A melanoma antigen recognized by T-cells 1, melanocyte marker

MMP matrix metalloproteinase

OLP oral lichen planus

OLL oral lichenoid lesion

OLR oral lichenoid reaction

OR odds ratio

PAMP pathogen-associated molecular pattern

PTPRC receptor-type tyrosine-protein phosphatase C (CD45 antigen),

lymphocyte marker

RCT randomized controlled trial

Tg, TG thyroglobulin

TLR toll-like receptor

TNF tumor necrosis factor

TPO thyroid peroxidase

TPSAB1 tryptase alpha/beta-1 (mast cell tryptase)

TSHR thyroid stimulating hormone receptor, thyrotropin receptor

UWS unstimulated whole saliva

VAS visual analogue scale

WHO World Health Organization

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Original publications

This thesis is based on the following publications, which are referred to throughout

the text by their Roman numerals:

I Siponen M, Huuskonen L, Läärä E, Salo T (2010) Association of oral lichen planus with thyroid disease in a Finnish population: a retrospective case-control study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 110(3): 319–324.

II Siponen M, Huuskonen L, Kallio-Pulkkinen S, Nieminen P, Salo T (2016) Topical tacrolimus, triamcinolone acetonide and placebo in oral lichen planus: a pilot randomized controlled trial. Manuscript.

III Siponen M, Kauppila JH, Soini Y, Salo T (2012) TLR4 and TLR9 are induced in oral lichen planus. J Oral Pathol Med 41(10): 741–747.

IV Siponen M, Kullaa A, Nieminen P, Salo T, Pasonen-Seppänen S (2015) Altered expression of hyaluronan, HAS1–2, and HYAL1–2 in oral lichen planus. J Oral Pathol Med 44(6): 401–409.

V Siponen M, Bitu CC, Al-Samadi A, Nieminen P, Salo T (2016) Cathepsin K expression is increased in oral lichen planus. J Oral Pathol Med 45(10): 758–765.

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15

Contents

Abstract

Tiivistelmä

Acknowledgements 9 

Abbreviations 11 

Original publications 13 

Contents 15 

Introduction 17 

1  Review of the literature 19 

1.1  Oral lichen planus ................................................................................... 19 

1.1.1  Definition...................................................................................... 19 

1.1.2  Epidemiology ............................................................................... 20 

1.1.3  Clinical features ............................................................................ 20 

1.1.4  Histopathological features ............................................................ 23 

1.1.5  Diagnosis ...................................................................................... 25 

1.1.6  Differential diagnosis ................................................................... 26 

1.1.7  Oral lichenoid lesions ................................................................... 26 

1.1.8  Associations with systemic diseases ............................................. 27 

1.1.9  Management ................................................................................. 28 

1.1.10 Outcome measures in OLP ........................................................... 31 

1.1.11 Prognosis ...................................................................................... 31 

1.1.12 Pathogenesis ................................................................................. 32 

1.2  Toll-like receptors 4 and 9 in immune-mediated conditions ................... 38 

1.3  Hyaluronan, its receptor CD44 and its metabolizing enzymes

HAS1–3 and HYAL1–2 in immune-mediated conditions ....................... 39 

1.4  Cathepsin K in immune-mediated conditions ......................................... 41 

2  Aims of the study 43 

3  Patients and materials 45 

3.1  Patients .................................................................................................... 45 

3.2  Medical records (I) .................................................................................. 45 

3.3  Medications in the clinical trial (II) ........................................................ 46 

3.4  Tissue specimens (II–V).......................................................................... 46 

4  Methods 47 

4.1  Epidemiological study (I) ........................................................................ 47 

4.2  Clinical trial (II) ...................................................................................... 47 

4.3  Statistical methods (I, II) ......................................................................... 47 

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4.4  Immunohistochemistry (III-V) ................................................................ 48 

4.5  Evaluation of the immunohistochemical staining ................................... 49 

4.6  Statistical methods (III–V) ...................................................................... 51 

5  Results 53 

5.1  Association of thyroid disease and OLP (I) ............................................ 53 

5.2  Topical tacrolimus and triamcinolone acetonide in the

management of symptomatic OLP (II) .................................................... 54 

5.2.1  The clinical score reliability and validity ..................................... 54 

5.2.2  Patient flow and characteristics .................................................... 54 

5.2.3  Primary and secondary outcomes ................................................. 54 

5.2.4  Adverse reactions to the study medications .................................. 55 

5.3  Immunohistochemical expression of the studied molecules in

OLP and the effect of tacrolimus (III–V) ................................................ 56 

5.3.1  Toll-like receptors 4 and 9 ............................................................ 56 

5.3.2  Hyaluronan, its receptor CD44, and its metabolizing

enzymes HAS1–3 and HYAL1–2 ................................................. 57 

5.3.3  Cathepsin K .................................................................................. 61 

6  Discussion 63 

6.1  Hypothyroidism and OLP ....................................................................... 63 

6.2  Topical tacrolimus and triamcinolone acetonide in the

management of OLP ............................................................................... 65 

6.3  The role of toll-like receptors 4 and 9 in OLP ........................................ 71 

6.4  The role of hyaluronan, its receptor CD44 and its metabolizing

enzymes HAS1–3 and HYAL1–2 in OLP ............................................... 73 

6.5  The role of cathepsin K in OLP .............................................................. 76 

6.6  The molecular level effect of tacrolimus on the studied

molecules ................................................................................................ 77 

6.7  Methodological considerations ............................................................... 78 

6.7.1  Epidemiologic study ..................................................................... 78 

6.7.2  Clinical trial .................................................................................. 79 

6.7.3  Immunohistochemistry ................................................................. 80 

7  Conclusions 83 

References 85 

Original publications 107 

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Introduction

Oral lichen planus (OLP) is an enigmatic disease that has puzzled countless

researchers, clinicians and patients alike. While extensive research has been

conducted regarding the pathogenetic mechanisms involved, we still do not know

the triggering factor(s) of OLP nor much about associated systemic factors (Kurago

2016). The management of symptoms of severe OLP often poses challenges for

clinicians, and may leave patients without adequate symptomatic relief thereby

lowering their quality of life. Hence, it is clear that more research on the

etiopathogenetic mechanisms underlying OLP is needed, with the aim of

developing better treatment options.

Several systemic conditions, like hepatitis C and diabetes, and autoimmune

diseases such as Sjögren’s syndrome are suggested to be associated with OLP

(Likar-Manookin et al. 2013, Lodi et al. 2005, Scully et al. 1998). With the

exception of hepatitis C in certain populations, conclusive evidence for these

associations is lacking (Eisen 2002, Lodi et al. 2010, Lopez-Jornet et al. 2014).

Based on our clinical experience, we postulated that the prevalence of thyroid

diseases is increased in OLP patients. To study the possible associated systemic

conditions, especially thyroid diseases in OLP, we planned an epidemiologic case-

control study.

Symptomatic OLP is typically treated with topical corticosteroids. Since the

late 1990s, reports have been published on the use of topical tacrolimus in the

management of OLP, mainly in cases not responding to topical corticosteroids

(Kaliakatsou et al. 2002, Lener et al. 2001, Morrison et al. 2002, Olivier et al. 2002,

Rozycki et al. 2002, Vente et al. 1999). Most of the previous studies of topical

tacrolimus in OLP have been open-label, non-randomized or non-controlled. In

general, there is only weak evidence for the effectiveness of any specific treatment

in OLP (Thongprasom et al. 2011, Thongprasom et al. 2013). Therefore, we

designed a randomized controlled trial (RCT) to study the efficacy and side-effects

of topical tacrolimus in the management of OLP.

To increase understanding and gain new insights into the molecular factors in

OLP pathogenesis, we studied the immunohistochemical expression of certain

biomarkers associated with innate immunity, hyaluronan metabolism and protein

degradation in archival OLP tissue samples. In addition, the effect of tacrolimus on

the immunohistochemical expression of these molecules was investigated in the

tissue samples collected in the clinical trial.

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19

1 Review of the literature

1.1 Oral lichen planus

1.1.1 Definition

Lichen planus (LP) is a chronic inflammatory mucocutaneous disease of unknown

etiology affecting mainly middle-aged women (Bolognia et al. 2012). The typical

oral manifestation of LP is the presence of symmetrical white reticular lesions in

the buccal mucosa, although diverse clinical features may be seen. The name lichen

means a slow-growing symbiotic organism (of a fungus or fungi and algae or

cyanobacteria) which “typically forms a low crust-like, leaf-like, or branching

growth on rocks, walls, and trees” (Oxford Dictionaries 2016, Spribille et al. 2016).

It derives from Greek leikhēn meaning tree-moss, originally “what eats around

itself”, probably from leichein “to lick”, and Latin planus meaning flat, level

(Harper 2001–2016, Stedman's 2006).

LP was initially described by the Austrian dermatologist Ferdinand von Hebra

who called the disease “lichen ruber” (Wilson 1866). The first clinical description

of LP was reported in 1866 by an English surgeon and dermatologist Erasmus

Wilson, who depicted in 4 patients a papular skin eruption that he called “lichen

planus” (Wilson 1866). In the same report he also described a 56-year old woman

with lesions in the oral mucosa. Later on, Dr. Wilson published a series of 50

patients with LP; three of these patients also had lesions in the mouth: white spots

on the tongue, the buccal membrane and the mucous lining of the fauces (Wilson

1869). It was not until 1906 that the microscopic features of OLP were first

described by Dubreuilh (Dubreuilh 1906).

Cutaneous lichen planus manifests as small violaceous, flat-topped, polygonal

papules, with a shiny or transparent surface and white or grey-white lines

(Wickham’s striae) or spots (Bolognia et al. 2012, Sachdeva et al. 2011). The

cutaneous lesions most frequently exist on the flexor surfaces of forearms, wrists,

dorsal surfaces of hands, anterior side of lower legs, neck and presacral skin

(Bolognia et al. 2012). Genital LP occurs in about 50% women and 25% of men

with cutaneous LP (Bolognia et al. 2012). Rarely, scalp, nails, esophagus, larynx

or conjunctiva may be affected (Eisen 1999, Gorouhi et al. 2014).

LP may appear only on the skin or on the oral mucosa but frequently both

lesions co-exist. Approximately 15% of OLP patients have cutaneous, and 20%

20

genital lesions, although in men the genital lesions are less prevalent (Eisen 1999).

On the other hand, oral lesions may occur in 70–77% of cutaneous LP patients

(Alrashdan et al. 2016). A form of LP with vulvovaginal and gingival involvement

has been termed vulvovaginal-gingival syndrome (Eisen 1994, Pelisse 1989).

Similarly, peno-gingival LP in men has been described (Petruzzi et al. 2005, Rogers

& Eisen 2003).

1.1.2 Epidemiology

About 0.22–1% of the population worldwide is affected by cutaneous LP (Bolognia

et al. 2012, Boyd & Neldner 1991). The prevalence of OLP is estimated to be about

0.2–2% (Regezi et al. 2017). Sixty-one to seventy-seven percent of OLP patients

are women (Bermejo-Fenoll et al. 2010, Carbone et al. 2009, Eisen 2002, Ingafou

et al. 2006, Thorn et al. 1988). The typical age at presentation of OLP is 30–60

years but the disease may start at any age (Regezi et al. 2017). Males may have a

younger age at onset of OLP (Ingafou et al. 2006).

1.1.3 Clinical features

Clinical manifestations of OLP are variable. Several clinical types, namely papular,

reticular, atrophic, plaque-like, erosive and bullous are recognized (Andreasen

1968, Pindborg et al. 1997, Regezi et al. 2017). The various clinical types of OLP

may be classified into two major groups: reticular and atrophic-erosive with or

without reticular lesions (Bagan-Sebastian et al. 1992, Neville et al. 2016). Often,

more than one clinical form manifests on the oral mucosa. OLP lesions are located

bilaterally, most often on the buccal mucosa, followed by the tongue and gingiva

(Carbone et al. 2009, Eisen et al. 2005).

The most common clinical form of OLP is reticular, which exhibits a lace-like

network of white striae, hence the term reticular (Figure 1a). The papular form of

OLP presents as small white papules (Figure 1b) and is quite rarely seen, which

may reflect the fact that it occurs mainly in the initial phase of the disease (Thorn

et al. 1988). Atrophic OLP displays an erythematous appearance of the oral mucosa,

often in combination with white lesions (Figure 1d). The atrophic form of OLP

affecting the gingivae may be called desquamative gingivitis (Figure 1c). Plaque-

like OLP lesions typically affect the dorsal tongue, and may clinically mimic

leukoplakia (Figure 1e). It has been reported that the plaque-like OLP is more

common in smokers than in non-smokers (Thorn et al. 1988). Erosive OLP

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manifests as ulcerative lesions, most commonly in the buccal mucosa (Figure 1f).

The rarest clinical form of OLP is the bullous form, with frank blisters (Cheng et

al. 2016). Bullae are typically located in the buccal mucosa.

The irritation of oral mucosa from sharp tooth edges, dentures, calculus etc.

may trigger OLP lesions, similarly to the isomorphic reaction to trauma in several

skin diseases called the Koebner phenomenon (Sagi & Trau 2011).

The majority of patients experience pain and tenderness associated with oral

lesions, especially in reaction to spicy, sour or hot food or drink (Carbone et al.

2009, Eisen 2002). Oral hygiene products may cause a burning sensation and

especially gingival lesions may impair oral hygiene practices. The oral mucosa may

feel rough or rigid. The signs and symptoms of OLP show natural fluctuation with

periods of exacerbations and quiescence, but typically persist for several years or

decades, in contrast to the self-limiting cutaneous LP (Carbone et al. 2009, Gorouhi

et al. 2014). The quality of life in patients with OLP is negatively affected by the

disease (Lopez-Jornet & Camacho-Alonso 2010b).

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Fig. 1. Clinical forms of oral lichen planus: a) reticular, b) papular, c) atrophic, d)

combination of reticular and atrophic forms, e) plaque-like and f) erosive-ulcerative.

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1.1.4 Histopathological features

Characteristic, although not specific, histopathological features of OLP include

epithelial hyperorthokeratosis or -parakeratosis, epithelial atrophy or acanthosis,

basal cell layer liquefaction (hydropic) degeneration, basal keratinocyte apoptosis,

a subepithelial eosinophilic amorphous band and a dense, band-like lymphocytic

inflammatory infiltrate in the superficial lamina propria that migrates to the lower

part of epithelium (Kramer et al. 1978, Neville et al. 2016, Pindborg et al. 1997).

Epithelial rete ridges are often missing or they may exhibit a “saw-tooth” pattern,

but this is more common in cutaneous LP (Cheng et al. 2016, Fernandez-Gonzalez

et al. 2011).

Intercellular spaces are widened in basal and spinous epithelial layers (Paul &

Shetty 2013, Shklar et al. 1978, Tyldesley & Appleton 1973). Ultrastructural

studies have demonstrated basal cells with cytoplasmic processes, which may be

longer and narrower in the erosive form of OLP compared to the non-erosive form

(Paul & Shetty 2013). Basal cells may also demonstrate acantholytic changes

(Shklar et al. 1978), cytoplasmic vacuolization (Paul & Shetty 2013, Tyldesley &

Appleton 1973), decrease or loss of desmosomal junctions, nuclear inclusions, and

lymphocyte entrapment (Paul & Shetty 2013). The basal and spinous epithelial

cells demonstrate thickening of tonofilaments (Hashimoto et al. 1966, Paul &

Shetty 2013, Pullon 1969). Reactive epithelial atypia is sometimes seen in OLP,

especially when superimposed candidiasis is present or when the biopsy is taken

adjacent to an existing ulceration.

The degree of liquefaction degeneration varies from slight to intense and is not

directly proportional to the number of apoptotic keratinocytes (Bascones-Ilundain

et al. 2007). The apoptotic basal keratinocytes manifest as homogenous

eosinophilic globules and are called colloid (hyaline, cytoid, Civatte) bodies

(Burgdorf & Plewig 2014).

The basement membrane zone (BMZ) may appear thickened and branching

and disruptions in its continuity are seen (Hashimoto et al. 1966, Jungell et al. 1989,

Paul & Shetty 2013, Pullon 1969). Disruptions of the anchoring structure of basal

keratinocytes including hemidesmosomes, filaments and fibrils have also been

demonstrated (Haapalainen et al. 1995). The basement membrane (BM) may show

separation from the overlying basal cells (Shklar et al. 1978). The structural

changes in the basal cell layer and BMZ make the epithelial-connective tissue

interface fragile and may occasionally result in cleft formation (Max-Joseph spaces)

that can clinically manifest as blisters (Sugerman et al. 2002).

