MODULATING THE CD8 PAPILLOMAVIRUS-INDUCED LESIONS: … · 2017-12-21 · arranja sempre que eu...
Transcript of MODULATING THE CD8 PAPILLOMAVIRUS-INDUCED LESIONS: … · 2017-12-21 · arranja sempre que eu...
Dissertação de candidatura ao grau de Mestre em Oncologia – especialização em Oncologia Molecular – submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Rui Gil da Costa Categoria – Investigador Afiliação – LEPABE, Faculdade de Engenharia da Universidade do Porto; Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Português de Oncologia do Porto Co-Orientador – Professor Doutor Rui Medeiros Categoria – Professor Convidado Associado Afiliação – Instituto de Ciências Biomédicas de Abel Salazar; Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Português de Oncologia do Porto Co-Orientador – Professor Doutor Manuel Vilanova Categoria – Professor Associado Afiliação – Instituto de Ciências Biomédicas de Abel Salazar
CARLOS EDUARDO REIS DOS SANTOS
MODULATING THE CD8+ T CELL RESPONSE IN HUMAN PAPILLOMAVIRUS-INDUCED LESIONS: STUDIES IN THE K14-HPV16 TRANSGENIC MOUSE MODEL
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Alguns dos resultados incluídos nesta dissertação de mestrado foram submetidos
para publicação:
- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha
A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Kinetics of CD8+ T
cells in human papillomavirus-induced lesions – data from K14-HPV16 transgenic mice.
Submitted to the journal Tumor Biology with the number TUBI-D-15-04094.
- Santos C., Neto T., Ferrerinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Oliveira
P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Celecoxib promotes
degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice. Submitted to the
journal Antiviral Research with the number AVR-D-16-00002.
Alguns resultados foram também publicados em atas de um congresso internacional:
- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha
A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Role of CD8+ T
cells in HPV-induced skin cancer: data from a K14-HPV16 transgenic mouse model;
Proceedings of the HPV2015 – 30th International Papillomavirus Conference & Clinical
and Public Health Workshops; 17th-21st September 2015, Lisbon, Portugal. (Best poster
presentation award)
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“When you want to succeed as bad as you want to bre athe then you’ll be successful” – Eric Thomas
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Agradecimentos Neste momento, que marca o final de mais uma etapa da minha vida, não poderia
deixar de agredecer às pessoas que de alguma forma contribuíram para a conclusão
desta etapa. Este último ano foi bastante desafiante e exigente e todo o conhecimento e
apoio que recebi foi fundamental para chegar até aqui.
Em primeiro lugar, gostaria de agradecer à pessoa que me recebeu e que me
acompanhou mais de perto no desenvolvimento deste trabalho, o meu orientador,
Professor Doutor Rui Gil da Costa. Agradeço a sua confiança em mim para levar a cabo
este trabalho, todos os conhecimentos que me transmitiu, a disponibilidade para ajudar
no que fosse preciso, todos os conselhos e a constante boa disposição.
Ao Professor Doutor Rui Medeiros, meu co-orientador, agradeço toda a ajuda
fornecida ao longo deste ano e por tudo o que me ensinou. Mesmo estando cheio de
trabalho arranjava maneira de me receber sempre que aparecia no seu gabinete do IPO
para me poder ajudar no que eu precisasse e sempre bem-disposto.
Ao Professor Doutor Manuel Vilanova, também meu co-orientador, por tudo aquilo
que continua a ensinar-me, pelo seu rigor que tanto aprecio, pela disponibilidade que
arranja sempre que eu preciso de falar consigo mesmo quando a quantidade de trabalho
não o “deixa respirar”.
A este conjunto de orientadores agradeço todas as discussões das quais surgiram
ideias de trabalho, agradeço tudo o que me ensinaram e, acima de tudo, agradeço a
compreensão e o apoio que sempre demonstraram perante a minha vida de estudante-
atleta. Não só compreendem como também valorizam o que faço e é realmente bom
sentir esse apoio.
Ao Pedro Ferreirinha, mais do que um co-orientador no passado, um amigo e
colega investigador no presente. Muito do que aprendi sobre trabalhar num laboratório
devo-o a ele. Para além de tudo o que me ensinou e continua a ensinar está sempre
disposto a ajudar no que for preciso. Não só é dedicado ao trabalho como também é uma
pessoa óptima para ir tomar um café e ter uma conversa animada.
Às restantes pessoas que trabalham no Laboratório de Imunologia Mário Arala
Chaves (ICBAS), em especial à Encarnação Rebelo, Joana Alves e Alexandra Correia,
agradeço o carinho e a boa disposição de todos os dias, a disponibilidade para ajudar no
que precisasse e as conversas alegres e enriquecedoras.
À Professora Paula Oliveira, à Ana Faustino-Rocha e ao Tiago Neto (todos da
Universidade de Trás-os-Montes e Alto Douro) agradeço todo o apoio que me deram
durante a realização dos trabalhos experimentais.
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Por último, mas muito importantes, a família e os amigos formam um conjunto de
pilares que servem de apoio nos momentos mais complicados e de diversão quando a
ocasião o permite. Àqueles que me acompanharam mais de perto durante este ano
mesmo, em alguns casos, estando longe (Tiago Silva, Beatriz Silva, Helena Pinheiro,
Mariana Pereira, Inês Sousa, Hildeberto Moreira, Ana Rosa, Luciana Leite, Rita Faria,
Miguel Gonçalves, Cátia Oliveira, Joana Pinto, Renata Lopes, Diogo Edi, Isabel Paiva,
Catarina Pereira e, claro, aos meus colegas de equipa) agradeço pela força que me
deram quando as coisas pareciam não correr bem, a motivação que me passaram
quando esta me faltava, as conversas enriquecedoras sobre o trabalho, os momentos de
descontração, gargalhada e diversão quando estes eram possíveis e, principalmente, por
serem verdadeiros amigos e compreenderem os sacrifícios que faço em prol dos meus
objectivos e ambições enquanto estudante-atleta.
Aos meus pais e irmã, agradeço por tudo aquilo que não é possível agradecer o
suficiente. Agradeço pela educação que me deram e por tudo o que me ensinaram, por
todas as condições que sempre me deram para o meu crescimento, pelas chamadas de
atenção, por exigirem o melhor de mim, por me apoiarem em todas as decisões, por me
orientarem. Se hoje posso dizer que tenho orgulho no que me tornei devo-o inteiramente
a eles. Obrigado.