24

The characteristic lymphocytic infiltrate in OLP is present immediately

subepithelially as a band-like zone. Intraepithelial lymphocytes are detected

between basal and spinous epithelial cells (Paul & Shetty 2013). The inflammatory

cell infiltrate in OLP consists mainly of T-lymphocytes (Walker 1976). Most T-cells

in the epithelium and in the disrupted BM are activated cytotoxic T-cell surface

glycoprotein CD8 alpha chain (CD8A)+ lymphocytes, while most T-cells in the

lamina propria are helper T-cell surface glycoprotein CD4 (CD4)+ lymphocytes

(Hirota et al. 1990, Sugerman et al. 2002). However, Khan et al. (2003) noted that

most subepithelial lymphocytes were CD8A+, and CD4+ lymphocytes were

located mainly in deeper lamina propria. Although B-cells are not considered to

have a major role in OLP pathogenesis, they are present in the inflammatory

infiltrate and may, in a proportion of cases be a dominant lymphocyte type (Mattila

et al. 2011). Plasma cells are typically not seen in any considerable numbers in OLP

inflammatory cell infiltrate, but may be present especially in biopsies from the

gingiva where nonspecific chronic inflammation is often present (Cheng et al.

2016). If plasma cells are seen in the infiltrate, a diagnosis of oral lichenoid lesion

(OLL) is usually favored (Mravak-Stipetic et al. 2014).

Besides lymphocytes, other leukocytes present in OLP lesions include

macrophages, mast cells and dendritic cells (Langerhans cells, LC) (Payeras et al.

2013). Macrophages are seen in close proximity to the basal cell layer in both

epithelium and lamina propria (Matthews et al. 1985, Paul & Shetty 2013).

Increased numbers of mast cells have been detected in OLP and most of them are

degranulated (Jontell et al. 1986, Sharma et al. 2011, Zhao et al. 1997, Zhao et al.

2001). Jontell et al. (1986) observed that most mast cells were located below the

subepithelial infiltrate while Zhao et al. (1997) found the greatest density of mast

cells in the superficial part of the subepithelial inflammatory infiltrate in OLP.

Degranulated mast cells were seen in the basal cell layer and juxtaepithelially by

electron microscopy (Paul & Shetty 2013). Langerhans cell density is increased

both in the epithelium and stroma in OLP compared to normal oral mucosa

(Gueiros et al. 2012, Gustafson et al. 2007, Hasseus et al. 2001, Santoro et al. 2005,

Villarroel Dorrego et al. 2002).

Direct immunofluorescence (IF) staining of lesional tissue shows a

characteristic linear or shaggy fibrinogen/fibrin deposition in the BMZ in almost

all OLP cases, however, this finding is not specific (Buajeeb et al. 2015, Laskaris

et al. 1982). Less often, colloid bodies and vessel walls show fibrin deposition

(Laskaris et al. 1982). Colloid bodies may also stain positive for immunoglobulin

M (IgM), immunoglobulin A (IgA) and complement 3 (C3) (Buajeeb et al. 2015).

25

Rarely, immunoglobulin G (IgG), IgM, IgA or C3 deposition may be found in the

BMZ (Hashimoto et al. 2015, Laskaris et al. 1982, Schiodt et al. 1981).

1.1.5 Diagnosis

The diagnosis of OLP can be made based on the typical clinical features, but may

require a histopathologic correlation (Neville et al. 2016). Occasionally, IF studies

are needed to rule out other immune-mediated mucosal diseases. Some authorities

recommend that both clinical and histopathologic criteria should be fulfilled for the

diagnosis of OLP (Cheng et al. 2016). Originally, the World Health Organization

(WHO) defined the clinical and histologic diagnostic features of OLP (Kramer et

al. 1978, Pindborg et al. 1997). In 2003, a paper proposed a modified set of

diagnostic criteria for OLP (van der Meij & van der Waal 2003). Recently, in an

American Academy of Oral and Maxillofacial Pathology (AAOMP) opinion paper

further amendments were suggested to the diagnostic criteria for OLP (Cheng et al.

2016) (Table 1).

The clinical and histopathologic features of OLP are variable and depend on

the site and type of OLP, as well as disease activity at the time of diagnostic

assessment (Cheng et al. 2016). In addition, other concurrent oral inflammatory

processes may alter the typical features of OLP. Furthermore, many clinical and

histopathological features of OLP are shared by a variety of other oral mucosal

diseases. It is therefore no wonder that inter- and intraobserver variability has been

demonstrated in the clinical and histopathologic assessment of OLP (van der Meij

et al. 1999, van der Meij et al. 2002, van der Meij & van der Waal 2003).

Hence, the accurate diagnosis of OLP can be challenging (Cheng et al. 2016).

The lack of a clear-cut and reproducible diagnosis is also a factor that may affect

the value of studies on OLP.

26

Table 1. AAOMP position paper diagnostic criteria for OLP (Cheng et al. 2016).

Criteria Features Exlusion criteria

Clinical Multifocal symmetric distribution

White and red lesions exhibiting one or

more of the following forms:

Reticular/papular

Atrophic

Erosive (ulcerative)

Plaque

Bullous

Lesions are not localized exclusively to

the site of smokeless tobacco placement

Lesions are not localized exclusively

adjacent to and in contact with dental

restorations

Lesion onset does not correlate with the

start of a medication

Lesion onset does not correlate with the

use of cinnamon-containing products

Histopathologic Band-like or patchy, predominantly

lymphocytic infiltrate in the lamina propria

confined to the epithelium-lamina propria

interface

Basal cell liquefactive (hydropic)

degeneration

Lymphocytic exocytosis

Absence of epithelial dysplasia

Absence of verrucous epithelial

architectural change

1.1.6 Differential diagnosis

The clinical differential diagnosis of OLP includes OLL, contact hypersensitivity,

chronic candidiasis, morsicatio, white sponge nevus, pemphigoid, pemphigus,

discoid lupus erythematosus, systemic lupus erythematosus, hairy leukoplakia,

graft-versus-host disease, chronic ulcerative stomatitis, multifocal leukoplakia,

proliferative verrucous leukoplakia and squamous cell carcinoma (Cheng et al.

2016, Regezi et al. 2017).

1.1.7 Oral lichenoid lesions

The terms oral lichenoid lesion (OLL) or oral lichenoid reaction (OLR) are used

for lesions that are similar to OLP but lack some of the typical clinical and/or

histopathologic features of OLP (Al-Hashimi et al. 2007, Cheng et al. 2016). OLL

include oral lichenoid contact lesion, oral lichenoid drug reaction, oral lichenoid

lesions of graft-versus-host disease, oral discoid lupus erythematosus, oral lesions

27

of systemic lupus erythematosus, erythema multiforme, paraneoplastic

pemphigus/paraneoplastic autoimmune multiorgan syndrome, chronic ulcerative

stomatitis and lichen planus pemphigoides (Khudhur et al. 2014).

The lichenoid tissue reaction, called “interface dermatitis” in dermatology is a

histopathological term originally defined by Pinkus (1973), and encompasses many

clinically diverse inflammatory skin diseases (Sontheimer 2009). Lichen planus is

the prototypic skin disease with interface dermatitis (Sontheimer 2009). Of note,

the chronic inflammation associated with preneoplastic or dysplastic lesions and

oral squamous cell carcinoma may sometimes exhibit a lichenoid tissue reaction

pattern (Fitzpatrick et al. 2014b).

In contrast to OLP, a causative factor for OLL should be sought and can

sometimes be found, most often being drugs or an amalgam filling (Al-Hashimi et

al. 2007, Cheng et al. 2016). A wide variety of medications are capable of inducing

OLL, including anti-hypertensives, non-steroidal anti-inflammatory drugs,

penicillamine and anti-malarials (Al-Hashimi et al. 2007, McCartan & McCreary

1997).

1.1.8 Associations with systemic diseases

Although no single systemic factor or disease is consistently associated with OLP,

hepatitis C virus (HCV) seropositivity has been demonstrated to be more common

in OLP patients (Alaizari et al. 2016, Lodi et al. 2010). This association is

especially present in certain geographic areas, being strongest in the Middle-East

(Baccaglini et al. 2013). HCV-RNA has been detected in OLP lesions and HCV

may attract HCV-specific T-lymphocytes (Kurokawa et al. 2003, Nagao et al. 2000,

Pilli et al. 2002).

The association of LP and various autoimmune diseases, including alopecia

areata and lupus erythematosus, is proposed (Chung et al. 2015, Scully et al. 1998).

Regarding OLP, no firm evidence linking it to autoimmune comorbidities exists

(Lopez-Jornet et al. 2014).

A few reports suggest that thyroid diseases could be associated with OLP. In a

study comparing small groups of HCV-negative and HCV-positive OLP patients,

thyroid dysfunction was found in 13% (3/23) of the HCV-negative cases and in 15%

(4/26) of the HCV-positive cases (Carrozzo et al. 1999). In addition, serum anti-

thyroid antibodies were found in 17% of the HCV-negative and in 4% of the HCV-

positive patients. Another study reported anti-thyroglobulin (Tg) antibodies in

21.4%, and anti-thyroid microsomal (TMA) antibodies in 24.4% of OLP patients

28

compared to 1.9% and 1.9%, respectively, in the healthy control subjects (Chang et

al. 2009). Furthermore, in a relatively large epidemiologic study, patients with LP

were found to have a higher prevalence of hypothyroidism than the age and sex

matched controls (10% vs. 5.7%) (Dreiher et al. 2009). In addition, the use of

thyroid therapeutics was reported to be more common in OLP patients than in

controls (4% vs. 0.5%), which suggests an association of OLP and hypothyroidism

(Kragelund et al. 2003).

Thymomas and Good syndrome (thymoma with hypogammaglobulinemia) are

associated with autoimmune conditions such as myasthenia gravis, thyroid diseases,

systemic lupus erythematosus, alopecia and lichen planus (Bernard et al. 2016,

Motegi et al. 2015, Seneschal et al. 2008). In particular, the erosive form of OLP

may be associated with thymoma (Bobbio et al. 2007, Kaku et al. 2011, Motegi et

al. 2015, Seneschal et al. 2008).

Stress is often attributed to the onset or exacerbation of OLP, even though no

causal relationship has been demonstrated. However, OLP patients are more likely

to suffer from anxiety, depression and low self-control than controls (Pippi et al.

2016).

1.1.9 Management

There is no curative treatment for OLP, so therapy is aimed at controlling symptoms

(Hodgson & Chaudhry 2010). Elimination of local irritating factors, treatment of

possible oral candidiasis and dental and periodontal treatment is important. In fact,

patients with gingival OLP typically benefit from calculus removal and rigorous

plaque control (Holmstrup et al. 1990, Salgado et al. 2013, Stone et al. 2013, Stone

et al. 2015). Regarding medical management, topical corticosteroids are considered

the mainstay of therapy of OLP, although the evidence-base for their effectiveness

is weak (Thongprasom et al. 2011). There are a wide range of other treatment

options available, both topical and systemic, but again with little evidence for their

effectiveness in reducing the signs or symptoms of OLP or for any one being better

than the others (Cheng et al. 2012, Suresh et al. 2016, Thongprasom et al. 2011).

Besides topical corticosteroids, most clinical research in recent years has been done

on topical tacrolimus. Conflicting evidence comparing the effectiveness of topical

corticosteroids and tacrolimus has been reported (Chamani et al. 2015, Guo et al.

2015, Hettiarachchi et al. 2016, Sivaraman et al. 2016).

29

Topical glucocorticoids

The mechanism of action of corticosteroids is believed to involve the binding of

the drug to steroid receptors in the cytoplasm, which results in inhibition of

synthesis of proteins responsible for induction of an inflammatory response (Goa

1988). Corticosteroids are known to suppress the production of inflammatory

humoral factors, hinder leucocyte migration to sites of inflammation, decrease T-

cell proliferation, increase T-cell apoptosis and interfere with the function of

endothelial cells, granulocytes, mast cells and fibroblasts (Brazzini & Pimpinelli

2002).

Topical corticosteroid potency is based on their skin vasoconstricting capacity;

however, comparable vasoconstricting ability does not denote therapeutic

equivalence. Topical corticosteroids are classified from very high potency to low

potency (classes I–VII) (Essential Medicines and Health Products Information

Portal. A World Health Organization resource 2016).

Several classes and potencies of topical corticosteroids are used in the

management of OLP (Thongprasom & Dhanuthai 2008). In Finland the available

mid-potency preparations include 0.1% triamcinolone acetonide as a rinse, spray

or paste with Orabase® gel, beclomethasonedipropionate or beclomethasone-

dipropionatemonohydrate as a spray, fluticasonepropionate spray; potent

preparations include 0.1% betamethasone 17-valerate; and high-potency

preparations include 0.05% clobetasolpropionate. Methylprednisolone is available

for intralesional injections.

The most common side effect of topical corticosteroid use in the oral cavity is

secondary oral candidiasis (Thongprasom & Dhanuthai 2008). A temporary

burning sensation is occasionally reported when topical corticosteroids are applied

on the oral mucosa (Laeijendecker et al. 2006, Sonthalia & Singal 2012). Other

possible adverse effects of oral topical corticosteroids include taste alteration,

xerostomia, nausea and sore throat (Thongprasom & Dhanuthai 2008). Systemic

transmucosal absorption of topical corticosteroids may occur (Varoni et al. 2012).

This can lead to adrenal suppression clinically manifesting as cushingoid symptoms

or Cushing’s syndrome (Decani et al. 2014, Gonzalez-Moles et al. 2002, Lehner &

Lyne 1969). However, most patients do not develop systemic side effects, even

after prolonged use of topical corticosteroids (Carbone et al. 1999, Plemons et al.

1990).

30

Topical tacrolimus

Tacrolimus (FK-506) is a macrolide obtained from a soil bacterium, Streptomyces

tsukubaensis (Kino et al. 1987). It is an immunosuppressant that was originally

developed for systemic administration to prevent rejection in organ transplants

(Fung & Starzl 1995). The mechanism of action of tacrolimus is based on the

inhibition of calcineurin, which results in inhibition of the activation and

proliferation of T-lymphocytes and suppression of synthesis of pro-inflammatory

cytokines (Reynolds & Al-Daraji 2002, Tsuda et al. 2012).

A topical formulation of tacrolimus was later developed for treatment of atopic

dermatitis (AD) (Ruzicka et al. 1999). Since the late 1990s, off-label use of topical

tacrolimus with favorable outcomes was reported in OLP not responding to

conventional treatment (Hodgson et al. 2003, Kaliakatsou et al. 2002, Lener et al.

2001, Morrison et al. 2002, Rozycki et al. 2002, Vente et al. 1999). In Finland,

topical tacrolimus is available as 0.03% and 0.1% ointment preparations.

Topical tacrolimus may cause transient local irritation at the site of oral

mucosal application in 16–56% of the patients (Byrd et al. 2004, Corrocher et al.

2008, Hodgson et al. 2003, Kaliakatsou et al. 2002, Laeijendecker et al. 2006). The

irritation is typically felt as a burning or stinging sensation, either when the drug is

administered, or is associated with eating or drinking. Other less commonly

encountered adverse effects of oral topical tacrolimus use include taste alterations,

oral dryness and headache (Hodgson et al. 2003, Kaliakatsou et al. 2002, Olivier

et al. 2002, Ribero et al. 2015). Systemic absorption of topical tacrolimus through

the oral mucosa resulting in detectable blood concentrations of the drug may occur

(Kaliakatsou et al. 2002). However, hematological renal and liver function tests

typically reveal no changes in these cases, and clinically significant adverse effects

have not been reported (Hodgson et al. 2003, Lopez-Jornet et al. 2010). The

possibility that topical tacrolimus could contribute to oral cancer development has

been suggested in a few case reports (Becker et al. 2006, Mattsson et al. 2010,

Morita et al. 2016). At the moment there is no evidence linking topical tacrolimus

to malignancies in cutaneous use (Chia & Tey 2015, Cury Martins et al. 2015,

Siegfried et al. 2016), and in mucosal use there is not enough data to make firm

conclusions. The long-term safety of topical tacrolimus use in the oral mucosa

remains to be established (Lopez-Jornet et al. 2010).

31

Other therapies

Besides topical corticosteroids and tacrolimus, numerous therapeutics in various

formulations and dosages are reported to have efficacy in OLP (Yang et al. 2016).