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Index
Agradecimentos .............................................................................................................. VII
Index ................................................................................................................................ IX
Abbreviations ................................................................................................................... XI
Resumo ......................................................................................................................... XIII
Abstract .......................................................................................................................... XV
Chapter 1: Introduction ..................................................................................................... 1
The Basis of HPV Infection and Cell Transformation ..................................................... 4
HPV Infection vs. Host Immune Response .................................................................... 5
HPV-associated Chronic Inflammation .......................................................................... 6
HPV-induced Carcinogenesis: The Best Models to Understand It ................................. 9
K14-HPV16 Transgenic Mice as a Model for Inflammation-associated Carcinogenesis12
Modulators of the immune response and their effect over CD8+ T cells ........................15
Chapter 2: Objectives ......................................................................................................17
Chapter 3: Kinetics of CD8+ T cells in human papillomavirus-induced lesions..................19
Introduction ..................................................................................................................21
Material & Methods ......................................................................................................22
Animals .....................................................................................................................22
Mice genotyping........................................................................................................22
Study Design ............................................................................................................22
Histology ...................................................................................................................22
Preparation of Single-Cell Suspensions ....................................................................23
Immunophenotyping .................................................................................................23
Statistical Analysis ....................................................................................................23
Results .........................................................................................................................24
Transgenic Mice Show Epidermal Hyperplasia and Dysplasia ..................................24
Increased CD8+ T lymphocytes numbers and activation in HPV16+/- mice ................24
Discussion ....................................................................................................................26
Conclusion ...................................................................................................................27
Chapter 4: Ptaquiloside inhibits tumour-infiltrating CD8+ T cells in HPV-transgenic mice .29
Introduction ..................................................................................................................31
Material & Methods ......................................................................................................32
Mice ..........................................................................................................................32
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Mice genotyping........................................................................................................32
Ptaquiloside isolation ................................................................................................32
Study Design ............................................................................................................33
Ptaquiloside administration and toxicity ....................................................................33
Skin histology ...........................................................................................................34
Isolation of a Single-Cell Suspension ........................................................................34
Flow Cytometry Analysis ...........................................................................................34
Statistical Analysis ....................................................................................................34
Results .........................................................................................................................35
General findings........................................................................................................35
Cutaneous lesions ....................................................................................................37
CD8+ T lymphocytes are present in ptaquiloside-treated and untreated animals .......37
Ptaquiloside decreases the number of CD8+CD107a+ T cells in HPV16+/- mice ........38
Reduced number of CD8+CD44+ T cells in ptaquiloside-treated transgenic animals .39
Discussion ....................................................................................................................40
Conclusion ...................................................................................................................42
Chapter 5: Celecoxib promotes degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice ................................................................................................................45
Introduction ..................................................................................................................47
Material & Methods ......................................................................................................48
Animals .....................................................................................................................48
Mice genotyping........................................................................................................48
Experimental Design .................................................................................................48
Celecoxib administration ...........................................................................................49
Histological Analysis .................................................................................................49
Preparation of Single-Cell Suspensions ....................................................................49
Immunophenotyping .................................................................................................49
Statistical Analysis ....................................................................................................50
Results .........................................................................................................................50
General findings........................................................................................................50
Histological analysis .................................................................................................50
CTL infiltration and activation in celecoxib-treated mice ............................................51
Discussion ....................................................................................................................53
Conclusion ...................................................................................................................54
Chapter 6: General Discussion and Conclusions .............................................................55
References ......................................................................................................................61
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Abbreviations 4-NQO – 4-Nitroquinoline 1-oxide
AP-1 – Activator Protein-1
APC – Antigen Presenting Cell
BPV – Bovine Papillomavirus
CD – Cluster of Differentiation
CIN – Cervial Intraepithelial Neoplasia
COX – Cyclooxygenase
CpG – cytosine-phosphate-guanine motif
CPV – Canine Papillomavirus
CRPV – Cottontail Rabbit Papillomavirus
CTL – Cytotoxic T Lymphocyte
CXB – Celecoxib
CXCL12 – C-X-C motif chemokine 12
DC – Dendritic Cell
FASL – FAS Ligand
FcγR – Fragment crystallizable gamma Receptor
H&E – Haematoxilin-Eosin
HIF-1 – Hypoxia Inducible Factor-1
HNSCC – Head and Neck Squamous Cell Carcinoma
HPV – Human Papillomavirus
IFN – Interferon
Ig – Immunoglobulin
IL – Interleukin
K14 – Keratin-14
LAMP1 – Lysosome-associated membrane protein 1
mAb – Monoclonal Antibody
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MC – Mast Cell
MHC – Major Histocompatibility Complex
miR – MicroRNA
MMP – Matrix Metalloproteinase
N – North
NF-κB – Nuclear Factor kappa-B
NK – Natural Killer
NMR – Nuclear Magnetic Resonance
ORF – Open Reading Frame
pRb – Retinoblastoma protein
PRR – Pattern Recognition Receptor
PTQ – Ptaquiloside
SCC – Squamous Cell Carcinoma
TAM – Tumour-associated Macrophage
TAM67 - Dominant-negative c-Jun
Th – T helper
TIMP – Tissue Inhibitor of Metalloproteinase
TLR – Toll-like Receptor
TNF-α – Tumour Necrosis Factor-alpha
TRAIL – TNF-related Apoptosis-Inducing Ligand
Treg – T regulatory
VEGF – Vascular Endothelial Growth Factor
VLP – Virus Like Particle
W – West
WT – Wild-type
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Resumo O Papilomavirus Humano (HPV) consiste em pequenos fragmentos circulares de
ADN de dupla cadeia sem invólucro, associado ao desenvolvimento de lesões benignas e
malignas na pele e mucosas queratinizadas. O murganho transgénico K14-HPV16 é um
modelo útil para estudar o processo de carcinogénese induzido por HPV, dado que se
assemelha ao processo que acontece em doentes oncológicos. Este processo é
acompanhado por um complexo infiltrado de leucócitos que desempenham um papel
decisivo quer na progressão quer na regressão das lesões. Os linfócitos T CD8+
citotóxicos (CTL) são uma parte importante deste infiltrado dado que são capazes de
destruir células infectadas por vírus e células tumorais. Neste trabalho, a infiltração e
actividade dos CTL foi analisada em diferentes tempos de vida e diferentes lesões. Ainda,
foi analisado o efeito de dois imuno-moduladores – uma toxina imunosupressora
(ptaquilosídeo – PTQ) e um fármaco anti-inflamatório (celecoxib – CXB, um inibidor
selectivo da ciclooxigenase-2) – na infiltração das células T CD8+ e na sua actividade.
Após o sacrifício dos murganhos, foram colhidas amostras de pele do peito de
murganhos wild-type (WT, HPV-/-) e transgénicos (HPV+/-), que foram tratados com PTQ,
com CXB ou não receberam tratamento. Os fragmentos de pele foram digeridos com
colagenase e a suspensão celular obtida foi analisada por citometria de fluxo. Para além
disso, foram colhidas amostras de pele correspondentes para análise histológica.
Todos os murganhos HPV-/- apresentam histologia da pele normal enquanto todos
os animais HPV+/- exibem lesões cuja agressividade aumenta através de etapas etárias
consecutivas. Verificou-se que os murganhos transgénicos apresentam maior infiltração e
actividade de células T CD8+ quando comparados com animais WT da mesma idade e,
que a activação destas células aumenta com a idade e acompanha a progressão das
lesões. Foram também estudados os efeitos de uma toxina de origem vegetal, o
ptaquilosídeo, que se pensa contribuir para a carcinogénese induzida por papilomavírus
em animais e em populações humanas. Os murganhos transgénicos tratados com PTQ
mostram menor percentagem de linfócitos T CD8+ activados quando comparados com os
animais HPV+/- não tratados, revelando um novo efeito imunossupressor desta toxina. Por
outro lado, os murganhos transgénicos tratados com CXB apresentam menor infiltração
de células citotóxicas comparativamente com os animais não tratados, mas apresentam
um aumento da percentagem de linfócitos T CD8+ activados. Esta observação sugere
possíveis abordagens terapêuticas às neoplasias induzidas pelo HPV, nomeadamente o
uso de inibidores selectivos da ciclooxigenase-2 para potenciar a actividade dos CTL.
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Globalmente, este trabalho levanta diversas questões sobre a função das células
T CD8+ nos cancros induzidos por HPV, abrindo o caminho para futuros estudos na área
da imunotoxicologia e da imunoterapia.
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Abstract Human papillomavirus (HPV) is a small non-enveloped double-stranded circular
DNA virus associated with the development of cutaneous and mucosal malignancies. The
K14-HPV16 transgenic mouse is an useful model to study the process of carcinogenesis
induced by a HPV infection as it closely mimics the occurring process in human patients.
This process is accompanied by a complex immune cell infiltrate which plays a key role in
either lesion progression or regression. Cytotoxic CD8+ T lymphocytes (CTL) are an
important part of this infiltrate as they target virus-infected and tumour cells for
destruction. Thus, in this work, CTL infiltration and activity was assessed and compared at
different time-points. In addition, the effect of two distinct immunomodulators – an
immunosuppressive toxin (ptaquiloside – PTQ) and an anti-inflammatory drug (celecoxib –
CXB, a selective inhibitor of cyclooxygenase-2) – over CD8+ T cell infiltration and activity
was examined.
Following the mice sacrifice, chest skin samples were collected from untreated,
PTQ-treated or CXB-treated wild-type (WT, HPV-/-) and transgenic (HPV+/-) mice. Skin
fragments were digested with collagenase and the obtained cell suspension was analysed
by flow cytometry. Additionally, matched skin samples were histologically examined.
All HPV-/- mice show normal skin histology whilst all HPV+/- animals present skin
lesions, with increased aggressiveness in older and PTQ-treated mice. Untreated
transgenic mice show increased CTL infiltration and activity when compared to untreated
WT animals. CD8+ T cell activation is higher in older HPV+/- mice than in younger
transgenic mice. Moreover, transgenic mice receiving PTQ treatment show reduced
percentage of activated CTL when comparing with untreated HPV+/- animals. Furthermore,
CXB-treated mice presented lower infiltration of cytotoxic cells when compared with the
respective controls. Despite the reduction regarding infiltrating cells, CXB-treated HPV+/-
mice showed increased percentage of activated CD8+ T lymphocytes than controls.
These findings suggest that CTL infiltrate HPV-induced lesions and their activity is
enhanced in more aggressive lesions. Moreover, the trafficking and activity of these cells
can be modified when exposed to immunomodulators. In this regard, ptaquiloside seems
to decrease CTL activity while celecoxib appears to enhance this activity.
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Chapter 1: Introduction
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Human papillomavirus (HPV) is the pathogen responsible for the most common
sexually transmitted infection among both men and women in the United States [1]. Most
sexually active people will be infected by HPV during their life. The vast majority of HPV
infections in immunocompetent individuals are cleared by the immune system, but in
approximately 10% of cases [2, 3] the persistence of this infection will cause the
development of disease and progression to cancer. HPV has tropism for skin and mucosal
epithelia especially from the anogenital and oropharyngeal areas. High-risk HPVs (mostly
types 16, 18, 31 and 33) act as crucial etiologic agents in cervical carcinoma [4] – which is
the sixth most frequent malignancy in women worldwide [5] – and are the cause of some
head and neck neoplasms where incidence depends on the country and other associated
risk factors like tobacco [6, 7].