Many are mentioned in cases refractory to topical corticosteroids.

Topical pimecrolimus, cyclosporine A, aloe vera, hyaluronic acid, chamomile,

curcumin, retinoids, tetracyclin, rapamycin, , thalidomide, different phototherapies

like psoralen and ultraviolet A radiation (PUVA) or low-level or CO2 laser therapy,

ozone, surgical excision, cryosurgery, and palatal graft are among the treatment

modalities reported (Jajarm et al. 2011, Kazancioglu & Erisen 2015, Kia et al. 2015,

Lopez-Jornet & Aznar-Cayuela 2016, Nolan et al. 2009, Pavlic & Vujic-Aleksic

2014, Thongprasom et al. 2013, Yang et al. 2016). However, in Finland these are

not commonly in use.

Systemic therapies for OLP reported include corticosteroids, retinoids,

thalidomide, mycophenolate mophetil, etanercept, curcumin, glycyrrhizin and

extracorporeal photochemotherapy (Yang et al. 2016). In Finland, mainly

prednisolone is used for systemic management of OLP.

1.1.10 Outcome measures in OLP

Different outcome measures, both investigator-assessed and patient-reported are

used in OLP intervention trials (Ni Riordain et al. 2015, Wang & van der Waal

2015). A recent review of the disease scoring systems identified 22 different scales

and a lack of universally accepted and validated measure for OLP (Wang & van der

Waal 2015). In addition, the measures used frequently lack the necessary sensitivity

to detect therapeutic efficacy vs. placebo (Zakrzewska et al. 2005).

1.1.11 Prognosis

No studies are available with a decades long follow-up of a large population of OLP

patients. However, it is acknowledged that OLP is typically a long-lasting disease,

with only a small proportion of patients experiencing recuperation (Carbone et al.

2009, Thorn et al. 1988). The malignant transformation rate of OLP is estimated to

be 1.09% (Fitzpatrick et al. 2014a). One study suggested that the malignant

transformation is associated with OLL, not OLP (van der Meij et al. 2007). A recent

study found that DNA ploidy analysis of a biopsy specimen was able to predict

malignant transformation in a proportion of OLP patients (Sperandio et al. 2016).

32

The time from diagnosis to transformation is on average 51.4 months (Fitzpatrick

et al. 2014a).

1.1.12 Pathogenesis

The current hypothesis of pathogenic mechanisms in OLP points to a state of

immune dysregulation (Kurago 2016, Nogueira et al. 2015, Payeras et al. 2013).

Antigen-specific, T-cell-mediated as well as non-specific immune mechanisms are

involved in OLP (Sugerman et al. 2002). (Figure 2: Schematic illustration of the

hypothetical pathogenesis of OLP).

Although OLP has many features of a true autoimmune disease, like disease

chronicity, female predilection, adult onset and autoreactive T-cells, other features

like circulating autoantibodies are not found consistently (Ingafou et al. 1997,

Sugerman et al. 2002). In addition, the target self-antigen in OLP is not known.

Although a lichen planus-specific antibody has not been found in OLP or in oral

lichenoid drug eruptions (McCartan & Lamey 2000), some investigators have

found basal cell cytoplasmic autoantibodies in OLR (Lamey et al. 1995). In

addition, occasional positivity for antiepithelial, and desmoglein 1 and 3 antibodies

using indirect IF has been reported (Ingafou et al. 1997, Kinjyo et al. 2015, Lukac

et al. 2006). Also autoantibodies against ETS-related transcription factor Elf-3

(ELF3), a transcription factor for keratinocyte differentiation, are present in a

proportion of OLP patients (Danielsson et al. 2013).

Although no firm conclusions about the possible genetic basis for OLP can be

made at this time, gene polymorphisms of cytokines interferon gamma (IFNG),

tumor necrosis factor (TNF), interleukins 4, 10 and 12A (IL4, IL10 and IL12A) may

contribute to OLP susceptibility (Jiang et al. 2015, Lu et al. 2015).

Antigen-specific immune mechanisms

The antigen or antigens triggering the T-cell mediated immune response are

suspected to include extrinsic factors like viruses, bacteria, drugs, dental materials

or intrinsic factors like self-peptides (Kurago 2016). The virus most commonly

linked to OLP is HCV, and in susceptible individuals, the HCV antigens expressed

on keratinocyte surfaces could act as targets for cytotoxic T cells (Lodi et al. 2005).

An interesting recent finding was the presence of bacteria in keratinocytes and T-

cells in OLP tissue (Choi et al. 2016). Altered expression of heat shock proteins

(HSP) have been found in OLP so it has been speculated that they could act as

33

autoantigens in OLP (Chaiyarit et al. 2009, Garcia-Garcia et al. 2013, Sugerman et

al. 1995).

There are two possible mechanisms suggested for the initiation of the T-cell

response in OLP. During the initial phase of OLP lesion formation, an antigen may

be expressed in association with the human leukocyte antigen (HLA) class I

histocompatibility complex on keratinocytes (Sugerman et al. 2002). Cytotoxic

CD8A+ T-cells may then, on routine surveillance (“chance encounter”) or via

attraction by keratinocyte-derived chemokines (“directed migration”), recognize

the antigen, become activated and trigger apoptosis of the basal keratinocytes

(Sugerman et al. 2000, Sugerman et al. 2002).

On the other hand, it seems that before CD8A+ T-cell-mediated cytotoxic

attack on keratinocytes can occur, CD8A+ T-cells require interaction with activated

CD4+ T-cells (Sugerman et al. 2002). Hence, early in the OLP disease process,

antigen-presenting cells (APC), including LCs or keratinocytes may present a HLA

class II histocompatibility complex associated antigen to CD4+ T-cells, which, with

the help of co-stimulatory molecules from keratinocytes and APCs, become

activated (Sugerman et al. 2002). The initial encounter of LCs with the triggering

antigen typically leads to LC maturation and migration to regional lymph nodes for

antigen presentation and subsequent T-cell activation. However, T-cell-LC

interactions leading to T-cell activation may possibly occur at a lesion site in OLP

(Kurago 2016). The crosstalk between activated CD4+ T-cells and CD8A+T-cells

is thought to result in apoptosis of the keratinocytes. The increased number of LCs

and CD4+T-cells in lesion sites suggests that they are indeed involved in OLP

pathogenesis. Furthermore, early OLP lesions may contain more CD4+ cells with

an increase of CD8A+ cells with progression of the disease (Sugerman et al. 2002).

Non-specific T-cells are also attracted to the lesion site by secretion of chemokines.

The role of cytokines

The immune dysregulation in OLP is in many ways related to abnormal production

of various inflammatory mediators, including cytokines and proteases, which are

present both in the lesions and in peripheral blood. These inflammatory mediators

orchestrate interactions between many cell types, like keratinocytes, T-cells, and

dendritic cells (DCs) in complex signalling networks (Lu et al. 2015). Depending

on the effect the mediators have on the cells, the resulting state may be pro-

inflammatory or anti-inflammatory.

34

Among the key molecules that regulate both innate and adaptive immune

responses are cytokines, small peptides that are synthesized and secreted by many

immune and non-immune cells (Lu et al. 2015). They act by binding to specific cell

surface receptors and, depending on the target cell type and the environment, exert

different effects on the cell phenotype and function (Lu et al. 2015). The expression

of a great range of inflammation-related cytokines in lesional tissue, saliva,

peripheral blood mononuclear cells and serum in OLP has been studied. In

particular, TNF, INFG and interleukins have been proteins of interest in OLP (Lu

et al. 2015).

Expression of TNF is increased in lesional tissue, saliva and serum in OLP

patients compared to controls (Pezelj-Ribaric et al. 2004, Rhodus et al. 2005,

Sklavounou et al. 2000, Sklavounou-Andrikopoulou et al. 2004, Younes et al.

1996). Moreover, increased production of TNF has been demonstrated by

keratinocytes, mast cells and CD4+ T-cells in OLP (Piccinni et al. 2014, Yamamoto

et al. 1994, Yamamoto & Osaki 1995, Zhao et al. 2001). TNF is thought to stimulate

adhesion of lymphocytes to the luminal surfaces of blood vessels and their

subsequent extravasation into the tissue (Zhao et al. 2002a). In addition, TNF may

stimulate the production of other inflammatory mediators, including C-C motif

chemokine 5 (CCL5) by T-cells, and matrix metalloproteinase 9 (MMP9) as well

as itself (Khan et al. 2003, Zhao et al. 2001, Zhou et al. 2001). One of the

mechanisms suggested to induce keratinocyte apoptosis in OLP involves TNF,

secreted by CD8A+ T-cells, which may bind to its receptor on the basal and

suprabasal keratinocyte surface and eventually activate the caspase cascade leading

to keratinocyte apoptosis (Younes et al. 1996).

IFNG positivity and overexpression compared to healthy controls has been

detected in OLP subepithelial mononuclear cells, suggesting a Th1 predominant

type immune response in OLP (Khan et al. 2003, Wang et al. 2015). IFNG may

thus be implicated in the activation of CD8A+ T-cells and subsequent keratinocyte

apoptosis (Kurago 2016, Lu et al. 2015). Decreased production of IFNG in

peripheral blood mononuclear cells in OLP patients has been observed, while

inconsistent results have been reported regarding the expression in saliva and serum

of patients (Lu et al. 2015).

Interleukins are produced by numerous cell types and participate broadly in

inflammatory and immune-mediated processes (Lu et al. 2015). A wide variety of

interleukins have been studied in OLP and implicated in its pathogenesis (reviewed

in Lu et al. 2015). It is suggested that, e.g. IL12 and IL18 may stimulate IFNG

expression and activate natural killer (NK) cell and T-cell cytotoxicity; in addition

35

IL18 may stimulate TNF expression (Kurago 2016). IL2 may participate in the

signaling between CD4+ and CD8A+ T-cells in OLP (Sugerman et al. 2002).

Among the chemokines implicated in OLP pathogenesis are CCL5 and

chemerin that are involved in the recruitment of various inflammatory cells in OLP

(Kurago 2016, Sugerman et al. 2002). CCL5 activates the C-C chemokine receptor

type 1 (CCR1) in mast cells and stimulates mast cell degranulation with subsequent

release of TNF and chymase. Cell surface receptors for CCL5 have been identified

in T-cells and mast cells in OLP (Zhao et al. 2002b). Chemerin may be involved in

attracting natural killer (NK) cells in OLP, since NK cells were found to express

the chemerin receptor in OLP (Parolini 2007).

The role of proteases

Matrix metalloproteinases are a family of zinc-containing endo-peptidases whose

main function is proteolytic degradation of extracellular matrix proteins. MMP1,

MMP2, MMP3, MMP7 and MMP9 may play a role in OLP (Ertugrul et al. 2013,

Rubaci et al. 2012, Zhou et al. 2001). MMP9 is seen mainly in the inflammatory

infiltrate in OLP, and it may be involved in the BM disruption (Sugerman et al.

2002, Zhou et al. 2001). Chymase, secreted by mast cells, may directly cause BM

disruption or activate T-cells to secrete MMP9, which causes the disruption

(Sugerman et al. 2002). T-lymphocytes may use the BMZ breaks to infiltrate the

epithelium (Hashimoto et al. 1966, Zhou et al. 2002).

The role of adhesion molecules

The migration of leukocytes to the OLP lesion site is mediated via chemokines and

cellular adhesion molecules. Intercellular adhesion molecule 1 (ICAM1)

expression is detected in keratinocytes, endothelial cells and leukocytes in OLP

(Little et al. 2003). The expression of other vascular cell adhesion molecules, e.g.

vascular cell adhesion protein 1 (VCAM1), is also increased in OLP (Regezi et al.

1996, Walton et al. 1994), and probably contributes to lymphocyte extravasation.

In addition, components of BM that are overexpressed, laminin, type IV collagen

and type VII collagen, in the junctional area may serve as ligands for lymphocyte

adhesion molecules in OLP (Eversole et al. 1994).

It is proposed that in OLP, keratinocytes may respond to lymphocyte-mediated

damage in the epithelium-connective tissue interface by increased expression of the

36

interface-associated adhesion molecules, and this could help in resisting epithelial

separation from connective tissue (Ramirez-Amador et al. 1996).

Other possible factors contributing to OLP pathogenesis

The specific mechanism that results in keratinocyte apoptosis in OLP is still unclear,

but several types of interactions between cytotoxic CD8A+ T cells and basal and

suprabasal keratinocytes may occur that activate the caspase cascade. These include

mainly TNF-TNF receptor interaction (mentioned above), perforin-1 (PNF1) and

granzyme B (GZMB) secreted by cytotoxic T-cells and tumor necrosis factor

receptor superfamily member 6–tumor necrosis factor ligand superfamily member

6 (FAS-FASLG) interaction (Sugerman et al. 2002). It has also been suggested that

the basal keratinocyte apoptosis may be related to cellular tumor antigen p53 (TP53)

or bcl-2-like protein 1 (BCL2L1) proteins (Dekker et al. 1997).

It has been proposed that the BM disruption seen in OLP may by caused by

apoptotic keratinocytes that are not able to perform their normal function in the

maintenance of BM structure. On the other hand, keratinocytes need a BM-derived

cell survival signal to avert the start of apoptosis. Hence, both BM and

keratinocytes are dependent on each other for normal function and the breakdown

of this equilibrium may lead to a cyclical, chronic disease process (Sugerman et al.

2002).

Keratinocyte differentiation is altered in OLP (Boisnic et al. 1995, Danielsson

et al. 2013, Danielsson et al. 2014, Jacques et al. 2009). In fact, several genes

involved in epithelial development and differentiation were differentially expressed

in OLP epithelium compared to healthy epithelium (Danielsson et al. 2014). The

altered keratinocyte differentiation suggests that the normal epithelial barrier

function is affected, and may contribute to the pathogenesis of OLP.

37

Fig. 2. A hypothetical model of pathogenesis of OLP. A. Antigen specific mechanisms.

B. Non-specific mechanisms. Please see text. Modified from Sugerman et al. (2002).

38

1.2 Toll-like receptors 4 and 9 in immune-mediated conditions

Toll-like receptors (TLR) are a family of pattern recognition receptors (PRRs)

expressed in various immune and non-immune cells that play a key role in the host

defense against infection by sensing invading pathogens (Kumar et al. 2011). The

specific recognition of the evolutionarily conserved molecules of pathogens known

as pathogen associated molecular patterns (PAMPs) by TLRs is essential in

initiating and controlling innate immunity and subsequent activation of the adaptive

immune responses (Kawai & Akira 2010). In addition to PAMPs, endogenous

metabolites such as nucleic acids, fatty acids and phospholipids, produced by tissue

injury or inflammation may act as ligands for TLRs (Miyake & Kaisho 2014).

In humans, 10 different TLRs have been identified (Kawai & Akira 2010, Sasai

& Yamamoto 2013). TLRs expressed on cell surfaces (e.g. TLR4) recognize mainly

microbial lipopolysaccharides (LPS), lipids and proteins (Kawai & Akira 2010).

On the other hand, TLRs that are expressed exclusively intracellularly (e.g. TLR9)

mostly sense microbial nucleic acids (Kawai & Akira 2010, Sasai & Yamamoto

2013). The non-microbial, endogenous ligands or self-molecules (constituents of

an organism that can be differentiated from foreign substances by the immune

system) capable of binding to TLRs include hyaluronan (HA), high mobility group

protein B1 (HMGB1), HSP, and defensin beta 4A (DEFB4A) (Lin et al. 2011, Yu

et al. 2010). After the stimulation of TLRs by their agonists, they recruit several

downstream adaptor molecules (mainly TIR domain-containing adapter molecule

(TICAM1) and myeloid differentiation primary response protein MyD88 (MYD88)

that mediate further immune signaling to produce inflammatory cytokines,

chemokines and co-stimulatory molecules by the immune cells (Gianchecchi &

Fierabracci 2015, Li et al. 2009, Lin et al. 2011). TLR activation and specificity

are regulated through complex signaling mechanisms (Qian & Cao 2013).

Based on the ability of TLRs to recognize a large range of host-derived

molecular patterns and their expression on a wide variety of cell types, it is probable

that TLRs participate in the development, progression and resolution of most

noninfectious inflammatory and immune diseases (Lin et al. 2011). TLR-mediated

immune responses are implicated in chronic epidermal inflammatory diseases such

as psoriasis, LP and AD (Ermertcan et al. 2011, Miller & Modlin 2007), as well as

mucosal diseases such as chronic rhinosinusitis (Ramanathan et al. 2007),

ulcerative colitis, coeliac disease (Ospelt & Gay 2010) and aphthous ulcers (Gallo

et al. 2012, Hietanen et al. 2012). Numerous studies have demonstrated the

39

involvement of various TLRs in initiation and progression of periodontitis,

reviewed by Song et al. (2016).