Since 1863 [8], when Virchow first hypothesised a link between inflammation and
cancer, many studies have been carried out in order to understand the underlying
mechanisms of this relation and its intervenients. The importance of this relation was
further reinforced by the award of the Nobel Prize to Robin Warren and Barry Marshall for
their work in discovering Helicobacter pylori as the etiologic agent of inflammation-
associated gastric cancer [9]. The tumour microenvironment is characterised by the
presence of a variety of leucocytes which have been shown to be attracted by
chemokines produced by tumour and stromal cells [10]. Lymphocytes are an important
part of inflammatory infiltrates in the tumour microenvironment [8, 11] and different
lymphocytic populations can play different roles. For instance, T helper (Th) 1, Tγδ and
cytotoxic T cells are associated with disease-free survival. In contrary, Th2, Th17 and T
regulatory (Treg) cells are allied with a poorer outcome [12]. Neutrophils, tumour-
associated macrophages (TAMs), dendritic cells (DCs) and mast cells represent other
leucocyte populations also frequently found in the tumour environment that may play dual
roles during carcinogenesis [8, 13]. These tumour-associated inflammatory cells, together
with the tumour cells, produce and secrete a wide set of cytokines (e.g.: tumour necrosis
factor (TNF)-α, interleukin (IL)-1, IL-6 and monocyte colony-stimulating factor) and
chemokines (e.g.: monocyte chemotactic protein-1) that may either help tumour
progression or contain it [14-17].
In the case of HPV-associated carcinogenesis, the virus infects keratinocytes and
an immunological response arises as cytokines (mostly TNF-α, IL-1 and type I and II
interferons – IFNs) are released by the infected keratinocytes and local immune cells [18].
However, HPVs may avoid the effector functions of pro-inflammatory cytokines by
different mechanisms [19-22]. Thus, if the infection persists, the chance of neoplastic
transformation rises [23]. Besides its importance in the early stages of carcinogenesis,
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innate and adaptive immune responses are critical factors involved in malignant
progression.
The Basis of HPV Infection and Cell Transformation
Human papillomaviruses have their genome divided in three main regions: the
non-coding upper regulatory region, the early region encoding six known oncoproteins
and the late region, which encodes the capsid proteins. The oncoproteins encoded in the
early region, mainly E6 and E7 but also E5, are key participants in the infectious cycle and
are the source of the genetic alterations which potentially lead to HPV-associated
carcinogenesis as reviewed elsewhere [24]. The E5 oncogene is believed to have low
impact in the maintenance of the malignant phenotype of cervical cancer cells as it
appears to be deleted in various carcinomas [25]. Nevertheless, when present, it takes an
important part in multiple mechanisms favouring neoplastic transformation. Some in vitro
studies have shown that the HPV16 E5 oncoprotein can promote cell cycle progression
and DNA synthesis by down-regulating the expression of tumour suppressor proteins p21
and p27 [26, 27]. Moreover, this oncoprotein is central to evasion of the host
immunosurveillance. In E5-expressing cell lines antigen-presenting major
histocompatibility complex (MHC) class I molecules appear to be trapped in the Golgi
apparatus which reduced their surface expression [28, 29]. The down-regulation of MHC
class I on cells expressing E5 ultimately impairs their recognition and clearance by
cytotoxic T lymphocytes (CTL). The early genes E6 and E7 are known to have great
oncogenic potential. They play a pivotal role in HPV-mediated carcinogenesis as they
react with important tumour suppressor proteins impairing their functions. Thus, E6 binds
to and promotes the degradation of the p53 tumour suppressor protein [30, 31], impairing
important mechanisms of defence such as DNA repair and apoptosis. Additionally, the E6
protein facilitates carcinogenesis by reducing cellular senescence (as a result of
telomerase activation [32]), activating the NF-κB pathway and evading the interferon
response [24]. The E7 oncoprotein binds to tumour suppressor proteins of the
retinoblastoma family of proteins (pRb) and induces their inactivation [33, 34], therefore
increasing DNA synthesis and boosting cellular proliferation. Furthermore, E7 is able to
promote genomic instability, which increases the likelihood of malignant progression [24].
Together, the E6 and E7 oncogenes are capable of transforming and induce the
immortalization of keratinocytes [35]. Until today, the three early proteins mentioned
above appear to be, of all HPV proteins, the ones with higher oncogenic potential. Given
their roles in evading host immunity, it is important to understand how the human immune
system responds to HPV infection.
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HPV Infection vs. Host Immune Response
Along the years, mammals have grown complex innate and adaptive immune
mechanisms to control autoimmunity and to protect the host against microbial infections.
Viruses can be detected by cellular receptors generally designated as pattern recognition
receptors (PRRs), which trigger innate immune mechanisms that subsequently may be
followed by a more specific adaptive immune response. These cellular receptors are
proficient at recognising several pathogen-associated molecular patterns, like viral DNA or
RNA or bacterial cell wall or intracellular components, and also interacting with danger-
associated molecular patterns like intracellular molecules released from damaged cells
and cells undergoing unprogrammed cell death [36]. HPVs are double-stranded DNA
viruses that are most likely to be recognized by Toll-like receptor 9 (TLR-9). This receptor
is intracellularly expressed in the membrane of endolysosomes and recognizes
unmethylated CpG-DNA sequences harboured by pathogens like viruses, bacteria and
protozoa [36]. However, HPVs appear to somehow surpass these PRRs. The
oncoproteins E6 and E7 may promote the down-regulation of TLR-9 as has been shown
in vitro for HPV16 [37]. Therefore, at the start, the HPV infection seems to occur without
the awareness of the host immune system [38]. Furthermore, in case of a persistent
infection, the virus impairs immune cell functions as well as several molecular pathways
involved in the immune response [39]. Also, other innate immune mechanisms, like the
IFN response, can be abrogated by the actions of some HPV oncoproteins, as mentioned
above.
HPV is transmitted through skin-to-skin contact, usually through micro wounds.
There exists a variety of immune cells divided between the dermis and epidermis. In the
epidermis, the main cellular population are keratinocytes but there can also be found
Langerhans cells and T lymphocytes [39]. Keratinocytes, the main targets of HPV, are
capable of some immune functions. These cells can produce and release cytokines,
induce the activation of memory T cells and may also act as non-professional antigen-
presenting cells (APCs) [40]. Hence, keratinocytes are of great importance in the initiation
of an immune response against HPV infections. On the other hand, the majority of the so-
called “classical” immune cells (macrophages, DCs, T lymphocytes and others) can be
found in the dermis [39]. Both keratinocytes and immune cells on site are capable of pro-
inflammatory cytokine production [41] and antigen presentation in response to several
pathogens. Although the innate immune response to HPV infections remains poorly
understood, the immunological processes described above are thought to be the main
ones responsible for the initiation of an adaptive immune response, which will determine
the clearance or persistence of the infection. In fact, only about 10% of HPV infections will
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develop clinical symptoms and possibly evolve to cancer [2, 3]. Therefore, warts and low
grade cervical intraepithelial neoplasms (CIN1) are in most cases forced to regress by the
cell-mediated adaptive immune response [42]. It has been shown that CD4+ and CD8+
HPV16-specific T cells migrate to the skin after intradermal challenge with HPV16
peptides [43]. This cellular migration event appears to be followed by an antibody-
mediated response, as observed in HPV-infected women [44]. Still, antibody titres are low
and many women may even not produce them.
HPV-associated Chronic Inflammation
The above described mechanisms are thought to explain how a human immune
system reacts since the beginning of a HPV infection until the moment of immunological
clearance. However, as seen earlier, HPV oncoproteins have ways of evading the host
immune response. This evasion favours the persistence of the infection, which in turn may
promote tumourigenesis.
In this context, the cellular content of the inflammatory infiltrate is decisive for
either neoplastic transformation or elimination of infected/transformed cells (Fig. 1). While
some cellular populations support HPV’s immune evasion and cell transformation
mechanisms, others have a role in inhibiting malignant progression or even in potentiating
tumour regression. For instance, T regulatory (Treg) cells facilitate the viral immune
evasion. These cells display the CD4+CD25+FoxP3+ phenotype and play an
immunosuppressive role important to prevent self-aggression by the immune system [45].
Because of their immunosuppressive function, Treg cells are often associated to
persistent infection and tumour progression. Indeed, studies have shown a correlation
between increased frequencies of FoxP3+ T cells at both systemic [46] and cervical [47]
level and HPV persistent infection. Furthermore, a study has shown the chemokine
CXCL12 (which is not expressed in normal skin) to act as a chemoattractant for Treg cells
[48]. This work shows evidence that HPV stimulates an increased production of CXCL12
which draws a rising number of FoxP3+ cells. Besides Treg cells, another subset of CD4+
T cells can have pro-tumour functions. Type-2 helper T (Th2) cells can also contribute to
impair the protective immune response, predominantly of the Th1 type, in the tumour
microenvironment as they produce IL-4, -5 and -13, as well as IL-10. As seen earlier, this
type of immune response facilitates malignant progression [12]. Furthermore,
macrophages are thought to be a major component of tumour infiltrates [8] and are highly
frequent in HPV-associated tumours [49]. In the presence of Th2-type cytokines like IL-4
and IL-13, tumour-associated macrophages (TAMs) are alternatively activated and
acquire the M2-phenotype [50]. Unlike the M1 macrophages, these M2 cells express
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various features and functions that are commonly associated with immune regulation and
tumour progression. For instance, M2 TAMs, together with mast cells, are an important
source of growth factors (e.g.: vascular endothelial growth factor – VEGF), cytokines (e.g.:
TNF-α, transforming growth factor-β), proteases (e.g.: matrix metalloproteinases) and
other molecules which in turn promote angiogenesis and tissue remodelling [51, 52].
These events facilitate the intake of nutrients and provide other stimuli which help tumour
growth, invasion and metastasis. On the other hand, TAMs can be classically activated
upon microbial stimuli or induced by pro-inflammatory cytokines, such as IFN-γ, to play
important anti-tumour functions [50]. M1-type TAMs can, therefore, act as inducer and
effector cells in Th1-type immune responses against tumours. They usually display an IL-
12high, IL-23high phenotype and produce high levels of pro-inflammatory cytokines (IL-1β,
IL-6 and TNF-α) and reactive nitrogen species [53]. Besides M1 TAMs, other cellular
populations play important anti-tumour roles among the largely heterogeneous tumour
inflammatory infiltrates. T lymphocytes are also important immune effectors. Type-1
responses, mediated by Th1 cells represent the prototypic response adequate to
eradicate cancer cells. The differentiation of this type of CD4+ T cells is driven by IL-12
and they are characterised by the production of pro-inflammatory cytokines like IFN-γ. Th1
cancer cells can directly induce the killing of tumour cells through TNF-related apoptosis-
inducing ligand (TRAIL) and/or Fas Ligand (FasL) pathways [54] and can induce the
cytotoxic activity of CD8+ T lymphocytes and other effector cells through the production of
specific cytokines [55]. In turn, CD8+ T cells, which may differentiate into cytotoxic T
lymphocytes (CTL), are commonly protective against intracellular pathogens, like viruses,
and tumour cells. These lymphocytes were observed in higher frequency in HPV-positive
carcinomas when compared with HPV-negative lesions [56]. Further, this difference
correlates with the patients’ prognosis as a higher CD8+ T cell frequency is associated
with augmented overall survival. Moreover, “natural killer” (NK) cells and NKT cells act
similarly to CTL but are part of the innate immune response. Unlike CTL, NK cells do not
require the presence of MHC I molecules on target cells to exert their cytotoxic function.
Thus, if HPV successfully down-regulates MHC I expression, thereby avoiding the action
of CTL, infected cells may still be targeted by NK cells. Like CTL, NK and NKT cells
express IFN-γ, perforin and granzyme and are thus capable effectors in the anti-viral
immune response [39] as well as being able to also participate in the killing of cancer cells
[57]. Additionally, several APCs (like DCs and Langerhans cells) are of great value and
are frequent in the tumour microenvironment. They are essential for the activation of
effector cells through antigen presentation and cytokine production [13].
8
Fig. 1: Schematic representation of the immune cell infiltr ate in HPV- induced carcinogenesis. From top to bottom, HPV infection induces neoplastic transformation with associated tissue remodelling events which lea d to host immune response. This response could eit her contribute to disease progression or regression which is reflected in the cellular populations recruited in either case. CD8 +, Th1-type CD4 + T, NK cells and M1 macrophages are commonly associa ted with tumour regression while, Treg, Th2-type CD4 + T cells and M2 macrophages are usually related to malignant progression.
9
The cross-talk of immune cells with HPV-infected cells is still not fully understood.
However, it is generally agreed that the inflammation associated with the viral infection
facilitates the development of HPV-associated cancers. In general terms, regulatory T
cells, M2 TAMs, mast cells and Th2 cells most likely represent the immune cells with the
greatest contribution to persistent infections and malignant progression. Additionally,
HPV-transformed keratinocytes were shown to produce the immunosuppressive cytokine
IL-10 in HPV-associated cervical cancer [58], which may be triggered by the virus
oncoproteins as an immune evasion mechanism. On the other hand, M1 macrophages,
NK and NKT cells, CTL and Th1 cells are, with the support of APCs, the major
participants in anti-viral responses and tumour regression. Still, it is important to
emphasize that this whole interaction in the tumour milieu is greatly complex due to the
heterogeneity of cellular populations therein, which likely justifies the difficulty in
characterising the relationship between inflammatory cells and HPV infection.
HPV-induced Carcinogenesis: The Best Models to Unde rstand It
Along the years, scientists have been improving our understanding of chronic HPV
infection and its association with carcinogenesis. In vitro and in vivo animal models are
key elements towards the evolution of scientific knowledge (Table 1) and the in vitro ones
are already widely used in HPV-induced carcinogenesis research. HPV-related in vitro
studies include, in most cases, the use of single-layer cultures of common cervical
carcinoma cell lines (HeLa, SiHa, CaSki and others). In the last two decades, a more
sophisticated type of in vitro assay was developed and used as an alternative model.
Organotypic (or raft) cultures allow the proliferation and differentiation of epithelial cells at
an air-liquid interface on a dermal-equivalent support [59-61]. Thus, these raft cultures are
useful to study the events occurring in human stratified epithelia in the course of HPV
infection and HPV-induced carcinogenesis.
Alongside with the cell culture technique, in vivo animal models are extensively
exploited for scientific purposes. On one hand, bovine cattle, dogs and rabbits can be
used to study bovine, canine oral and cottontail rabbit papillomaviruses, respectively [62,
63]. The study of these animal papillomaviruses is important as they are etiologic agents
of diseases in farm and companion animals, provide in vivo models for HPV research and
help to discover new subjects to study on HPV. However, these large animals are not
common objects of study because they are associated with a great deal of financial costs
and ethical issues. In turn, the mouse is the most commonly used test subject in health-
associated studies. Mice display a great similarity to humans in terms of anatomy,
physiology, including the immune one, and genetics which, in addition to its cost-
10
effectiveness and a wide set of other advantages, make it applicable for the study of
human diseases [64]. In the past couple of decades, new HPV transgenic mouse models
have been created to more accurately infer on the function of some virus’ oncogenic
proteins. The first transgenic mice were reported in 1993 and incorporated the E6 and E7
open reading frames (ORFs) in their genome [65]. In this study, the authors targeted the
oncogene expression to the ocular lens (through the αA crystallin promoter) and showed
that these genes potentiate cell proliferation and impair cell differentiation. At the same
time, another study reported the development of neuroepithelial tumours in HPV16 E6/E7
transgenic mice that employed the human β-actin promoter to target the expression of
oncogenes [66]. However, these models failed to adequately target the expression of HPV
oncogenes to relevant tissues and were unsuitable for the study of HPV-associated
cancers occurring in patients. Besides these, other in vivo animal models were created
and ameliorated. Thus, mice expressing the early proteins E5, E6 or E7 alone were
created under the control of the keratin-14 (K14) promoter, targeting gene expression to
keratinocytes [67-69]. The targeting of HPV DNA to keratinocytes allows a great similarity
with the multistep process of HPV-associated carcinogenesis that occurs in the clinical
disease in patients. These models allow a better understanding of these proteins’
functions in vivo, which are expected to be similar to their roles in humans. For instance,
E5 transgenic mice treated with estrogen showed that this gene is capable of inducing
cervical cancer alone and, when E6 and E7 are present, these act synergically with E5 to
induce more severe lesions [70]. Additionally, E5 collaborated with the E7 oncogene to
increase the number and size of tumours. This alerts to the importance of the E5
oncogene, which may have a finer role in human cancers than is usually assumed and,
therefore, requires further investigation. In vivo animal models have also been playing a
part in the study of other HPV-associated malignancies like head and neck [71] or anal
[72] neoplasia. With the aid of K14-E6 and -E7 transgenic mice, it has been shown that E7
is the prevailing oncogene in promoting the development of 4-NQO-initiated head and
neck squamous cell carcinoma (HNSCC) [71]. In addition, K14-E6E7 double-transgenic
mice developed more and larger tumours and with superior histopathological severity than
K14-E7 single-transgenic mice. This result highlights the role of E6 which acts in synergy
with E7 to develop HNSCC.