Regarding TLR4 and TLR9 expression in oral diseases, both have been found

in the epithelial and connective tissue of healthy gingivae and periodontitis, with a

propensity for stronger expression in inflamed tissues (Beklen et al. 2008, Rojo-

Botello et al. 2012, Sugawara et al. 2006). In contrast to these findings, Ren et al.

did not find expression of TLR4 in healthy periodontal tissue (Ren et al. 2005).

TLR4 and TLR9 expression with variable intensities has been reported also in oral

chronic hyperplastic candidiasis, leukoplakia and healthy oral sulcular mucosa (Ali

et al. 2008).

In previous literature on TLRs and LP, toll-like receptor 9 protein and mRNA

expression was shown to be increased in skin lesions compared to healthy skin (Li

et al. 2007). In OLP patients, enhanced expression of TLR2 in mucosal lesions as

well as in peripheral blood monocytes was reported (Ohno et al. 2011). In contrast

to these findings, another research group reported a reduced toll-like receptor 2, but

increased toll-like receptor 4 proteins and transcripts expression in lesional

epithelium and unstimulated whole saliva (UWS) epithelial cells from OLP patients

(Janardhanam et al. 2012). In addition, soluble forms of TLR4 were found to be

increased and functional in UWS, but TLR4 mRNA levels of salivary epithelial

cells were reduced in OLP patients compared to controls (Zunt et al. 2009).

Two earlier reports have described the effect of tacrolimus on toll-like receptor

expression. Kis et al. (2006) reported that TLR4 expression in human keratinocytes

was not influenced by tacrolimus and Antiga et al. (2011) showed that topical

tacrolimus therapy did not have an impact on the immunohistochemical expression

of TLR4 and TLR9 in AD.

1.3 Hyaluronan, its receptor CD44 and its metabolizing enzymes

HAS1–3 and HYAL1–2 in immune-mediated conditions

Hyaluronan (HA) is a large polysaccharide composed of alternating N-acetyl-

glucosamine and glucuronic acid units that is found ubiquitously in human

extracellular matrix tissues (Dicker et al. 2014). HA has diverse biological

functions in both normal and pathological processes: it can bind large amounts of

water and form viscous gels, function as a filter, and act as a signaling molecule.

HA is involved in regulating cell functions, tissue development, embryogenesis,

inflammation, wound healing, tumor progression and metastasis (Dicker et al.

2014). It also contributes to the maintenance of tissue homeostasis.

40

HA is recognized as a significant and active molecule in many aspects of

inflammation, participating in the regulation of expression of inflammatory genes,

the recruitment of inflammatory cells and the release of inflammatory cytokines

(Heldin et al. 2008, Jiang et al. 2011, Petrey & de la Motte 2014). Increased

accumulation of HA is noted after tissue injury and during inflammatory conditions

(Heldin et al. 2008, Jiang et al. 2011, Monslow et al. 2015, Petrey & de la Motte

2014). Immune cell interactions with HA increase upon an inflammatory response.

This may help recruit immune cells to the site of inflammation as well as keep cells

at the site and may facilitate their survival and function (Lee-Sayer et al. 2015).

During infection and tissue injury, HA becomes fragmented, and the fragments

accumulate extracellularly, which provokes an inflammatory response (Lee-Sayer

et al. 2015).

HA is synthesized on the plasma membrane by three hyaluronan synthase

isoenzymes (HAS1-3), of which HAS2 is the most active and abundant in many

cells (Vigetti et al. 2014). An inactive reservoir of HAS is present intracellularly

that may be activated upon various stimuli and transported to the plasma membrane

for HA synthesis (Rilla et al. 2005). Dysregulation of hyaluronan synthases occurs

during tissue injury and inflammation (Siiskonen et al. 2015).

Enzymatic degradation of HA occurs by hyaluronidases (HYAL). Human

HYAL1 and HYAL2 are the two major hyaluronidases cleaving HA in somatic

tissues (Stern & Jedrzejas 2006). HYAL1 (serum hyaluronidase) is an acid active

endo/lysosomal enzyme, while HYAL2 is attached to the plasma membrane via a

glycosylphosphatidylinositol anchor (Harada & Takahashi 2007). In addition to

HYALs, HA can be degraded by reactive oxygen species created under stress

conditions (Dicker et al. 2014). Cleavage of HA increases the permeability of

connective tissue and lowers the viscosity of body fluids. Hence, HYALs have been

implicated to have a role in bacterial pathogenesis, spread of toxins and progression

of cancer, as well as in facilitating direct contact between microbes and host cell

surfaces (Girish & Kemparaju 2007).

CD44 is a cell surface adhesion molecule that is widely expressed in a variety

of cells, including leukocytes, fibroblasts, epithelial and endothelial cells

(Goodison et al. 1999). CD44 may occur as a transmembrane cell surface receptor,

in the extracellular matrix as well as in a soluble form, e.g. in saliva (Pure & Cuff

2001). The diverse roles of CD44 include cell–cell and cell–matrix interactions

comprising cell proliferation and migration, hematopoiesis and lymphocyte

activation, homing and extravasation (Jordan et al. 2015). CD44 is a principal HA-

binding receptor (Heldin et al. 2008).

41

Previous studies have shown that healthy oral epithelium expresses HA both

on the cell surface and in the intercellular space in the basal and spinous cell layers

(Kosunen et al. 2004, Tammi et al. 1990). The expression of hyaluronan synthases

and hyaluronidases in oral mucosal tissue has not been described before, but

cultured oral mucosal epithelial cells demonstrated HAS1–3 mRNA (Yamada et al.

2004). CD44 is known to be expressed in normal squamous epithelium, locating

on cell membranes of the basal two-thirds of the epithelium (Kosunen et al. 2007).

In OLP specimens, Neppelberg et al. (2007) demonstrated that CD44

immunoexpression was comparable to that in healthy control tissue. On the other

hand, Chaiyarit et al. (2008) found a decreased expression of the isoform CD44v6

in OLP epithelium compared to healthy oral epithelium.

1.4 Cathepsin K in immune-mediated conditions

Cathepsin K (CTSK) is an acidic cysteine endoproteinase belonging to the papain-

like cysteine peptidase family (Novinec & Lenarcic 2013). CTSK was initially

identified as an osteoclast-specific lysosomal protease, but has since been found to

be expressed in many different cell types and to be involved in various

physiological and pathological processes (Novinec & Lenarcic 2013). Experimental studies have found that CTSK contributes to autoimmune

inflammation. CTSK is required for TLR9 signaling by DCs in autoimmune

arthritis (Asagiri et al. 2008) and for induction of inflammation and bone erosion,

as well as for expression of TLRs 4, 5 and 9 in rheumatoid arthritis (Hao et al.

2015b). In a mouse model of psoriasis, CTSK was involved in the development of

psoriasis-like skin lesions through TLR7-dependent Th17 activation (Hirai et al.

2013).

In oral diseases, CTSK was first found to be involved in peri-implantitis and

periodontitis (Mogi & Otogoto 2007, Strbac et al. 2006). Further studies have

corroborated the important role of CTSK in periodontal disease (Beklen et al. 2015,

Chen et al. 2016, Hao et al. 2015b). CTSK is also involved in the bone loss and

inflammation associated with periapical disease (Gao et al. 2013, Hao et al. 2015a),

and in diffuse sclerosing osteomyelitis (Montonen et al. 2006).

42

43

2 Aims of the study

The objectives of this thesis included investigating the prevalence of systemic

conditions, especially thyroid diseases, in OLP, and comparing the effectiveness of

topical tacrolimus, triamcinolone acetonide and placebo in the management of

symptomatic OLP. To increase our understanding of the etiopathogenesis of OLP,

the immunohistochemical expression of specific, previously uninvestigated

molecules were studied in OLP, as well as the effect of tacrolimus treatment on

their expression.

The more specific research questions were:

1. Are thyroid diseases or thyroxin medication more common in OLP and OLL

patients?

2. Are topical tacrolimus and triamcinolone acetonide more effective than

placebo in the management of symptomatic OLP, and is there a difference in

the effectiveness between tacrolimus and triamcinolone acetonide?

3. Are toll-like receptors 4 and 9 (TLR4 and TLR9) associated with OLP

pathogenesis?

4. Could hyaluronan (HA), its receptor CD44 antigen (CD44) and its

metabolizing enzymes hyaluronan synthases 1–3 (HAS1–3) and

hyaluronidases 1-2 (HYAL1–2) be involved in OLP pathogenesis?

5. Is cathepsin K (CTSK) expressed in OLP mucosa, and is its expression

associated with TLR4 or TLR9?

6. Does topical tacrolimus have an effect on the expression of 1) TLR4 and TLR9

2) HA, CD44, HAS1–2, and HYAL1–2, and 3) CTSK?

44

45

3 Patients and materials

Patients, materials and methods are described in more detail in the original articles

(I, III–V) and manuscript (II). The study protocols were approved by The Regional

Ethics Committee of the Northern Ostrobothnia Hospital District (I, II, III, IV–V).

The Research Ethics Committee of the Northern Savo Hospital District (Kuopio)

was notified of the study II. Permission to use archival tissue material (III–V) was

given by the National Supervisory Authority for Welfare and Health (VALVIRA).

An approval for work II was granted by the Finnish Medicines Agency. The clinical

trial (II) was registered at clinical.trials.gov (identifier NCT01544842).

3.1 Patients

The number of cases and controls, and other demographic data of the patients in

the studies are shown in table 2. In studies III and IV, the same group of specimens

was used.

Table 2. Basic characteristics of the case and control subjects in studies I-V.

Characteristic I

II III

IV

V

Sample size

Cases

Controls

222

222

27

n/a

50

5

55

23

25

14

Female %

Cases

Controls

71

71

85

n/a

n/a

n/a

n/a

n/a

55

71

Age mean (range)

Cases

Controls

50 (17–77)

50 (13–80)

56 (37–71)

n/a

n/a

n/a

n/a

n/a

53 (9–88)

30 (22–59)

OLP diagnostic criteria Cl+Hp Cl+Hp Hp Hp Hp

n/a not applicable, Cl=clinical, Hp=histopathologic

3.2 Medical records (I)

Patient files of subjects diagnosed with OLP or OLL from 1992 to 2001 at the

Institute of Dentistry, University of Oulu and/or the Oral and Maxillofacial

Department, Oulu University Hospital were investigated. The control group

consisted of age and sex matched persons, who had visited the Institute of Dentistry,

46

University of Oulu, for general dental care as undergraduate teaching patients

during the same period, and their patient files were investigated also.

3.3 Medications in the clinical trial (II)

Interventions in the clinical trial included 0.1% tacrolimus ointment (Protopic®,

Astellas Pharma, Tokyo, Japan), 0.1% triamcinolone acetonide paste (Kenacort-T®

/Kenacort-A® Orabase, Bristol–Myers Squibb, New York, USA/Dermapharm,

Grünwald, Germany) and Orabase paste (ConvaTec, Deeside, UK).

3.4 Tissue specimens (II–V)

Tissue specimens in the clinical trial (II) were collected from 24 patients before the

intervention and after using the study medication for 3–6 weeks. Half of each

specimen was fixed in 10% neutral buffered formalin and half was snap frozen in

liquid nitrogen and stored at -70°C.

For studies III–V, formalin-fixed, paraffin-embedded tissue specimens (FFPE)

diagnosed as OLP were retrieved from the archives of the Department of Pathology,

Oulu University Hospital.

As controls in studies III–V, we used a) healthy oral mucosa tissue samples

from the archives of the research laboratory of the Institute of Dentistry, University

of Oulu, b) healthy buccal mucosal samples obtained from investigators (MS, AK)

and from students in Kuopio University Hospital and c) histologically normal

looking oral mucosal tissue from oral squamous cell cancer resection specimens

(distant from the tumor) from the archives of the Department of Pathology, Kuopio

University Hospital.

47

4 Methods

4.1 Epidemiological study (I)

Study I was conducted as a retrospective case-control study. Age, gender, medical

history (medical diagnoses and regular medications), allergies, smoking, oral

diseases, main OLP/OLL clinical type and OLP/OLL location were recorded for

the cases and relevant corresponding variables also for the controls.

4.2 Clinical trial (II)

The clinical trial (II) was conducted as a double-blind, randomized, placebo-

controlled trial.

To measure the outcome of the interventions, a scoring system was developed

for the trial, modified from a previously reported assessment method in mucous

membrane pemphigoid (Setterfield et al. 1998). This clinical score (CS) was used

to measure the clinical signs and subjective symptoms at different time points. The

primary outcome variable was the change in CS from baseline to week 3, and the

primary research question was whether or not there are differences between the

treatment arms.

If a ≥20% decrease in the CS was noted at week 3, it was considered a response

to treatment, while <20% decrease or increase in CS was considered a non-response

to treatment.

4.3 Statistical methods (I, II)

I: The clinical features of the patients’ OLP/OLL lesions were presented using

frequency and percentage distributions. Cross-tabulation was used to compare

patient characteristics between OLP, OLL and control cases. In addition, a

multivariable logistic regression method was applied to analyze the associations of

selected patient characteristics with the patient groups. Odd ratios (ORs) with 95%

confidence intervals (CIs) adjusted by age and gender were reported. Statistical

analyses were performed using the R software.

II: The inter-rater reliability of the CS measurements was evaluated with the

intraclass correlation coefficient (ICC). The relationships between clinical signs

48

(1A and 1B of CS) and subjective symptoms (VAS) were analyzed using the

Pearson correlation coefficient.

The development of CS and VAS scores in different treatment groups during

the study was visualized graphically with line plots. Repeated measures t-test was

performed to evaluate the statistical significance of the changes in the CS and VAS

values between the different time points.

The main outcome variable was the change in CS values between the baseline

and week 3 measurements. To estimate if there were statistically significant

differences in CS changes between the treatment groups, analysis of variance

(ANOVA) was performed. Tukey's test was applied to identify pairwise differences.

Differences in the treatment responses between the interventions at week 3

were analyzed using cross-tabulation and the statistical significance of the

differences was evaluated with a chi-square test.

Missing values (5%) were imputed with the nearest neighbor method. The

primary analysis was performed according to the intention-to-treat principle.

The statistical analyses were carried out using IBM SPSS Statistics version 22.

G*Power and SamplePower 3.0 software were used for sample size and power

calculations. OriginPro software for scientific graphics was used to provide the line

plots.

4.4 Immunohistochemistry (III-V)

Immunohistochemical staining methods were used in studies III–V. The primary

antibodies used are shown in Table 3. Briefly, 5 µm sections were cut from the

FFPE tissue blocks, and the sections were deparaffinized and rehydrated. Antigen

retrieval, blocking of endogenous peroxidase and non-specific immunoglobulin

binding was done when indicated. For the negative controls, either 1) non-immune

serum was used, 2) the primary antibody was omitted, 3) the specimens were

predigested with Streptomyces hyaluronidase in the presence of protease inhibitors

(HA), or 4) the primary antibodies were treated with corresponding peptides (HAS).

Positive controls included tissues previously shown to be positive for the studied

antibodies. The bound antibodies were visualized with commercial detection

systems.

In the double immunoenzymatic staining, localization of the CTSK antibody

with various other antibodies (Table 3) was studied using a sequential staining

method. Visualization of the primary antibody reactions was done with a

peroxidase-based system combined with red and blue-gray chromogens.

49

In the double IF staining, sequential and simultaneous staining methods were

employed. Fluorescein-conjugated secondary antibodies were used to visualize the

antibody reactions and the slides were examined with a fluorescence microscope

(Zeiss Imager.D2).

Table 3. Antibodies used in the immunohistochemical staining.

Antibody Clone or code Mouse/rabbit/goat/

mono/polyclonal

Dilution Incubation time

/temperature

Manufacturer

Standard

CTSK E-7 Mouse/monoclonal 1:400 60 min/RT Santa Cruz

CD44 Hermes-3 Mouse/monoclonal 1:200 overnight/+4°C Dr. Sirpa Jalkanen

HA bHABR n/a 1:30 30 min/+4°C ref. Tammi 1994

HAS1 G-17 Goat/polyclonal 1:75 overnight/+4°C Santa Cruz

HAS2 S-15 Goat/polyclonal 1:120 overnight/+4°C Santa Cruz

HAS3 E-15 Goat/polyclonal 1:75 overnight/+4°C Santa Cruz

HYAL1 HPA002112 Rabbit/polyclonal 1:100 overnight/+4°C Atlas Antibodies

HYAL2 ab68608 Rabbit/polyclonal 1:100 overnight/+4°C Abcam

TLR4 H-80 Rabbit/polyclonal 1:50 overnight/+4°C Santa Cruz

TLR9 IMG-305A Mouse/monoclonal 1:100 30 min/RT Imgenex

Double IE

CTSK E-7 Mouse/monoclonal 1:400 30–45 min/RT Santa Cruz

CD1A 010 Mouse/monoclonal 1:100 45 min/RT Dako

PTPRC 2B11+ PD7/26 Mouse/monoclonal 1:400 45 min/RT Dako

CD68 KP1 Mouse/monoclonal 1:10 000 30 min/RT Dako

Melan-A A103 Mouse/monoclonal 1:50 45 min/RT Leica Biosystems

TLR4 3B6 Mouse/monoclonal 1:1500 60 min/RT Abnova

TLR9 26C593.2 Mouse/monoclonal 1:150 60 min/RT Novus Biologicals

Double IF

CTSK E-7 Mouse/monoclonal 1:50 overnight/+4°C Santa Cruz

CTSK N-20 Goat/polyclonal 2µg/ml overnight/+4C Santa Cruz

CD68 KP1 Mouse/monoclonal 1:1000 overnight/+4°C Dako

TPSAB1 AA1 Mouse/monoclonal 0.1µg/ml overnight/+4°C AbD Serotec

4.5 Evaluation of the immunohistochemical staining

The immunohistochemical staining was evaluated using light microscopy. In study

III the scoring of the staining was done by three of the investigators (MS, TS, JK),

and in studies IV (MS, AK, TS) and V (MS, CCB) by two of the investigators in a

consensus manner. In study V, the samples before and after tacrolimus treatment

50

were assessed individually and blinded to the medication status by two of the

investigators (MS, CCB).

III: In both OLP and control samples, the staining intensity of TLR4 and TLR9

in the basal, intermediate and superficial layers of the epithelium as well as

subepithelial inflammatory infiltrate was rated using the following categories:

absent (0), weak (1), moderate (2) or strong (3). The site of the densest subepithelial

inflammatory infiltrate in OLP was selected for the assessment. In each sample, the

cell (e.g. mast cell) that showed the strongest staining intensity in the sample was

regarded as an intrinsic reference for strong (3) staining.

IV: In OLP and control specimens, the staining intensity was assessed in the

basal, intermediate, and superficial layers of the epithelium. A three-level scale (0

= no staining, 1 = some staining, 2 = considerable staining) was used for HA, CD44,

and HAS1-2 scoring and a four-level scale (0 = no staining, 1 = weak staining, 2 =

moderate staining, 3 = strong staining) was used for HYAL1-2 scoring. HAS3

staining was almost uniformly negative, so it was not scored.

V: Epithelium as a whole was scored by evaluating the number of CTSK-

positive cells using grades 1, 2, 3, 4, and 5 corresponding to 0–5%, 6–25%, 26–

50%, 51–75%, and 76–100% of cells positive, respectively. Stroma as a whole was

scored by assessing the intensity of staining using grades 0 = no staining, 1 = mild,

2 = moderate, 3 = strong, and 4 = very strong.

The specimens before and after tacrolimus treatment were evaluated

quantitatively. Three photographs were taken of each slide (left, middle, and right

side of the section) with a 200 x magnification and the images were scored utilizing

ImageJ software (Rasband). A grid with area per point of 3.00 inchesᶺ2 was placed

on the image and the number of cells in the squares was counted using a cell counter:

in the epithelial area, the middle right square of the grid was used as a starting point,

continuing in a counterclockwise manner until at least 100 epithelial cells were

counted. The cells positive for CTSK were counted in the same squares to yield a

percentage value of positive cells in the epithelium. The stroma was scored in a

similar fashion using the square in the middle right as a starting point and

continuing down left diagonally and from there counterclockwise until at least 100

cells in the stroma were counted. The numbers of CTSK-positive cells in the same

squares were then counted, yielding a percentage count of positive cells in the

stroma. A mean count of the three measurements of each slide was used in the

analysis.

51

4.6 Statistical methods (III–V)

III: The distributions of the TLR4 and TLR9 staining intensity by layers of

epithelium were illustrated graphically. A non-parametric Mann-Whitney test was

used to evaluate the statistical significance between the control, white lichen and

atrophic lichen groups. The Wilcoxon paired samples test was used to evaluate the

statistical significances between layers of samples so that layers from the same

sample were treated as pairs.

Statistical analyses were done using IBM SPSS Statistics for Mac version 16.0

and Prism 4 for Macintosh (GraphPad, La Jolla, CA, USA).

IV–V: Frequency and percentage distributions of the staining intensity between

control and OLP groups were compared using cross-tabulation and presented also

graphically. The statistical significance of the differences between distributions was

evaluated with a chi-square test.

The agreement between the scores of the investigators among the tacrolimus-

treated cases (V) was evaluated using ICC. Repeated-measures t-test was used to

evaluate the change in the percentage of CTSK-positive cells before and after

treatment with tacrolimus.

The statistical analyses were performed using IBM SPSS Statistics version 21.

52

53

5 Results

5.1 Association of thyroid disease and OLP (I)

Fifty-three percent of the cases (both OLP and OLL) and 49% of the controls

reported having at least one general medical condition. Regular medications were

used by 52% of the cases (51% OLP/53% OLL) and by 49% of the controls.

Thyroid diseases were found to be one of the most common medical conditions

in OLP/OLL patients. They were reported by 14% (n=31) of the OLP/OLL patients

compared to 8% (n=18) of the control subjects. The odds ratio (OR) for thyroid

diseases was 1.95 (95% CI 1.04 to 3.73) when adjusted for age and sex. Looking

separately at the OLP/OLL cases, 15% (n=22) of the OLP and 13% (n=9) of the

OLL patients had thyroid disease the estimated ORs (with 95% CIs) being 2.12

(1.06 to 4.21) for OLP and 1.57 (0.62 to 3.73) for OLL.

Sixty-eight percent (n=21) of the OLP/OLL cases and 61% (n=11) of the

controls with thyroid gland diseases had hypothyroidism. The association of

hypothyroidism with OLP seemed stronger than the association with OLL, since

the estimated ORs (with 95 % CIs) were 2.39 (95% CI 1.05–5.61) for OLP and

1.73 (0.56–4.90) for OLL. However, the sizes of pertinent subgroups of cases and

controls were rather small, and the CIs quite wide, so this difference in the OR

estimates could be explained by random variation. Thyroxin medication use per se,

analyzed separately, was not associated with OLP/OLL (OR 0.94, 95% CI 0.27 to

5.20).

Diabetes was reported by 4% (n=8) of the cases (2 with OLL and 6 with OLP)

and 4% (n=8) of the controls.

A history of allergy was reported more frequently by the OLP/OLL patients

(n=91) than by the controls (n=69). The overall OR for the association of reported

allergy and OLP/OLL was 1.58 (95% CI 1.07 to 2.35).

54

5.2 Topical tacrolimus and triamcinolone acetonide in the

management of symptomatic OLP (II)

5.2.1 The clinical score reliability and validity

The inter-rater reliability of CS was found to be strong (ICC 0.959). On the other

hand, the correlation between clinical signs (1A+1B of the CS) and symptoms

(VAS) was very weak (Pearson correlation coefficient 0.180).

5.2.2 Patient flow and characteristics

The baseline clinical and demographic data of the study groups are presented in

tables in the study II manuscript (table 1, supplementary table 1 and supplementary

figure). A total of 27 patients were randomized and allocated to receive either 0.1%

tacrolimus ointment (n=11), 0.1% triamcinolone acetonide paste (n=7) or Orabase

(placebo) paste (n=9). The patient flow is illustrated in the flow chart (study II

manuscript, figure 1). Due to slow participant intake and lack of financial resources

and collaborators, the trial was ended before the target sample size was reached.

Therefore, the actual power of the study is 0.70.

5.2.3 Primary and secondary outcomes

The results of all the primary and secondary outcome analyses are presented in the

study II manuscript. All the participants were analyzed for the primary outcome

variable (change in CS from baseline to week 3). CS from baseline to week 3

decreased 37% (mean decrease 10.5, SD 8.6, p=0.002) in the tacrolimus group

(n=11) compared to 32% (mean decrease 10.3, SD 6.8, p=0.007) in the

triamcinolone group (n=7). In the triamcinolone group that responded to

intervention (n=4), and that did not respond to intervention (n=3), the CS decreased

41% (p=0.027) and 19% (p=0.026), respectively. The CS increased slightly (0.8%)

in the placebo group (p=0.927). Tacrolimus (p=0.012) and triamcinolone (p=0.031)

were more effective than placebo in reducing the CS at week 3. There was no

difference in the efficacy between tacrolimus and triamcinolone (p=0.997).

The response to intervention (≥20% decrease in CS from baseline) in the

intervention groups was assessed at week 3. Tacrolimus (p=0.005) and

triamcinolone (p=0.019) groups responded to treatment more often than the

55

placebo group, and tacrolimus and triamcinolone groups were equally effective in

producing a treatment response (study II manuscript, table 2).

Regarding the effect of interventions on symptoms, tacrolimus and

triamcinolone seemed to reduce pain more than placebo at week 3, but the

differences were not statistically significant (study II manuscript, figure 2,

supplementary tables 2 and 3).

Some secondary outcome analyses could not be completed for all the patients.

At week 3, the triamcinolone group was divided into responders and non-

responders to treatment, resulting in small sample sizes for further measures.

Therefore, although an obvious effect was seen in many of the outcome measures

in those intervention groups, a statistical significance was not observed (study II

manuscript, figure 2, supplementary tables 2 and 3).

5.2.4 Adverse reactions to the study medications

Altogether 74% of the participants experienced local side effects in association with

the use of study medications. During tacrolimus, triamcinolone acetonide or

placebo use, 73%, 43% and 33% of the patients, respectively, reported local adverse

effects. The most common local adverse effect in tacrolimus users was a burning

or smarting sensation either when administering the drug on the oral mucosa or

when eating or drinking. This occurred in 57% (n=12) of the patients. Forty-three

percent (n=9) of tacrolimus users experienced increased sensitivity to hot, cold or

spicy food, and 19% (n=4) reported altered taste sensation. Other less common side

effects in tacrolimus users were an unpleasant sensation in the mouth, xerostomia

and hoarseness (1 patient each). Triamcinolone acetonide use was associated with

a smarting feeling in the mouth (n=1) and tenderness in the gingiva (n=2). Placebo

users experienced burning and sensitivity to hot food or drink, tenderness in the

gingiva and increased salivary flow after administering the paste (1 patient each).

Before the trial started, oral candidiasis was diagnosed in 37% of the participants

(36%, 29% and 44% in the tacrolimus, triamcinolone acetonide and placebo

groups). None of the participants who used tacrolimus during the initial trial period

of 6–9 weeks were diagnosed with secondary oral candidiasis. On the other hand,

50% (n=2) of those who used triamcinolone acetonide for 6 weeks, 33% (n=1) of

those who switched from triamcinolone to tacrolimus at week 3, 25% (n=2) of those

who switched from placebo to tacrolimus at week 3 and 1 patient who used placebo

for 6 weeks developed oral candidiasis.

56

Two (18%) patients in the initial tacrolimus group reported systemic adverse

effects: nausea (n=2), headache (n=1) and nightmares (n=1).

5.3 Immunohistochemical expression of the studied molecules in

OLP and the effect of tacrolimus (III–V)

5.3.1 Toll-like receptors 4 and 9

TLR4 and TLR9 immunoreactivity was observed mainly intracellularly in both

normal and OLP mucosa. In normal oral epithelium, TLR4 was expressed only in

the basal layer (Figure 3a). The expression was weaker than in the basal layer of

OLP epithelium (p<0.001). In OLP, TLR4 staining intensity decreased gradually

from the basal to the superficial layer of the epithelium (Figure 3b), and the

differences between the basal and intermediate layers and basal and superficial

layers in both clinically white and atrophic OLP were substantial (p<0.001). The

difference in TLR4 staining intensity between the intermediate and superficial

layers of the epithelium in clinically atrophic OLP was considerable (p<0.05),

while in the clinically white OLP, the difference did not reach statistical

significance. In the subepithelial inflammatory cells, TLR4 staining intensity did

not differ markedly between OLP and normal oral mucosa. The subepithelial

inflammatory infiltrate showed weaker TLR4 expression compared with the basal

layer of the epithelium (P<0.001) in clinically atrophic and white OLP but when

compared with other layers of the epithelium, no notable differences were found.

TLR9 immunoreactivity in normal oral mucosa was only observed in the

superficial layer of the epithelium and the staining intensity was weaker than in the

superficial epithelial layer in OLP (p<0.001). In OLP, the distribution of TLR9

staining exhibited a reversed pattern compared to TLR4, as TLR9 staining intensity

increased gradually from basal to the superficial layer of the epithelium. The

difference in the staining intensity in clinically white and atrophic OLP was

substantial between basal, intermediate and superficial layers (p<0.001). The

subepithelial inflammatory infiltrate in OLP demonstrated weaker TLR9

expression compared to the superficial layer of the epithelium (p<0.001) and

stronger expression than the basal layer of the epithelium (p<0.001). No

appreciable difference in the TLR9 staining intensity was found between the

intermediate layer and the subepithelial inflammatory infiltrate (p<0.33).

57

No change in the TLR4 and TLR9 staining intensities were seen in a patient

case where samples before and after treatment with topical 0.1% tacrolimus

ointment were examined.

Fig. 3. Schematic illustration of TLR4 and TLR9 staining in healthy oral mucosa (a, c)

and OLP (b, d).

5.3.2 Hyaluronan, its receptor CD44, and its metabolizing enzymes

HAS1–3 and HYAL1–2

Hyaluronan and its main receptor CD44 were expressed homogeneously on the

plasma membrane mostly in the basal and intermediate layers of the epithelium in

both normal (control) and OLP epithelium (Figure 4a–d). However, HA staining

intensity was stronger in the basal layer in OLP compared to normal oral epithelium

(p<0.001). In the intermediate epithelial layer, more control than OLP samples

showed considerable staining, fewer control than OLP samples showed some

staining, and 17 % of the controls showed no staining compared to none of the OLP

samples (p<0.001). HA expression did not differ markedly in the superficial

58

epithelial layer in normal and OLP epithelium. In the subepithelial connective

tissue, intense HA immunoreactivity was found in both controls and OLP samples.

CD44 expression localization and intensity in the epithelium did not show

considerable differences between normal and OLP samples (Figure 4c and d).

However, in the subepithelial tissues OLP specimens demonstrated stronger CD44

staining than the control samples, probably due to increased numbers of

subepithelial inflammatory cells expressing the receptor in OLP. It is also possible

that more mesenchymal cells (e.g. fibroblasts) express CD44 in OLP than in normal

mucosa. CD44 staining in some subepithelial connective tissue fibers in control

samples presumably represents soluble CD44 shed by epithelial cells.

HAS1 and HAS2 expression of variable intensity was seen in the cytoplasm

and occasionally on the plasma membrane in both OLP and control samples (Figure

5a–d). HAS1 was not detected in the basal epithelial layer of healthy controls.

HAS3 immunoreactivity was low in both control and OLP mucosa, showing only

faint staining in some nuclei of the epithelial and inflammatory cells, and this was

regarded as non-specific.

The staining intensity of HAS1 (p=0.001) and HAS2 (p<0.001) was stronger

in the basal layer of the epithelium in OLP than in the control samples. In the

intermediate epithelial layer, there was no difference in the HAS1 or HAS2 staining

intensity between OLP and controls. In the superficial epithelial layer, both HAS1

and HAS2 showed greater staining intensity in control than in OLP samples

(p<0.001). Notably, HAS1 and HAS2 showed a strong cytoplasmic granular-like

staining pattern in the upper stratum intermedium and stratum superficiale in the

normal epithelium that was not detectable in OLP samples nor in HAS3-stained

control specimens. Inflammatory cells in the epithelium and in the connective

tissue expressed HAS1 and HAS2 in OLP. Fibroblasts in the lamina propria showed

HAS1 and HAS2 immunoreactivity in both control and OLP specimens.