The K14-HPV16 is another transgenic mouse model that has been explored in the
study of inflammation-driven carcinogenesis associated with HPV infection. These mice
incorporate in their genome the entire early region of the human papillomavirus type
16DNA and the expression of these HPV genes is also targeted to basal keratinocytes.
This model is more adequate to study the events of carcinogenesis because it comprises
all the virus oncogenes that control the early stages of the disease whilst the previously
11
described models target oncogene expression to uncharacteristic locations or are
transgenic for only certain oncogenes, thus helping to study the role played by the
respective protein. The K14-HPV16 mouse model was created by Arbeit et al. in 1994 [73]
and further characterized in 1996 [74]. A landmark study demonstrated that chronic
estrogen exposure induces the malignant transformation of squamous epithelium of the
cervix and vagina in transgenic mice [75]. In fact, a cross-species comparison study
showed great similarity in the features of carcinogenesis between human cervical disease
and K14-HPV16 transgenic mice [76]. Both patients and transgenic mice present
histological stages characteristic of squamous carcinogenesis marked by an increase of
VEGF expression and an up-regulation of inflammation and angiogenesis during
squamous carcinogenesis stages. This evidence strengthens the pertinence of this animal
model to study the multistage process of carcinogenesis and the roles of innate and
adaptive immunity. Recently, immunocompromised mice receiving tumour xenografts
have been used as a model to study novel therapeutics, new molecular targets and to
understand the biologic mechanisms of treatment sensitivity and/or resistance [77, 78].
However, these mice do not have a functional immune system and thus cannot mount an
effective immune response against the tumour. In these mice, the tumour growth is
heterotopic, instead of orthotopic, due to its atypical location. Additionally, in this model,
the lesions do not develop through a multi-step process like the ones occurring in HPV-
induced carcinogenesis but appear with a malignant phenotype at time of transplantation.
Therefore, immunocompromised mice xenografts do not seem reliable to be used in the
proposed HPV-associated studies as they do not mimic the carcinogenesis process
occurring in clinical disease. The K14-HPV16 transgenic mouse model should be a fitting
alternative to gain knowledge of these matters.
Table 1: In vitro and in vivo models for HPV research. Abbreviations: CRPV – Cot tontail Rabbit Papillomavirus; CPV-1 – Canine Oral Papillomavirus; BPV – Bovine Pa pillomavirus.
Model Main Characteristics References
In vitro
HeLa, SiHa, CaSki Cell lines obtained from cervical cancer patients, expressing HPV-18 (HeLa) or HPV-16 (SiHa and CaSki).
[79-84]
Organotypic cultures
Proliferating keratinocytes cultured in a dermal-equivalent support; Allows some degree of histologic interpretation; May be used to mimic HPV-induced squamous lesions.
[60, 61]
In vivo
Cottontail Rabbit, Dog, Bovine
Naturally or experimentally infected with CRPV, CPV and BPV, respectively; Allows comparative studies with HPV; Their use as models is limited by ethical and financial edges.
[62]
12
Mice
αA-HPV16-E6/E7 HPV oncogenes are expressed in the mice ocular lenses; Does not affect the animal viability.
[65]
hAc-HPV16-E6/E7 Develop neuroepithelial tumours; Is not possible to define a pre-neoplastic phase; Exhibit rapid malignant progression.
[66]
αHPV18-E6/E7
HPV-18-E6/E7 oncogenes are targeted to the ocular lenses; May be useful to study some factors that play a part in carcinogenesis, namely the roles of p53 and pRB proteins.
[85]
UC-HPV11-E2 The HPV-11-E2 gene is expressed in several cell types; Useful for the study of the E2 protein.
[86]
HPV18-URR, HPV11-URR
The upstream regulatory region of HPV type-18 or -11 is expressed only in epithelial cells; For both types, HPV is active in gland cells which may originate adenocarcinomas.
[87, 88]
NOD-scid -
IL2Rgamma null (NSG), Nude-Fox
n1null
Immunodeficient mice used for human head and neck tumours xenografts; These models skip the carcinogenic process; May be useful for testing new therapeutic strategies.
[77, 78]
K14-HPV16-E5/E6/E7
HPV-16 oncogenes are expressed only in keratinocytes, mimicking the site of clinical disease; These single-transgenic mice are useful to study the role of the respective oncoprotein in HPV-induced carcinogenesis.
[67-69]
K14-HPV18-E7, K14-HPV8-E2
Allow the study of the function of each oncogenic protein from the respective type of HPV.
[89, 90]
K14-HPV8 The HPV-8 early genes are targeted to be expressed in basal keratinocytes; Is appropriate to the study of HPV8-induced carcinogenesis.
[91]
K14-HPV16
The HPV-16 early genes are expressed in basal keratinocytes; Is very useful to reproduce the multi-stage carcinogenic process occurring in human disease; Shares histological features with human lesions; Shares patterns of angiogenesis with human cervical carcinogenesis.
[73, 74, 76]
K14-HPV16 Transgenic Mice as a Model for Inflammati on-
associated Carcinogenesis
In K14-HPV16 transgenic mice, inflammation rises at the sites where HPV is
expressed and progresses along the time while playing a part in carcinogenesis. For that
reason, this in vivo animal model is suitable for studying the features involved in
inflammation-associated carcinogenesis. As so, this model is also adequate to study the
13
effect of anti-inflammatory drugs as possible therapeutic agents to treat inflammation and
its associated malignancies.
The K14-HPV16 mouse model helped to understand that CD4+ T lymphocytes
may promote the development of dysplasia in the skin instead of having an immune
surveillance function [92]. Mice with a genetic deletion of CD4+ T cells presented a
reduced incidence of carcinomas when compared with controls. These results are in
agreement with the events occurring in human patients, where women with cervical
intraepithelial neoplasia (CIN) 3 lesions had a higher frequency of CD4+CD25hi Treg cells
when compared to patients with CIN0 and CIN1/2 [46]. However, not only CD4+ T cells
may contribute to increased incidence of carcinomas. The absence of B lymphocytes
impairs the ability of innate immune cells to infiltrate and become activated in neoplastic
skin [93]. B cells do not infiltrate the tumour site but they secrete immunoglobulins (Ig)
which are deposited in the neoplastic tissue. The reduction in infiltrating innate immune
cells in premalignant skin results in deficient angiogenesis and diminished keratinocytes
hyperproliferative activity. Therefore, carcinogenesis promoted by chronic inflammation
requires the presence of B cells. In addition, also mast cells (MCs) may be important in
malignant progression. The absence of these cells seems to compromise the initial
features of malignant progression in vivo, while its presence is usually associated with
increased angiogenesis [94], therefore suggesting a pro-tumour role of MCs during cancer
progression. Mast cells, among other immune and stromal cells, are an important source
of the matrix metalloproteinase 9 (MMP9) which seems to play a pro-tumour role as it
increases the incidence of carcinomas in HPV-transgenic mice [95]. However, in the
presence of MMP9 the arising tumours are less aggressive than in its absence. MMP9
appears to act as an important mediator of angiogenesis and regulator of epithelial
proliferation and therefore permits the faster tumour progression when compared to
MMP9-deficient mice [96]. Paradoxically, tissue inhibitors of metalloproteinases (TIMPs),
namely TIMP-1, which should oppose the action of MMPs, also appear to enhance
epithelial carcinogenesis [97]. It is reported that the expression of TIMP-1 increases
during the course of malignant progression in vivo promoting carcinogenesis. This
promotion is most likely due to the TIMP-1 capacity to potentiate the proliferation of
keratinocytes. In addition, also the complement system is activated during multi-step
carcinogenesis, albeit its component C3 is not responsible for recruiting immune cells to
the tumour milieu [98]. In fact, extensive regions of IgG deposition were observed in
hyperplastic and dysplastic skin of HPV16 mice which was comparable to HPV16 mice
lacking the complement protein C3. This outcome suggests an antibody-mediated
mechanism, independent of complement activation, to mediate the recruitment of immune
cells during carcinogenesis. A more recent study suggests that the peripheral activation of
14
an antibody-mediated response promotes the development of squamous cell carcinoma
(SCC) by local binding of the Fc γ-receptor (FcγR) on resident and recruited immune cells
[99]. Else, the dominant-negative c-Jun (TAM67) blocks the activation of the transcription
factors AP-1 (activator protein-1) and NF-κB (nuclear factor-kappa B) which are central in
the process of tumour promotion and progression [100]. Using HPV16 transgenic mice, it
has been shown that TAM67 also inhibits the action of the E7 oncogene upon certain
genes that may enhance carcinogenesis [101]. Additionally, the same study also
describes that TAM67 down-regulates the expression of the pro-inflammatory enzyme
cyclooxygenase-2 (COX-2), therefore acting as a tumour suppressor. Furthermore,
hypoxia is a known feature commonly linked to poor prognosis in most cancers. Hypoxia
induces changes in cancer tissues which may lead to inhibition of apoptosis, genomic
instability, angiogenesis, cellular differentiation, invasion, radio-resistance and other
alterations [102] and so, can be used as a prognostic and therapeutic factor. The hypoxic
cellular response is coordinated by the hypoxia-inducible factor-1 (HIF-1). This
transcription factor promotes the expression of vascular endothelial growth factor (VEGF)
which in turn increases angiogenesis, a known feature related with poor prognosis. In fact,
in vitro studies showed that HPV oncoproteins E6 and E7 enhance VEGF expression
through a HIF-1α-dependent mechanism, thus contributing to increased angiogenesis
[103, 104]. This is in agreement with the observed in K14-HPV16 transgenic mice, where
HIF-1 is up-regulated during the stages of carcinogenesis [105], suggesting that HIF-1
promotes local growth and invasion of cervical cancer, similarly to the natural events in
clinical disease [106]. Furthermore, also integrins play an important role in cancer
progression and, especially, in metastasis, as they are mediators of cell adhesion [107].