HYAL1 and HYAL2 expression was localized mainly intracellularly, and

HYAL2 also occasionally faintly on the plasma membrane (Figure 5e–h). HYAL1

and HYAL2 staining intensity were stronger in all layers of the epithelium in OLP

than in control samples (p=0.011, p=0.004 and p<0.001 in the basal, intermediate

and superficial layers, respectively, for HYAL1 and p=0.001, p=0.175 and p=0.008

in the basal, intermediate and superficial layers, respectively, for HYAL2). HYAL1

and HYAL2 were expressed in the subepithelial inflammatory cells and fibroblasts

in OLP while in controls, their expression was detected mainly in stromal

fibroblasts and endothelial cells.

59

The clinically white (reticular or plaque-like) and red (atrophic, erosive) types

of OLP did not show marked differences in the immunoexpression of HA, CD44,

HAS1–2, and HYAL1–2.

In the patient cases (n=3) treated with topical 0.1% tacrolimus, the samples

taken after treatment showed that CD44 staining was decreased in the stroma and

HYAL2 staining was slightly increased in the basal layer of the epithelium

compared to the samples taken before the treatment. HA, HAS1–2 and HYAL1 did

not show a change in immunoexpression before and after tacrolimus treatment.

Fig. 4. Schematic illustration of HA and CD44 staining in healthy oral mucosa (a, c) and

OLP (b, d).

60

Fig. 5. Schematic illustration of HAS1–2 and HYAL1–2 staining in healthy oral mucosa

(a, c, e, g) and OLP (b, d, f, h).

61

5.3.3 Cathepsin K

In general, CTSK was expressed with variable intensity as small granules in the

cytoplasm of macrophages, fibroblasts, melanocytes, a few endothelial cells and

probably some basal keratinocytes in OLP (Figure 5b), as demonstrated in part by

double IHC. In addition, CTSK was noted extracellularly. In the normal oral

mucosa, the epithelium was negative for CTSK immunostaining while some

stromal fibroblasts expressed CTSK (Figure 5a).

CTSK positive cells were more numerous in OLP epithelium than in normal

(control) oral epithelium (p<0.001). In the stroma, CTSK staining intensity was

greater in OLP compared to control samples (p<0.001).

Langerhans cells, lymphocytes and mast cells did not show immunoreactivity

for CTSK, as indicated by double IHC and IF. CTSK co-localized with TLR4 and

TLR9 in some mucosal cells, presumably macrophages and melanocytes.

In the tacrolimus treated OLP patient cases (n=9), the number of CTSK positive

cells decreased in the epithelium after treatment with tacrolimus. However, the

observed changes were appreciable only as evaluated by one of the two

investigators. In the stroma, there was a tendency for the number of CTSK positive

cells to increase after treatment with tacrolimus, but the change did not show

statistical significance.

Fig. 6. Schematic illustration of CTSK staining in healthy oral mucosa (a) and OLP (b).

62

63

6 Discussion

6.1 Hypothyroidism and OLP

A few previous studies indicated that thyroid disorders may be associated with OLP.

In a small sample of HCV-negative and HCV-positive OLP patients, thyroid

dysfunction was found in 13% of the HCV-negative patients and in 15% of the

HCV-positive patients (Carrozzo et al. 1999). Another study reported increased

prevalence of serum anti-thyroid antibodies in OLP patients compared to controls

(Chang et al. 2009). Furthermore, an epidemiologic study suggested an association

of LP and hypothyroidism (Dreiher et al. 2009). In addition, thyroxin medication

was found to be more common in OLP patients compared to controls (Kragelund

et al. 2003). After our finding of the association of hypothyroidism and OLP was

published (study I), several other groups have reported also increased prevalence

of thyroid diseases in patients with OLP in Northern and Southern Europe and

Brazil (Ebrahimi et al. 2012, Garcia-Pola et al. 2016, Hirota et al. 2011, Lauritano

et al. 2016, Lo Muzio et al. 2013, Robledo-Sierra et al. 2013, Robledo-Sierra et al.

2015). Interestingly, studies from Iran, Sicily and Croatia/Australia found no

significant association of OLP and hypothyroidism (Compilato et al. 2011, Lavaee

& Majd 2016, Vucicevic Boras et al. 2014).

Autoimmune thyroid diseases (AITD) are multifactorial T- and B-cell mediated

organ-specific diseases thought to result from immune dysregulation (Lee et al.

2015, Ramos-Levi & Marazuela 2016). Two main AITDs are Graves' disease and

Hashimoto's thyroiditis, manifesting as thyrotoxicosis and hypothyroidism,

respectively (Antonelli et al. 2015). Both genetic and environmental factors trigger

AITDs (Antonelli et al. 2015). AITD are associated with other autoimmune

diseases such as rheumatoid arthritis, Sjögren’s syndrome and systemic lupus

erythematosus (Antonelli et al. 2015, Lee et al. 2015).

The mechanism behind the association of thyroid disease and OLP is unknown.

However, it has been proposed that circulating thyroid antibodies could trigger,

even in subclinical asymptomatic chronic autoimmune thyroiditis, an organ-

specific autoimmune response also in the oral mucosa (Lo Muzio et al. 2013). This

hypothesis is based on a finding that most patients presumably developed OLP after

the onset of thyroid dysfunction (Lo Muzio et al. 2013). Also in a study by Robledo-

Sierra et al. (2015) the vast majority of OLP patients reported that the diagnosis of

thyroid disease and start of thyroxin medication antedated the onset of OLP.

64

However, the prevalence of elevated serum antithyroid antibodies thyroid

peroxidase (TPO) and thyroglobulin (Tg) did not differ between OLP patients

without thyroid disease and the general population in a study done by the same

group (Robledo-Sierra 2015). Of note however, they found that OLP patients

without previously diagnosed thyroid disease had elevated levels of thyroid

stimulating hormone (TSH), and low levels of free thyroxine 4 (FT4) more often

than the general population, indicating a higher prevalence of hypothyroidism in

OLP patients (Robledo-Sierra 2015). Furthermore, the expression of thyrotropin

receptor (TSHR) was found in the basal epithelial layer in OLP lesions but not in

the healthy oral mucosa, and thyroid stimulating hormone receptor (TSHR)

expression had a tendency to be higher in OLP than in healthy oral mucosa,

suggesting their possible association with OLP disease mechanism (Robledo-Sierra

2015). Also skin keratinocytes express functional TSHR and TG (Slominski et al.

2002). In our material we could not reliably determine if thyroid dysfunction was

preceded by OLP.

The association of thyroid disorders and OLP or OLL raises the question of

whether thyroid medication could be the causative factor of the oral disease.

However, when only patients with a history of thyroid disease were analyzed,

thyroxin medication per se was not associated with OLP or OLL in our data. Of

note, lichenoid drug reactions caused by thyroid hormone supplementation are

exceedingly rarely reported. In fact, only one case report describes a lichenoid

eruption of the skin in association with thyroxin overdose (Kaur et al. 2003). Also

the authors of the study where thyroid hormone supplementation use was associated

with OLP, conclude that the thyroid disease is probably behind the association

rather than the medication (Robledo-Sierra et al. 2013).

Furthermore, the association of thyroid dysfunction and OLP could be

explained by impaired immune system function associated with genetic factors.

However, only a few of the same genes are implicated in both diseases. Genes

linked to susceptibility to AITD include 1) MHC class II, especially human

leukocyte antigen (HLA)-DRB3, 2) immune-regulatory genes CD40 molecule

(CD40), cytotoxic T-lymphocyte associated protein 4 (CTLA4), protein tyrosine

phosphatase, non-receptor type 22 (PTPN22), forkhead box P3 (FOXP3) and

interleukin 2 receptor subunit alpha (IL2RA) and 3) thyroid-specific genes TG and

TSHR (Hasham & Tomer 2012). In OLP, increased prevalence of several MHC

genes has been reported, namely HLA-Bw57 (Porter et al. 1993), HLA-DRw9

(Watanabe et al. 1986), HLA-DRB3 (Jontell et al. 1987) and HLA-DRB9 and HLA-

65

Te22 (Lin & Sun 1990). In addition, FOXP3 gene expression levels in OLP patients

are higher than in controls (Lei et al. 2014).

6.2 Topical tacrolimus and triamcinolone acetonide in the

management of OLP

Topical corticosteroids have long been the drug of choice for treating patients with

symptomatic OLP (Bagan et al. 2012). Even though placebo-controlled RCTs,

albeit with their limitations, are considered the gold standard in intervention trials

(Bothwell et al. 2016), we have not identified any previous studies comparing

topical triamcinolone acetonide or tacrolimus with placebo in OLP. A few placebo-

controlled trials of topical corticosteroids in the management of OLP do exist, but

many of them suffer from methodological issues related to outcome measures and

reporting (Cilurzo et al. 2010, Kazancioglu & Erisen 2015, Tyldesley & Harding

1977, Voute et al. 1993) and blinding (Carbone et al. 1999) (Listed in table 4, in

comparison with the present study II). The latest Cochrane review of interventions

for treating OLP states that no RCTs that compare steroids with placebo in patients

with symptomatic OLP were found (Thongprasom et al. 2011). Also relatively few

RCTs that compare topical tacrolimus with corticosteroids in OLP are available,

some showing no difference in the efficacy, some showing that tacrolimus is more

effective, and one reporting that topical corticosteroids are more effective (Azizi &

Lawaf 2007, Corrocher et al. 2008, Laeijendecker et al. 2006, Radfar et al. 2008,

Revanappa et al. 2012, Sonthalia & Singal 2012, Sivaraman et al. 2016) (Listed in

table 5, in comparison with the present study II). Some of these studies also have

the above-mentioned methodological issues. In general, evaluation and comparison

of the results of the published RCTs in OLP is challenging due to heterogeneous

reporting (Lopez-Jornet & Camacho-Alonso 2010a).

66

Ta

ble

4. P

lac

eb

o-c

on

tro

lle

d R

CTs

of

top

ica

l c

ort

ico

ste

roid

s i

n t

he

ma

nag

em

en

t o

f O

LP

(o

nly

th

os

e p

ub

lish

ed

in

En

gli

sh

are

inc

lud

ed

).

Au

tho

r (y

ea

r)

n

Thera

py

and it

s du

ratio

n

Main

fin

din

gs

Tyl

de

sle

y &

Ha

rdin

g (

19

77

) 2

3/2

0*

Act

ive

: beta

meth

aso

ne v

ale

rate

aero

sol (

n=

11)

2 p

uffs

4 x

daily

(to

tal d

aily

dose

800µ

g)

8 w

eeks

, m

ost

patie

nts

did

not ta

ke the full

do

se

Pla

cebo

: aero

sol c

onta

inin

g A

rcto

n -

11 a

nd -

12 (

n=

9)

2 p

uffs

4 x

daily

8 w

eeks

Resp

onse

to t

reatm

ent

better

in

the b

eta

meth

aso

ne v

ale

rate

gro

up

Vo

ute

et al.

(19

93

)

40

Act

ive

: 0.0

25%

flu

oci

nonid

e in

an a

dhesi

ve b

ase

(n=

20

) at le

ast

6 x

da

ily 6

weeks

Pla

cebo

: adhesi

ve o

intm

ent

base

with

40%

hyp

rom

ellu

lose

in p

ara

ffin

(n

=20)

at

least

6 x

da

ily 6

weeks

Flu

oci

nonid

e m

ore

effect

ive tha

n

pla

cebo in

re

duci

ng s

igns

and

sym

pto

ms

Carb

one e

t al.

(19

99

) 6

0/5

0*

Act

ive

: 0.0

5%

clo

beta

sol p

ropio

nate

oin

tment (n

=2

0)

2 x

daily

4 m

onth

s +

1 x

daily

2 m

onth

s or

0.0

5%

flu

oci

nonid

e o

intm

ent (n

=20)

3 x

daily

2 m

onth

s +

2 x

daily

2

month

s +

1 x

daily

2 m

onth

s, b

oth

tx

in 4

% h

ydro

xyeth

yl c

ellu

lose

gel +

mic

onazo

le

gel +

CH

X r

inse

Pla

ceb

o: 4%

hyd

roxy

eth

yl c

ellu

lose

gel (

n=

10)

2 x

daily

6 m

onth

s

Clo

beta

sol m

ore

effect

ive than

fluoci

non

ide o

r pla

cebo in

reduci

ng s

igns

an

d s

ympto

ms.

No

diff

ere

nce

betw

ee

n flu

oci

non

ide

and p

lace

bo.

Cilu

rzo e

t a

l. (2

01

0)

48

Act

ive: 24µ

g c

lobe

taso

l-17 p

ropio

nate

in m

uco

adh

esi

ve table

t (n

=16)

or

clobeta

sol

oin

tment m

ixe

d w

ith O

rabase

123 µ

g/a

pplic

atio

n m

ixed w

ith O

rabase

TM (

n=

16)

3 x

daily

4 w

eeks

Pla

cebo

: poly

sodiu

m m

eth

acr

ylate

, m

eth

ylm

eth

acr

ylate

+

hyd

roxy

pro

pyl

meth

ylce

llulo

se (

HP

MC

)+/M

gC

l 2)

mu

coa

dhesi

ve tab

lets

(n

=16)

3 x

daily

4 w

eeks

Clo

beta

sol m

ore

effect

ive than

pla

cebo in

re

duci

ng s

igns

and

sym

pto

ms

67

Au

tho

r (y

ea

r)

n

Thera

py

and it

s du

ratio

n

Main

fin

din

gs

Ka

zan

cio

glu

& E

rise

n (

20

15

) 1

20

A

ctiv

e: 0.0

5m

g/m

l dexa

meth

aso

ne m

outh

wash

+ n

ysta

tin s

olu

tion

4 x

daily

1 m

onth

(n=

30)

or

low

leve

l lase

r th

era

py

(LLL

T)

(n=

30)

or

ozo

ne

(n=

30),

both

2 x

we

ekl

y, m

ax

10 s

ess

ions

Pla

cebo

: S

olu

tion w

ith b

ase

oin

tment

(n=

30)

rinse

4 x

daily

1 m

onth

Dexa

meth

aso

ne a

nd o

zone m

ore

effect

ive tha

n L

LL

T a

nd p

lace

bo in

reduci

ng s

igns.

De

xam

eth

aso

ne,

ozo

ne a

nd L

LLT

more

effect

ive

than p

lace

bo in

re

duci

ng

sym

pto

ms

Stu

dy

II m

an

usc

rip

t (2

01

6)

27

A

ctiv

e:

0.1

% t

riam

cinolo

ne a

ceto

nid

e p

ast

e (

n=

7)

or

0.1

% t

acr

olim

us

oin

tment

(n=

11)

3 x

daily

6-9

weeks

+ 6

-month

follo

w-u

p w

ith s

pora

dic

use

for

tacr

olim

us

and

inte

rmitt

ent use

for

topic

al c

ort

icost

ero

id if

neede

d

Pla

cebo

: O

rabase

past

e (

n=

9)

3 x

daily

3-6

weeks

Triam

cinolo

ne a

nd

tacr

olim

us

equally

an

d m

ore

effect

ive tha

n

pla

cebo in

re

duci

ng s

igns

and

sym

pto

ms

*com

ple

ted the s

tudy

68

Ta

ble

5. R

CTs

of

top

ical

tac

rolim

us

(TA

C)

an

d c

ort

ico

ste

roid

s i

n t

he

ma

na

gem

en

t o

f O

LP

(o

nly

th

ose

pu

bli

sh

ed

in

En

gli

sh

are

inc

lud

ed

).

Auth

or

(year)

n

Thera

py

and it

s du

ratio

n

Main

fin

din

gs

Laeije

nde

cker

et

al.

(20

06

) 4

0

TA

C: 0.1

% o

intm

ent (n

=20)

4 x

da

ily 6

weeks

Contr

ol:

0.1

% tria

mci

nolo

ne a

ceto

nid

e in

hyp

rom

ello

se 2

0%

oin

tment

(n=

20)

4 x

daily

6 w

eeks

TA

C m

ore

effect

ive than triam

cino

lone in

reduci

ng s

igns

Azi

zi &

La

wa

f (2

00

7)

60

T

AC

: 0.1

% o

intm

ent (n

=30)

4 x

da

ily 4

weeks

Contr

ol:

0.1

% tria

mci

nolo

ne in

ora

base

(n=

30)

4 x

daily

4 w

eeks

Both

as

effect

ive in

reduci

ng s

igns

and

sym

pto

ms

Radfa

r et al.

(20

08

) 3

0

TA

C: 0.1

% o

intm

ent (n

=15)

4 x

da

ily 2

weeks

+ 3

x d

aily

2 w

eeks

+ 2

x

daily

1 w

eek

+ 1

x d

aily

1 w

eek

+ n

ysta

tin r

inse

1 x

daily

Contr

ol:

0. 05%

clo

beta

sol o

intm

ent

(n=

15)

4 x

daily

2 w

eeks

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69

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ms

70

One of the most important aspects of intervention trials is the assessment of

outcome. Numerous different OLP scoring systems have been created, but none

have gained universal acceptance (Chainani-Wu et al. 2008, Escudier et al. 2007,

Lopez-Jornet & Camacho-Alonso 2010a). Especially for future research purposes,

a validated, commonly approved outcome measure for OLP would be useful (Wang

& van der Waal 2015). Recently, it has been proposed that quality of life-measures

could be applied to assess the outcome of intervention in OLP (Wang & van der

Waal 2015). Furthermore, the amount of change in the outcome measures regarded

as indicative of therapeutic effect seems to vary between the publications. For

example, Laeijendecker et al. (2006) graded ≥30% improvement in the extent and

the severity of the lesions as improved and Sonthalia & Singal (2012) defined >50%

improvement in their net clinical score (CS) as partial improvement. In our novel

CS (study II), we set a ≥20% decrease in the score value as an indicator of

therapeutic response. This cut-off value, similar to previously published ones, was

not based on any further analyses or calculations.

In our CS the inter-rater reliability was strong, but correlation of clinical signs

and symptoms reported by the patients was weak. Similarly, a previous study

showed that the correlation of symptoms and clinical signs scores in OLP varied

from minimal to high (Chainani-Wu et al. 2008). Visual analogue scale (VAS), one

part of our CS has been shown to be a valid measure for symptom assessment

(Flaherty 1996, Miller & Ferris 1993). Furthermore, the expected decrease in CS

values in the active comparator groups compared to placebo comparator group

supports the validity of our CS. For this reason, we believe that the present CS is a

valid instrument in evaluating treatment response in OLP and could well be used

in the future intervention trials.

One of the strengths of the clinical trial (II) is its design, but unfortunately it is

underpowered, and is therefore considered a pilot study. However, the results

indicated, for the first time, that topical 0.1% triamcinolone acetonide or 0.1%

tacrolimus are more effective than placebo in reducing the signs and symptoms of

OLP after three weeks of use. Both drugs showed equal efficacy. Interestingly,

during the first week of intervention, placebo or triamcinolone acetonide produced

a greater reduction in pain/discomfort than tacrolimus, probably reflecting 1)

placebo effect and 2) tacrolimus’ known ability to cause burning at the initial phase

of use. Although both active drugs demonstrated a similar decrease in CS and VAS

from baseline to 6 months, the change was statistically significant only in the

tacrolimus group, reflecting the small sample size in the topical corticosteroid

71

group during the 6-month follow-up. However, the VAS values assessed at home

during the 6-month follow-up period showed that tacrolimus had a tendency to

provide a better long-term pain reduction than topical corticosteroid.

In conclusion, the findings of the study II corroborate the results of a few

previous clinical trials (Azizi & Lawaf 2007, Radfar et al. 2008, Sonthalia & Singal

2012). Topical tacrolimus may be considered an effective alternative for

management of symptomatic OLP, especially in cases refractory to topical

corticosteroids. Although the adverse effects of topical tacrolimus use in OLP are

typically local and benign, the long-term safety of the drug remains to be

established.

6.3 The role of toll-like receptors 4 and 9 in OLP

Pathogenetic mechanisms of many chronic inflammatory and autoimmune diseases

are believed to associate with inappropriate response of the TLR, due to particular

genetic factors, or from inadequate quantity or quality of ligands (Gianchecchi &

Fierabracci 2015, Ospelt & Gay 2010).

Literature on the participation of TLRs 4 and 9 in the pathogenesis of LP or

OLP is relatively scarce. Earlier, elevated expression of toll-like receptor 4 protein

and transcripts was reported in OLP epithelium as well as in UWS keratinocytes

compared to controls (Janardhanam et al. 2012). Therefore, our finding of

increased immunohistochemical expression of TLR4 in OLP (study III) confirms

the results of Janardhanam et al. (2012). In their report, normal epithelium

expressed TLR4 mainly in the lower spinous epithelial layer, while in our study it

was located only in the basal epithelial layer. Recently, equal TLR4 cytoplasmic

immunostaining was observed in the lower aspect of epithelium in OLP and non-

specific inflammatory control tissue, but stromal inflammatory infiltrate contained

more TLR4 staining cells in OLP than in the non-specifically inflamed controls

(Sinon et al. 2016). We observed that while OLP samples had more inflammatory

cells in the lamina propria than healthy controls, the TLR4 staining intensity in the

inflammatory cells was only slightly greater in OLP sections.

The present study shows, for the first time, increased immunohistochemical

expression of TLR9 in OLP compared with healthy oral mucosa. This finding is in

accordance with a previous report of elevated toll-like receptor 9 protein and

mRNA expression in cutaneous LP compared with controls (Li et al. 2007).

In contrast to our finding that TLR9 staining was seen only in the superficial

epithelial layer in healthy controls, an earlier report described that TLR 9

72

immunoreactive cells were more numerous in the basal epithelial layer in healthy

gingival tissue, and gradually decreased toward the superficial layer (Beklen et al.

2008). That study and our results are similar in the weaker expression of TLR4 and

TLR9 in healthy oral mucosal connective tissue than in inflamed tissue. Weak

immunodetection of TLRs 4 and 9 in the healthy oral mucosa observed in the

present study may be due to a protective mechanism to prevent constant activation

of the immune system in an environment replete with micro-organisms, as

suggested in the case of skin (Ospelt & Gay 2010).

The mechanism behind TLR 4 and TLR9 induction in OLP could be associated

with bacteria or viruses. Lipopolysaccharide in the outer wall of Gram-negative

bacteria is a known ligand for TLR4 (Molteni et al. 2016). Previously, activation

of TLR2 and TLR4 by periodontal Gram-negative bacteria was reported (Kikkert

et al. 2007). Interestingly, increased numbers of Gram-negative bacteria have been

detected (samples collected by non-invasive methods) in gingival LP lesions

compared with non-LP gingival sites in the same person (Bornstein et al. 2008). In

addition, bacteria have been reported to be present in OLP lesions in the epithelium

and lamina propria, suggesting that in OLP the epithelial barrier is breached

enabling the invasion of bacteria into tissue (Choi et al. 2016). In the same study

also the bacteria found to be increased in OLP mucosa were periodontitis-

associated, often gram-negative bacteria (Choi et al. 2016). Hence, the observed

increased expression of TLR4 in OLP could be due to the increase in Gram-

negative bacteria in OLP lesion sites.

Bacterial or viral DNA-containing unmethylated (deoxycytidyl-phosphate-

deoxyguanosine) CpG motifs are recognized by TLR9 (Ospelt & Gay 2010). Also

TLR4 is able to bind viral structural proteins (Kawai & Akira 2010, Molteni et al.

2016). At the moment, the association of viral or bacterial DNA in the oral mucosa

and TLR activation in OLP is unexplored. However, HCV-RNA strands have been

detected in oral mucosa in patients with chronic HCV-infection both in OLP and

non-OLP cases (Arrieta et al. 2000, Kurokawa et al. 2003). As mentioned above,

bacteria were demonstrated in epithelial and T-cells in OLP (Choi et al. 2016).

Taken together, the role of micro-organisms in the initiation of OLP is still

hypothetical and their detection in OLP lesions does not prove a causal relationship,

but could be the result of altered mucosal surface or increased permeability of the

mucosa in OLP (Choi et al. 2016).

Endogenous ligands that can activate TLR-mediated signaling include

molecules that are derived either from the extracellular matrix or from cells, either

73

actively secreted or passively released (Molteni et al. 2016). Some of these

molecules or proteins have been studied in OLP, namely HSPs, DEFB4A and HA.

Increased expression of the antimicrobial peptide DEFB4A has been reported in

OLP epithelium (Abiko et al. 2002). HSPs are released by cells after cellular

damage or under stressful conditions (Calderwood 2007). Many reports have

shown altered HSPs immunoexpression in OLP (Bramanti et al. 1995, Chaiyarit et

al. 1999, Chaiyarit et al. 2009, Garcia-Garcia et al. 2013, Sugerman et al. 1995),

although contrasting results have also been published (Seoane et al. 2004). Of note,

the role of HSP70 as an endogenous TLR4 ligand has been questioned, since many

studies describing this ligand receptor interaction used a recombinant protein which

may have been contaminated by endotoxin (LPS) (Ospelt & Gay 2010). HA is a

large extracellular matrix molecule that is degraded into smaller fragments during

tissue injury or inflammation, and these fragments may bind to TLR4 (Lee-Sayer

et al. 2015, Termeer et al. 2002). As we observed increased expression of HA in

OLP (study IV), it cannot be excluded that HA acts as a TLR ligand in OLP.

Genetic variation in TLRs is associated with susceptibility to infectious and

autoimmune diseases (Netea et al. 2012). In OLP, TLR3 gene polymorphism

associated with an increased risk of the disease (Stanimirovic et al. 2013). In

addition, downregulation of TLR-mediated signaling pathways in OLP was

recently reported (Sinon et al. 2016). In cutaneous LP, impaired TLR signaling

pathways are also suggested to play a role; agonists of TLR7/TLR8 or TLR9 were

shown to overcome impaired IFN-α secretion in LP (Domingues et al. 2015). In the

future, in TLR gene variations in OLP may provide more interesting findings.

6.4 The role of hyaluronan, its receptor CD44 and its metabolizing

enzymes HAS1–3 and HYAL1–2 in OLP

Previously, the expression of HA and its metabolizing enzymes have not been

described in LP or OLP. The study IV demonstrates the distribution of HA, its

synthases (HAS1, 2 and 3), and degrading enzymes (HYAL1 and 2) in OLP by

immunohistochemistry. HA was expressed homogeneously on the plasma

membrane mostly in the lower part of epithelium in OLP and the staining intensity

of HA was strongest in the basal layer. In the healthy control epithelium, HA

immunoreactivity was detected similarly mostly in the basal and intermediate

epithelial layers confirming the findings of previous studies (Kosunen et al. 2004,

Tammi et al. 1990). In contrast to OLP however, the staining intensity was most

74

intense in the intermediate epithelial layer in the control samples. Subepithelial

connective tissue exhibited strong HA staining in both OLP and healthy controls.

The more intense staining intensity of HA in the basal epithelial layer compared

to healthy epithelium may indicate increased synthesis of HA in that location in

OLP, as we also observed increased staining of HAS1 and HAS2 in OLP in the

same location. However, although the upper intermediate and superficial epithelial

layers exhibited stronger immunoreactivity for HAS1 and HAS2 in control samples

compared to OLP, a corresponding increase in HA immunoreactivity was not seen

in those areas. This discrepancy between HAS and HA expression could possibly

be explained by HAS not being enzymatically active (Rilla et al. 2005).

Furthermore, HAS1 and HAS2 staining was observed in a granular or vesicular

pattern intracellularly in the upper intermediate and superficial layers in healthy

epithelium, probably located in intracellular glycogen vesicles. Interestingly, in

OLP epithelium these vesicles were not seen.

We observed elevated HYAL immunoreactivity in OLP epithelium compared

to healthy epithelium; only for HYAL2 in the intermediate layer of the epithelium,

the increase in immunohistochemical expression did not reach statistical

significance. Increased HYAL expression could be expected to lead to decreased

HA expression, but this was not seen in the present study. However, as both

accelerated HA synthesis and degradation with increased HA accumulation is often

noted in pathological conditions, such as in the tumor microenvironment (Schmaus

et al. 2014), these could similarly occur in OLP.

Although HA expression was increased basally in OLP epithelium, expression

of CD44, the main receptor for HA, was not altered in OLP epithelium compared

to control tissue, which confirms the results of a previous study (Neppelberg et al.

2007). Contrary to these findings, decreased expression of the isoform CD44v6

(Chaiyarit et al. 2008), and increased expression of CD44 in OLP lesional

epithelium compared to healthy controls has been reported (Santarelli et al. 2015).

However, in the study IV, stronger CD44 immunoreactivity was noted in the stroma

in OLP compared to controls, probably due to an increased number of inflammatory

cells and maybe mesenchymal cells expressing CD44 in OLP. The binding avidity

of CD44 for HA may increase during inflammation as a consequence of

simultaneous elevation of HA synthesis and release of inflammatory mediators

(Heldin et al. 2008). For example, activated T-cells are able to bind HA when

stimulated by cytokines IL2 and TNF and chemokines such as IL8 and CCL5, and

monocytes may be activated to bind HA by TNF, IFNG and IL1A (Lee-Sayer et al.

75

2015, Petrey & de la Motte 2014). Enhanced HA interaction by immune cells

during inflammation may assist in recruiting more immune cells to the site of

inflammation, keep the cells at the site as well as increase their survival and

function (Lee-Sayer et al. 2015).

In tissue homeostasis and resolution of inflammation, HA exists as high

molecular weight (HMW)-HA and has protective and anti-inflammatory effects. At

sites of inflammation and injury, HA is fragmented to smaller sizes leading to

increased amounts of HA and different biological functions compared to HMW-

HA (Monslow et al. 2015, Petrey & de la Motte 2014). In general, low molecular

weight (LMW)-HA enhances the inflammatory response by promoting

angiogenesis, inflammation, maturation of APCs and cell migration (Bollyky et al.

2009, Termeer et al. 2000), mediated via, for example, MMPs and pro-

inflammatory cytokines such as TNF and IL12. In addition to purely CD44-

mediated effects of HA fragments, they may mediate pro-inflammatory or anti-

inflammatory signals via TLR2 and/or TLR4 (Lee-Sayer et al. 2015, Monslow et

al. 2015). Furthermore, HMW-HA is capable of promoting immunosuppression via

binding to TLR4, leading to enhanced release of the immunosuppressive cytokine

IL10 (Asari et al. 2010). In fact, it was demonstrated in a mouse model that oral

administration of HA900 modulated Th-1-type autoimmune disease and

inflammation via TLR4 in intestinal epithelial cells (Asari et al. 2010). As we and

others have reported increased expression of TLR4 in OLP epithelium, HA–TLR

interaction may have a role in OLP pathogenesis (Janardhanam et al. 2012).

However, in the present study, we did not assess the molecular size of HA nor the

possible co-localization of HA and TLR in OLP; these investigations would provide

interesting research topics for the future.

Interestingly, the management of OLP with topical hyaluronic acid has shown

efficacy in reducing pain and lesion size in two RCTs (Nolan et al. 2009, Shetty et

al. 2016). In light of the known HA–TLR4 interaction, it is interesting to speculate

that topical HA could have produced immunosuppressive effects and clinical

improvement in OLP. The molecular weight of HA defines its function, and the

different size fragments of the HA molecule present in the tissue can exert alternate,

often contrasting effects (Monslow et al. 2015). Even small changes in the

proportion of the different size HA fragments may shift the outcome of cellular

functions. It is therefore possible that therapies may change the ratio of the active

HA fragments and change the balance of signaling in favor of restoring homeostasis

(Monslow et al. 2015).

76

Overall, the results from the present study indicate that HA metabolism is

altered in OLP, as signs of both increased and decreased HA synthesis, and

increased HA degradation were seen in the epithelium.

6.5 The role of cathepsin K in OLP

Study V showed that CTSK immunoexpression was significantly increased in OLP

compared to healthy oral mucosa. CTSK staining was most intense around the

epithelial-stromal interface where some of the characteristic pathologic changes in

OLP occur. The cells mainly expressing CTSK were macrophages and epithelial

DCs, some of which were identified as melanocytes. Although melanocytic tumors

express CTSK, its expression in melanocytes in inflammatory mucocutaneous

disease has not been reported before (Rao et al. 2014). We suspected that the

CTSK-positive, morphologically dendritic cells in the epithelium would be LCs,

although we could not confirm this by double immunostaining. Previously however,

bone marrow-derived DCs were shown to express CTSK (Asagiri et al. 2008).