Also, the reduced expression of α2β1 integrin resulted in diminished progression from
papillomatosis to dysplasia and decreased tumour growth [108]. These results suggest
that the α2β1 integrin is important in regulating and promoting the initial steps of
carcinogenesis. More, cathepsin L – a lysosomal cysteine endoprotease – can influence
tumour progression in vivo [109]. When cathepsin L is absent in K14-HPV16 mice these
have a higher proliferative activity of keratinocytes, faster development of dysplasias and
increased frequency of metastasis when compared to animals in the presence of this
endoprotease [110]. Therefore, these data are suggestive of an anti-tumour role for
cathepsin L during carcinogenesis. In conflict with this conclusion, another study suggests
that cysteine cathepsins, including cathepsin L, promote tumour progression in vivo [111].
This report shows augmented progression from dysplasia to SCC, enhanced cathepsins
expression, reduced apoptosis, increased cell proliferation and promotion of angiogenesis
in K14-HPV16 mice with cystatin C deficiency. Cystatin C is the most abundant of all
cystatins which are endogenous inhibitors of cathepsins. Therefore it seems paradox that
15
both cathepsin L and cystatin C present anti-tumour functions. Else, it has been reported
that neuropilins – transmembrane receptors for the VEGF family of angiogenic proteins
and the class 3 semaphorin family of guidance proteins – are over-expressed in samples
of dysplasia and SCC from K14-HPV16 transgenic mice when compared to controls [112].
Nevertheless, neuropilins are only expressed in differentiated tumour cells. More recently,
the expression levels of the microRNA (miR)-155 were measured in K14-HPV16
transgenic mice in order to understand the possible influence of HPV with this biomarker
[113]. In this study, the HPV-positive mice presented significantly lower levels of miR-155
when compared with HPV-negative controls, suggesting that low levels of miR-155 may
play a role in early phases of HPV-associated carcinogenesis. The same group of
researchers also analyzed the expression of miR-21 in ear and chest skin samples of
HPV16-/- and HPV16+/- mice [114]. Levels of miR-21 expression were higher in less
aggressive lesions (hyperplasia) when compared with more aggressive ones (carcinoma
in situ), which is in agreement with the anti-inflammatory properties of miR-21 and
suggests that higher levels of this molecule may counteract malignant progression.
Modulators of the immune response and their effect over CD8 + T
cells
As seen earlier, the process of carcinogenesis induced by HPV infection is closely
associated with an intricate immune cell infiltrate and its soluble mediators [115]. Some of
these cell populations are commonly linked with tumour regression, as is the case of M1
macrophages, Th1 and cytotoxic T lymphocytes [12]. The latter, CTL, are specialists in
virus-infected and tumour cell destruction and, therefore, represent a key population in
anti-tumour immunity. These cells become activated when viral or tumour antigens are
presented in MHC I molecules together with the required co-stimulatory signals [116, 117].
Upon activation, CD8+ T cells exert their functions either directly, by releasing cytotoxic
granules (rich in perforin and granzymes) or cell-cell contact (e.g. FAS-FASL); or
indirectly, following an IFN-γ-dependent mechanism [118, 119]. In fact, CTL have already
been shown to infiltrate the tumour microenvironment and promote its regression in
several malignancies including HPV-associated cancers [56, 120, 121].
The occurring immune response can suffer alterations in the presence of
immunomodulatory stimuli which can be induced, for instance, by toxins or medical drugs.
Hence, immunomodulators can promote either immunosuppression or immunostimulation
and, therefore, alter the balance between regulatory and effector cells, resulting in
improved or impaired immune defensive mechanisms [122]. For instance, ptaquiloside, a
carcinogenic toxin found in bracken (Pteridium spp.), has some immunomodulatory effects
16
[123]. Bracken toxins are hypothesized to drive the malignant progression of
papillomavirus-induced upper digestive lesions in cattle and human populations, as
remarked by Chang et al. [124]. Ptaquiloside is a chemically unstable compound that has
been shown to induce splenic white pulp atrophy, neutropenia, reduced NK cells activity
and a B-cell lymphoproliferative malignancy [125-128]. However, until now, there are no
reports of an association between ptaquiloside and CD8+ T lymphocytes. In turn,
celecoxib (CXB) is an immunomodulatory drug which specifically inhibits cyclooxygenase-
2 (COX-2). This enzyme plays a key role in the development of an inflammatory response
[129] and is over-expressed in several types of cancer [130-134]. By selectively inhibiting
COX-2, celecoxib diminishes the production of prostaglandins without significantly
impairing the COX-1 isoform [135]. Actually, a few studies have already enlightened the
effect of this selective COX-2 inhibitor over cytotoxic T cells [136-140]. However, whether
celecoxib enhances or decreases CD8+ T lymphocytes infiltration and activity varies
between studies. Still, none of these studies analysed the effect of celecoxib over CTL in
HPV-associated lesions.
17
Chapter 2: Objectives
The purpose of this work was to assess the infiltration and activity of cytotoxic
CD8+ T lymphocytes in HPV-induced skin lesions, using the K14-HPV16 mouse model.
Thus, the work was focused in some more specific aims:
- To perform a kinetic study of the CTL infiltration and activation by comparing
lesions from different-aged mice;
- To analyse the immunomodulatory effect of ptaquiloside over CD8+ T cell
infiltration and activation;
- To analyse the immunomodulatory effect of celecoxib over CD8+ T cell infiltration
and activation.
18
19
Chapter 3: Kinetics of CD8 + T cells in human
papillomavirus-induced lesions 1
1 The contents of this chapter were adapted from:
- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Kinetics of CD8+ T cells in human papillomavirus-induced lesions – data from K14-HPV16 transgenic mice. Submitted to the journal Tumor Biology with the number TUBI-D-15-04094.
20
21
Introduction
Human papillomavirus (HPV) is the main etiologic agent of cervical cancer [4], but
can also originate anal, skin and head and neck malignancies [6, 141, 142]. Only a
persistent infection by an oncogenic HPV type (mainly HPV 16 and 18) can induce
malignant transformation. In approximately 90% of HPV infected individuals the host
immune system is able to fight the virus and hinder its dissemination. However, in the
remaining 10%, HPV can effectively evade immune defenses and lead to clinical disease
[2, 3].
K14-HPV16 transgenic mice, created nearly two decades ago [73], are an useful in
vivo animal model for the study of HPV-induced carcinogenesis. This model shares a
number of morphologic and molecular similarities to HPV-related human disease [76],
thus functioning as an excellent replica of the multi-stage process of carcinogenesis.
Targeting of HPV16 oncogenes to keratinocytes by the keratin-14 (K14)
promoter/enhancer is the key characteristic of this model [73].
HPV-induced carcinogenesis is associated with progressively intense chronic
inflammation. Therefore, a great diversity of immune cells and a multiplicity of soluble
mediators can be found within the tumour microenvironment [18]. The inflammatory
infiltrates provide pro- and anti-tumour stimuli, which will favour either the development or
the regression of the lesion [55, 143].
CD8+ T cells are restricted to major histocompatibility complex class I (MHC I)
molecules which can present peptides generated from intracellular viruses and/or tumour
cells [117]. Upon recognizing specific peptides presented on MHC I molecules on the
surface of professional antigen presenting cells, which also display co-stimulatory
molecules, these T cells can differentiate into cytotoxic T lymphocytes (CTL), helped by
cytokine stimuli [116]. CTL are an important part of tumour-specific immunity. They may
eliminate target cells either directly, through the release of lytic granules containing
several enzymes (such as perforin and granzyme) or by the engagement of death
receptors (e.g.: FAS-FASL), and indirectly, following an interferon-γ-dependent
mechanism which leads to cell cycle inhibition, apoptosis and stimulation of macrophage
anti-tumour activity [118, 119].