In line with our findings, activated cultured monocyte-derived but not normal

resident macrophages, were shown to express CTSK (Punturieri et al. 2000). We

observed that only a few basal keratinocytes and endothelial cells stained for CTSK

in OLP. In periodontitis however, gingival keratinocytes and endothelial cells were

found to express CTSK (Beklen et al. 2015). The same study also showed that more

mucosal macrophage-like cells and fibroblast-like cells stained for CTSK in

periodontitis compared to normal oral mucosa, and that TNF enhanced the

secretion of active CTSK from fibroblasts in culture. Moreover, CTSK was

expressed in macrophages and DCs and implicated in inflammation and bone

erosion in a mouse model for periodontitis (Hao et al. 2015b). We could not detect

CTSK in lymphocytes, and this finding is in line with a study on experimental

osteoarthritis where splenic T-cells did not show CTSK expression (Asagiri et al.

2008).

CTSK involvement in the innate immune processes may be associated with its

ability to proteolytically process TLRs, so that their structure becomes suitable for

antigen recognition (Ewald et al. 2011). CTSK may also participate in the cleavage

of proteins that interfere with the association of pathogen-derived unmethylated

DNA with TLR9 (Asagiri et al. 2008, Takayanagi 2010). In addition, TLR4

activation may be required for CTSK expression (Abdollahi-Roodsaz et al. 2009,

Hirabara et al. 2013). These findings are interesting in relation to earlier reports of

77

toll-like receptors 4 and 9 induction in OLP (Ge et al. 2012, Janardhanam et al.

2012). Our observation that CTSK was co-located with TLR4 and TLR9 in some

oral mucosal cells, likely in macrophages and melanocytes, suggests that these

molecules could be interacting in OLP.

Although primarily implicated for lysosomal proteolysis, cysteine cathepsins

are known to participate also in the extracellular degradation of proteins (Fonovic

& Turk 2014). During physiological as well as pathological conditions, cysteine

cathepsins can be secreted into the extracellular space, where their activity and

stability are modified by glycosaminoglycans (Fonovic & Turk 2014). We noted

that CTSK was located also extracellularly in OLP. This could be due to, for

example, macrophages or fibroblasts releasing the enzyme into the extracellular

milieu. Since our results suggest that HA metabolism is altered in OLP, it is an

intriguing possibility that HA and CTSK could interact in and contribute to the

pathogenesis of OLP.

CTSK is a powerful elastinolytic protease. Although CTSK is not required for

extracellular elastin degradation (Punturieri et al. 2000), it may have a role in ECM

degradation. Expression of ECM proteins, including collagen type 1, are altered in

OLP (Becker & Schuppan 1995). In addition, elastin in the connective tissue is

required for the non-keratinized phenotype of normal oral epithelium, and aberrant

keratinization of the oral mucosa in reticular OLP may be related to elastolysis by

elastases (Hsieh et al. 2010, Liao et al. 2012).

Taken together, our results suggest that macrophages, melanocytes and to some

extent keratinocytes and DCs could be activated in OLP possibly partly due to the

involvement of CTSK in OLP. Based on our findings, CTSK may be implicated in

OLP pathogenesis, although we have no proof that CTSK is active and functioning

in OLP. The role of CTSK in OLP should be more thoroughly investigated in the

future.

6.6 The molecular level effect of tacrolimus on the studied

molecules

Topical tacrolimus seemed to decrease the expression of CD44 in the stroma and CTSK

expression in the epithelium, and increase slightly the expression of HYAL2 in the basal

cell layer and CTSK expression in the stroma. However, these observations must be

interpreted with caution because of the small sample size, and ideally should be

repeated with a larger sample size. The decrease in CD44 expression after tacrolimus

treatment is probably associated with a reduced number of lymphocytes and

78

macrophages since T lymphocytes, after activation by antigen, and monocytes after

stimulation by inflammatory agents, are induced to bind CD44 to HA (Johnson &

Ruffell 2009). In addition, experiments using monoclonal antibodies against CD44

in mouse models of chronic inflammatory disease have demonstrated reduced

disease severity that is believed to result from decreased leukocyte recruitment

(Johnson & Ruffell 2009). In addition, the reduction of soluble CD44 in connective

tissue fibers may have accounted for the decrease of CD44 expression, although a

previous study did not find reduced CD44 expression in cultured rat cardiac rejection

or normal fibroblasts after tacrolimus treatment (Johnsson et al. 2004). The observed

slight increase of HYAL2 expression in the basal epithelial layer may suggest that

during tacrolimus treatment and subsequent resolution of inflammation in OLP, the

degradation of HA is increased. The tendency of CTSK expression to diminish in the

epithelium and increase in the stroma after treatment could be explained by the fact that

tacrolimus reduces the amount of epithelium infiltrating macrophages that express

CTSK, and the number of stromal lymphocytes (negative for CTSK), leading to a

relative increase in CTSK-positive cells (fibroblasts, endothelial cells, and

macrophages) in the stroma.

6.7 Methodological considerations

6.7.1 Epidemiologic study

Because of the retrospective nature of the case-control epidemiologic study (I),

some studied characteristics could not be controlled or reliably measured. In

particular, the medical histories in the patient files where the data were collected

could have been incomplete due to inaccurate reporting by the patients, doctors or

students. However, normally the medical histories are recorded very carefully in

the hospital and university clinics where the data were gathered. In addition, it is

reasonable to assume that there was no difference in the accuracy of the medical

histories between cases and controls.

Diagnosing patients as having OLP or OLL based on the information in the

patient files includes a possibility for error. The reporting of clinical features of oral

disease is likely to be inaccurate in some cases. The histopathologic diagnosis is

subjective and inter- and intra-rater variability is noted (van der Meij et al. 1999).

However, both clinical and histopathological diagnoses were combined, and the

79

final diagnosis was rendered by using the suggested modified WHO criteria (van

der Meij & van der Waal 2003).

6.7.2 Clinical trial

In RCTs there are a multitude of patient-, investigator-, intervention- and trial

design-related factors that can never be fully controlled. Regarding the treatment

groups, the patients in study II were randomly allocated to any one of groups.

However, because of the small sample size, the baseline characteristics of the

groups were somewhat dissimilar. In this kind of clinical trial, it would be difficult

to have totally comparable baseline characteristics for the study groups.

Although a placebo comparator is considered necessary in medication

intervention trials, we know that a pure placebo effect is difficult to measure

(Finniss et al. 2010). The placebo response includes also the natural healing that

may occur. It is also known that if the participant is aware that there is an active

comparator, and if he/she believes he/she is receiving it, that probably affects the

results. Therefore, it would be more ideal to compare placebo medication to no

medication at all (Hrobjartsson & Gotzsche 2010). In a disease like OLP, the

symptoms are known to fluctuate naturally. However, in this study we did not have

a no treatment group. When comparing the active and placebo treatments, a recent

systematic review and meta-analysis observed that active and placebo treatments

often have similar effect sizes (Howick et al. 2013). On the other hand, a systematic

review of randomized trials with a placebo group versus no intervention group,

found that placebo interventions had no clinically important effect in general

(Hrobjartsson & Gotzsche 2010). However, in some trials the placebo may have

produced some effect for patient-reported outcomes, especially for pain and nausea

(Hrobjartsson & Gotzsche 2010). Indeed, in this study (II) we detected an initial

effect in the patient-assessed VAS scores for discomfort/pain in the placebo group,

but the effect diminished at week 3, whereas the placebo had no effect on the CS.

Giving placebo medicine to a symptomatic patient when a therapy that is likely

to relieve his/her symptoms is available, may be considered unethical. In the

present trial, we partly resolved this problem by switching the medication to

tacrolimus at week 3 if no treatment response was apparent. In addition, the patients

were informed that they could withdraw from the trial at any point.

Blinding in clinical trials is important to increase objectivity of assessment of

outcomes. In the present trial, for reasons mentioned above, we could hold the

design strictly double-blind up to week 3, when those patients who did not respond

80

to intervention were switched to tacrolimus. The patients were told they were

switched to active medicine, but the investigator was aware that it was tacrolimus.

The patients that responded to intervention at week 3 continued to use the same

medication for another 3 weeks and thus, for those patients the trial design was

double-blind up to 6 weeks. This difference in the blinding among the treatment

groups could have affected the results.

The medicines used in the trial were prepared by the hospital pharmacists, and

even though they were blinded so that the containers were identical and the labels

had no indication of the intervention, they could not be made exactly identical in

consistency or color. This could have affected the patients’ perceptions of what

intervention they were receiving.

We found that patient co-operation in general was good, and we lost contact

with only one patient at 6 months. However, the assessment of discomfort/pain by

patients at home was quite varied, and shows that detailed patient instruction in the

beginning and revision at visits, as well as checking the assessments is extremely

important during a clinical trial.

Unfortunately, the sample size of the present trial was below optimal, which

lowered the power of the study. The problem with accruing the pre-specified

number of patients was mainly due to strict inclusion criteria, in which only patients

with relatively severe disease were considered for inclusion. In addition, we had

difficulties to engage collaborators from Finland. Of note, for a period, funding for

the study was cut off, which also affected the final number of patients included.

The aforementioned reasons underline the challenges of investigator-initiated

clinical trials of a relatively rare disease, in a small country and in the present

research environment. Future RCTs of OLP definitely need multicenter

collaboration and funding (Pihlstrom BL & Curran AE 2008).

The primary outcome measure (CS) used was similar to many that were

previously employed (Wang & van der Waal 2015). To date, none of the scoring

systems of OLP is widely accepted for use. Therefore, for future research purposes

and comparison of results of clinical trials, it would be beneficial to have a uniform

outcome measure for OLP.

6.7.3 Immunohistochemistry

IHC is a highly valuable tool for direct visualization of biomarker expression in

different tissue levels. At best, it confirms the presence and localization of an

81

antigen in the tissue and gives an indication of the amount of antigen. However,

IHC cannot determine if the observed biomarkers are active and functional in the

tissue. Moreover, the validity of IHC depends to a great extent on the quality of the

immunostaining. Major factors affecting this quality are antibody quality, tissue

fixation and processing, antigen retrieval (unmasking of epitope) and sensitivity of

the detection method (Werner et al. 2000). Dilution of the antibody also affects the

results of IHC, and may greatly influence quantification (Walker 2006). The tissue

used in the IHC studies was archival, formalin-fixed, paraffin-embedded, and the

oldest blocks were approximately 20 years old. The antigenicity of the tissue may

be altered during the long storage, thus possibly affecting the results. However, the

risk of degradation of epitopes is mainly associated with stored, precut unstained

slides (Bertheau et al. 1998, Grillo et al. 2015). In this study we used freshly cut

slides from the blocks, so the possibility of false negative or positive results due to

decreased immunoreactivity of the tissue is estimated to be low. However, we do

not have records of the exact time interval between cutting of the paraffin slides

and their staining. Also pre-staining conditions might affect the results of IHC. The

formalin fixation of tissue by itself is able to cause epitope masking or denaturation

(Werner et al. 2000). Too short or too long a fixation time, old tissue processing

solutions, and excessive paraffin temperature during embedding may affect antigen

expressivity (Werner et al. 2000). Although we don’t have exact information about

these factors for the specimens used, the biopsies were taken in the hospital or

university clinic where the fixation and processing of tissue is done by a fixed

routine. The storing conditions (e.g. temperature, humidity) of the specimens are

expected to be fairly stable in the hospital pathology department.

The use of positive and negative controls is of paramount importance for the

validity of immunohistochemistry (Hewitt et al. 2014, Torlakovic et al. 2015). In

the present study, negative and positive controls (tissue previously shown to

express the antigen) were employed in order to ensure the specificity of the staining.

However, the negative controls were not always ideal, as omission of the primary

antibody instead of non-immune IgG was used.

Different antigen retrieval methods were tested for some antibodies until the

staining result was considered optimal. Mainly routinely used detection methods

were applied for the staining, but some required additional testing.

Double staining is a useful tool for studying two different antigens in a single

tissue section. Using this method, we determined which cells express CTSK, and

whether it co-localized with TLR4 and TLR9 in OLP tissue. In any double staining,

the characteristics of the primary antibodies determine the protocol used, and

82

should be optimized first (van der Loos). Cross-reactivity of primary antibodies is

a potential problem, and may be overcome by different procedures (van der Loos

2008). If a simultaneous staining protocol is used, the primary antibodies should,

in general, be from different species. We used a sequential staining method to be

able to use two mouse antibodies. The chromogen combination is of utmost

importance since optimal contrast is needed between the two color reagents and in

the mixed color at sites of co-localization (van der Loos). Normally, sequential

double staining is applied for the identification of two different cell types or cell

components. It is not recommended for the observation of co-localization by mixed

colors (van der Loos 2008). However, we used a red-bluegray-color combination,

which yielded a fairly good contrast-mixed color at sites of suspected co-

localization. In addition to the selection of chromogens, the order of use of the

chromogens requires consideration. We observed that use of the red color first

yielded the best results. The use of IF double staining has additional drawbacks

compared to the immunoenzymatic method, namely quenching and fading of the

signal and autofluorescence due to formaldehyde fixation (van der Loos).

The interpretation of immunohistochemical staining is traditionally done

visually by a pathologist and is therefore subjective. In research, it is preferable to

have at least two investigators evaluate the staining to increase objectivity

(Fedchenko & Reifenrath 2014), as we also did in this thesis. The scoring of

staining is often done semi-quantitatively yielding ordinal variable data. Currently,

digital image analysis systems are also used to facilitate the scoring process and to

yield more accurate data of the scoring intensity with continuous variable data. The

parameters that are chosen for scoring should reflect the research question and take

into account the expression pattern of the studied biomarker. Therefore, the scoring

method and interpretation of staining is usually designed by the investigators for

each antibody individually, as was done in this thesis.

83

7 Conclusions

The present study revealed new findings regarding OLP etiopathogenesis and

management. First of all, we found that the prevalence of thyroid diseases,

especially hypothyroidism, is significantly higher in OLP patients compared to an

age- and sex-matched population without OLP from the same Northern Finland

area. Secondly, in a clinical intervention trial comparing topical 0.1% tacrolimus,

0.1% triamcinolone acetonide and placebo, we observed that both active

comparators were more effective than placebo in reducing the signs and symptoms

associated with OLP at week 3. In fact, the efficacy of topical tacrolimus and

triamcinolone acetonide compared to placebo is shown for the first time in OLP.

Thirdly, the immunohistochemical expression of TLR9, HA, HAS1–3, HYAL1–2

and CTSK is described for the first time in OLP, and was found to be altered

compared to healthy oral mucosa. Fourthly, we observed that treatment with topical

tacrolimus affected the expression of CD44 and CTSK in OLP.

In conclusion, our findings provide evidence for a multi-factorial etiologic

basis in OLP, reflected by the association with the autoimmune disease

hypothyroidism found in some patients. The observed involvement of several

previously undescribed molecules related to innate and adaptive immunity and

extracellular matrix modulation suggests their role in the pathogenesis of OLP. In

the management of symptoms and lesions associated with OLP, topical tacrolimus

seems equally effective compared to triamcinolone acetonide, but may provide

better long-term pain control.

84

85

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Original publications

I Siponen M, Huuskonen L, Läärä E, Salo T (2010) Association of oral lichen planus with thyroid disease in a Finnish population: a retrospective case-control study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 110(3): 319–324.

II Siponen M, Huuskonen L, Kallio-Pulkkinen S, Nieminen P, Salo T (2016) Topical tacrolimus, triamcinolone acetonide and placebo in oral lichen planus: a pilot randomized controlled trial. Manuscript.

III Siponen M, Kauppila JH, Soini Y, Salo T (2012) TLR4 and TLR9 are induced in oral lichen planus. J Oral Pathol Med 41(10): 741–747.

IV Siponen M, Kullaa A, Nieminen P, Salo T, Pasonen-Seppänen S (2015) Altered expression of hyaluronan, HAS1–2, and HYAL1–2 in oral lichen planus. J Oral Pathol Med 44(6): 401–409.

V Siponen M, Bitu CC, Al-Samadi A, Nieminen P, Salo T (2016) Cathepsin K expression is increased in oral lichen planus. J Oral Pathol Med 45(10): 758–765.

The original publications have been reprinted with permission of Elsevier (I) and

John Wiley & Sons (III-V). In addition, this thesis contains unpublished data.

Original publications are not included in the electronic version of the dissertation.

108

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1394. Eranti, Antti (2016) The role of electrocardiographic abnormalities, obesity, anddiabetes in risk stratification for sudden cardiac death in the general population

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1402. Hynninen, Nina (2016) Ikääntyvä muistisairas potilas kirurgisella vuodeosastolla

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ORAL LICHEN PLANUS – ETIOPATHOGENESISAND MANAGEMENT

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