Thus, using the K14-HPV16 transgenic mouse model, the aim was to examine the
kinetics of CD8+ T cell infiltration in HPV-induced lesions during multi-step carcinogenesis
at different time points. The goal was also pointed to understand whether these cells were
activated by determining the presence of the lysosome-associated membrane protein 1
(LAMP1), also known as CD107a, at the cell surface.
22
Material & Methods
Animals
Generation of K14-HPV16 mice on a FVB/n background has been previously
reported [73]. K14-HPV16 transgenic mice were generously donated by Drs. Jeffrey Arbeit
and Douglas Hanahan (University of California) through the USA National Cancer Institute
Mouse Repository. The animal experiments were approved by the Universidade de Trás-
os-Montes e Alto Douro ethics committee (10/2013) and the Portuguese Veterinary
Directorate (0421/000/000/2014). Animals were maintained and bred according to
Portuguese (Decreto-Lei 113/2013, August 7th) and European (EU Directive 2010/63/EU)
legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark cycle (12h
light/12h dark) and relative humidity (50 ± 10 %), using hardwood bedding. Food and
water were provided ad libitum.
Mice genotyping
Animals were genotyped at weaning, using tail tip samples as described previously
[113, 114]. Briefly, nucleic acids were extracted and DNA quality and purity were
assessed. HPV16-E6 and -E2 genes were amplified to confirm the presence of HPV DNA
and mouse β-globin was used as control. Amplicons lengths were confirmed by agarose
gel electrophoresis. Only hemizygous females were used for the transgenic mouse groups
in the experiment.
Study Design
Ten wild-type (WT) (HPV16-/-, Group 1) and twelve transgenic (HPV16+/-, Group 2)
female mice were euthanized at 24-26 weeks of age. Latter, six HPV16-/- (Group 3) and
six HPV16+/- (Group 4) female mice were sacrificed when 28-30 weeks-old. The mice were
humanely sacrificed by intraperitoneal pentobarbital overdose, followed by exsanguination
through cardiac puncture. Chest skin samples (approximately 4 cm2) were collected for
cell isolation and flow cytometry analysis. Matched samples were collected for histological
examination.
Histology
Skin samples were fixated in 10% neutral buffered formalin for 48h. Samples were
dehydrated through graded alcohols and xylene and paraffin-embedded in an automatic
STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with
haematoxylin-eosin (H&E) for histological examination on light microscopy. Skin samples
23
were classified as normal skin, epidermal hyperplasia or epidermal dysplasia. Minor
dysplastic foci on a hyperplastic background were also recorded.
Preparation of Single-Cell Suspensions
Chest skin samples were cleaned from excessive blood vessels and fat tissue and
cut into small pieces. These fragments were incubated for 2 hours with 125 U/ml type I
collagenase (Gibco, Life Technologies, Paisley, UK) in RPMI-1640 medium
complemented with 1% glutamine, 1% penicillin-streptomycin-amphotericin B, 1% HEPES
buffer (all from Sigma, St. Louis, MO) and 10% foetal bovine serum (BioWest, Nuaillé,
France) at 37 ºC and 150 rpm in a 3031 orbital incubator (GFL, Burgwedel, Germany).
The resulting cell suspension was then filtered and centrifuged at 300 � for 10 min at 4 ºC.
Cells were then ressuspended in phosphate buffered saline containing 1% bovine serum
albumin and 20 mM sodium azide followed by extracellular staining.
Immunophenotyping
Following cell isolation, the surface phenotype of the collected cells was assessed
by flow cytometry using specific monoclonal antibodies (mAb). To prevent non-specific
antibody binding, cells were incubated with anti-mouse CD16/CD32 mAb for FcγR
blocking. This was followed by incubation with anti-CD8 mAb phycoerythrin-cychrome 5-
conjugate (clone 53-6.7, BD Biosciences, San Diego, CA) and anti-CD107a mAb
phycoerythrin-conjugate (clone eBio1d4b, eBioscience, San Diego, CA). Following
extracellular staining, the cells were washed, fixed in 2% formaldehyde and washed with
phosphate buffered saline containing 1% bovine serum albumin and 20 mM sodium azide.
Antibody-labelled cells were analysed in an EPICS XL flow cytometer using the
EXPO32ADC software (Beckman Coulter, Miami, FL). The collected data files were
analysed using the FlowJo software v10.0.7 (FLOWJO, LLC, Ashland, OR).
Statistical Analysis
Statistical analyses were executed in the GraphPad software (version 6.0,
GraphPad Software, Inc. La Jolla, CA). Statistical analysis between group pairs was
performed using the Mann-Whitney test.
24
Results
Transgenic Mice Show Epidermal Hyperplasia and Dysp lasia
Analysis of skin samples showed that the totality of WT mice (groups 1 and 3)
presented normal skin histology (Fig. 2a) whilst all transgenic mice presented skin lesions.
In all 12 mice composing group 2 it was possible to observe simple to papillary, diffuse,
variably severe epidermal hyperplasia and papillomatosis with orthokeratotic
hyperkeratosis (Fig. 2b). Inflammation was mild, with a few macrophages, lymphocytes
and mast cells present in the superficial dermis. Also in this group, 2 animals (16.7%)
presented small epidermal dysplastic foci. In group 4, 3 animals (50.0%) presented diffuse
epidermal dysplasia (Fig. 2c); other 3 (50.0%) showed multifocal epidermal dysplasia in a
hyperplastic background. Sub-epidermal angiogenesis and dermal inflammatory infiltrates
were prominent, showing numerous mixed mononuclear leukocytes and neutrophils.
Fig. 2: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – WT animal; Normal skin histology, ��� �. b – 24-26 week-old HPV16 +/- animal; Epidermal hyperplasia extending to the fol licular infundibulum and isthmus, ��� �. c – 28-30 week-old HPV16 +/- animal; Epidermal dysplasia, ��� �. Note enhanced sub-epidermal inflammatory cell infiltration.
Increased CD8 + T lymphocytes numbers and activation in HPV16 +/- mice
In order to assess the presence of CD8+ T cells in HPV-associated lesions,
lymphoid cells were isolated from chest skin tissue and analysed by flow cytometry (Fig.
3a). As shown in Fig. 3b, chest skin samples from HPV16+/- mice (groups 2 and 4)
presented a significantly higher percentage of CD8+ T cells when compared with those of
WT animals (groups 1 and 3). Although the percentage of skin CD8+ T cells was slightly
higher in group 4 than in group 2, it did not reach statistical significant difference.
In order to determine if the CD8+ T cells found in the skin of HPV16+/-
mice
presented evidence of cytotoxic activity, the surface expression of CD107a was
evaluated. In CD8+ T lymphocytes CD107a reaches the cell surface when lytic granules
suffer exocytosis, thus exposing its membrane proteins [144]. Therefore, this lysosome-
associated membrane protein is a commonly used surrogate marker of CTL degranulation
[145]. As shown in Fig. 3c, the percentage of CD107a+CD8+ T cells in chest skin samples
25
of mice from groups 2 and 4 were found significantly higher when compared with
respective controls of groups 1 and 3. This indicates that in the HPV16+/- mice skin
infiltrating CTL released cytotoxic granules. Moreover, in group 4 mice, 100% of which
show multifocal or diffuse dysplasia, a markedly higher percentage of CD8+ T cells
express CD107a (P � 0.001) than in group 2 mice, of which only 16.7% show focal
dysplastic lesions. This result shows a positive correlation in the proportions of activated
CD8+ T cells and lesion severity.
Fig. 3: Percentage of CD8 + and CD8+CD107a+ T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analysis of the gating strategy empl oyed. Numbers within graphs correspond to the percentage of the gated population. b – Percentages of CD8 + T cells and c – percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after sk in tissue collection and digestion with collagenase. Chest sk in samples were collected from 24-26 weeks-old HPV -/- and HPV+/- mice (Groups 1 and 2, respectively) and 28-30 week s-old HPV -/- and HPV+/- mice (Groups 3 and 4, respectively). Group 1, n = 10; Group 2, n = 12; Gr oup 3, n = 6; Group 4, n = 6. Each dot represents a n individual animal. Bars represent the mean value in each group . ** P � 0.01; *** P � 0.001.
26
Discussion
HPV is considered to be the cause of virtually all cases of cervical cancer [146],
therefore playing a central role in carcinogenesis. Despite the continuous progress in the
understanding of the immune response against HPV and the development of effective
vaccines, much is still ignored about the cell-mediated response against HPV-induced
lesions. HPV-mediated carcinogenesis is commonly associated with chronic inflammation
[18]. Nevertheless, the complex interactions within the inflammatory infiltrate remain not
fully understood [39].
The K14-HPV16 transgenic mouse model appears as an appropriate in vivo
animal model to improve the knowledge on HPV-induced carcinogenesis and elicited
immune response. Besides cervical cancers, these mice develop aggressive skin lesions
with greater incidence in the ear and the chest [74]. As expected, it was possible to detect
a higher incidence of aggressive lesions (epidermal dysplasia) in older transgenic mice
than in younger ones, which presented mostly hyperplastic lesions. The development of
dysplastic lesions was accompanied by a dramatic intensification of sub-epidermal
inflammation. As this process of carcinogenesis is associated with progressive chronic
inflammation, this model will allow the study of the intervening immune effectors.
CD8+ CTL are important players in tumour-specific immunity [147]. When CD8+
CTL recognize their targets they release lytic granules containing a variety of enzymes
(perforin, granzyme and other) which allow the killing of virus-infected and tumour cells
[118]. During exocytosis, CD107a reaches the cell surface as the membrane of lysosomes
disrupts, thus helping to determine whether the CD8+ T cells are activated [145].
In this work, it was possible to observe that CD8+ T cells heavily infiltrate HPV-
induced chest skin lesions in K14-HPV16 transgenic female mice. This data is in
agreement with previous studies which described the presence of CD8+ T cells in HPV-
induced cervical and skin lesions in human patients [43, 148]. According to one of these
studies, the presence of CD8+ T cells in cervical tissue was associated with tumour
regression [148]. The results presented here suggest a trend towards the increase of
infiltrating CD8+ T cells in chest skin samples of 28-30 weeks-old mice when compared to
24-26 week-old animals. Although indicating that CD8+ T lymphocytes migrate in greater
number towards more severe lesions than to less aggressive ones, these results should
be further confirmed. Nevertheless, this work’s data clearly show that a significant
proportion of the infiltrating CTL are activated and degranulate. Whether these cells were
activated at the lesion site or at regional lymph nodes would be an appealing point to
address hereafter. CD8+ T lymphocytes isolated from older animals present a high degree
of activation when compared to that isolated from younger mice. This suggests that more
27
CD8+ T cells become activated in response to a more severe stimulus than when
confronted with a less aggressive lesion. However, despite the increase in number of
cytotoxic cells and its enhanced activation, the lesions still tend to progress to a poor
phenotype with aging. Low tumour infiltration by activated CTL has been associated with
the limiting efficacy of the immune response in eliminating established tumours [149]. The
results reported herein indicate that however important, CTL recruitment and
degranulation into lesion areas do not suffice to prevent progressive carcinogenesis in the
K14-HPV16 transgenic mice. Many immunoevasive strategies have been identified
preventing the effectiveness of tumour immunity mediated by CTL [150]. However, as a
high proportion of skin infiltrating CD8+ T cells showed evidence of degranulation in the
older K14-HPV16 transgenic mice, it would be worth exploring whether the transgenic
cells might present an intrinsic resistance to cytotoxic mechanisms dependent on CTL
degranulation.
Conclusion
The results presented here clearly show that CD8+ T cells infiltrate HPV-induced
lesions, but indicate their activation is not enough to stop malignant progression in this
model. These results also support the use of K14-HPV16 mice as an adequate model to
study the host immune response associated with HPV-induced lesions and help
developing immunotherapeutic strategies that could prevent carcinogenesis.
28
29
Chapter 4: Ptaquiloside inhibits tumour-infiltratin g CD8+
T cells in HPV-transgenic mice
30
31
Introduction
Papillomavirus cause benign and malignant lesions in many wild and domestic
animal species, as well as in human populations. Human papillomavirus (HPV) is the
source of several diseases, including many anogenital and some upper digestive cancers
[151]. The growing incidence of HPV-positive oropharyngeal cancer is now of particular
concern [152]. Several bovine papillomavirus (BPV) types, such as BPV1, BPV2 and
BPV4 are associated with cutaneous and upper digestive papillomatosis, as well as with
abortive urinary bladder infections [63]. Interestingly, digestive tract cancers induced by
HPV and BPV share many common features and, possibly, some environmental co-
factors [124].
A persistent infection by an oncogenic viral type is mandatory for malignant
progression to take place [3]. Viral persistence is determined by a complex interplay
between the host immune system and the virus ability to evade it. In this context,
environmental immunosuppressants are likely to play an important role.
Bracken (Pteridium spp.), is a fern belonging to the Pteridaceae family with a
worldwide distribution [123]. It is known for its ability to facilitate BPV persistence in
infected cattle, promoting the malignant transformation of upper digestive papillomas into
squamous cell carcinomas [153]. Importantly, exposure to bracken and its toxins has also
been associated with an increased risk of developing upper digestive malignancies in
human populations (reviewed in [123]). Ptaquiloside, the main bracken toxin, is an
unstable nor-sesquiterpene glycoside of the illudane family, with important immunotoxic
properties. It has the ability to induce neutropenia and inactivate natural killer (NK) cells,
which play an important role in innate immunity, by killing virus-infected and neoplastic
cells [126, 127]. Therefore, it might be hypothesized that these immunosuppressive
effects could facilitate viral persistence and the progression of early-stage papillomavirus-
induced lesions.
Cytotoxic CD8+ T lymphocytes (CTL) are another cell population involved in host
immunity to virus-infected and neoplastic cells as the main responsible for cytotoxic
functions in an adaptive immune response. CTL target virus-infected and tumour cells via
antigen presentation by Major Histocompatibility Complex class I (MHC I) molecules [117],
in the presence of other co-stimulatory signals [116]. These cells may directly induce the
killing of target cells, by releasing lytic granules or through death receptor engagement
(e.g. FAS-FASL) or indirectly, by producing IFN-γ, which leads to multiple anti-tumour
activities [118, 119]. Several HPV and BPV types (including HPV16 and BPV1 and 4) are
able to down-regulate MHC I expression through their E5 oncoprotein, as an immune
evasion strategy [154]. However, recent findings confirm that, even under conditions of
32
low MHC I expression, CD8+ T cells play a decisive role in fighting HPV-induced
oropharyngeal cancers [155].
In the present work, we hypothesize that ptaquiloside, the bracken toxin, exerts its
immunosuppressive effect by counteracting the action of CD8+ T cells against
papillomavirus-induced lesions. This would facilitate viral persistence and the progression
of early HPV- and BPV-induced lesions, posing major risks for human and animal health.
In order to test that hypothesis, we employed HPV16-transgenic mice and analysed the
effect of ptaquiloside on the population of skin infiltrating CD8+ T cells.
Material & Methods
Mice
Construction of K14/HPV16 mice on a FVB/n background has been previously
reported [73]. These animals develop characteristic multi-stage cutaneous and uterine
cervical carcinogenesis, and were generously donated by Drs. Jeffrey Arbeit and Douglas
Hanahan (University of California) through the USA National Cancer Institute Mouse
Repository. The animal experiments were approved by the Universidade de Trás-os-
Montes e Alto Douro ethics committee (10/2013) and the Portuguese Veterinary
Directorate (0421/000/000/2014). Animals were maintained and bred according to
Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive 2010/63/EU)
legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark cycle (12h
light/12h dark) and relative humidity (50 ± 10 %), using hardwood bedding. Food and
water were provided ad libitum.
Mice genotyping
Animals were genotyped at weaning, using tail tip samples as described previously
[113, 114]. Briefly, nucleic acids were extracted and DNA quality and purity were
assessed. HPV16-E6 and -E2 genes were amplified to confirm the integration of HPV
DNA into the mouse genome and a fragment of mouse β-globin was also amplified to
confirm the quality of the extracted DNA. Lengths of the fragments were confirmed by
agarose gel electrophoresis. Only hemizygous female mice were used for the transgenic
mice groups in the experiment.
Ptaquiloside isolation
Ptaquiloside was isolated from bracken as previously described [156] with minor
modifications. Briefly, 1000 g (dried weight) bracken crosiers were harvested at Arcos de
Valdevez, Portugal, 41º 49´ 12´´ N, 8º 24´ 11´´ W) and a sample was deposited at the
33
Universidade de Trás-os-Montes e Alto Douro herbarium (reference no. 18248). Bracken
was blended in distilled water (10 L), stirred at room temperature for 1 hour and the
extract was adsorbed on 3 L of XAD-2 resin (Supelco, Sigma, St. Louis, MO). The resin
was eluted with methanol (10 L) and the methanol extract was concentrated, dissolved in
water (400 ml) and extracted with butanol (5�500 ml). The butanol extract was
chromatographed on silica gel (Merck, Kenilworth, NJ). Fractions containing ptaquiloside
were separated twice on octadecyl-sylane silica gel (Fujy-Silysia, Kasugai Aichi, Japan)
using methanol-water mixtures to obtain pure ptaquiloside. The compound was
distinguished from other closely-related bracken illudane toxins on the basis of its
characteristic 1H and 13C nuclear magnetic resonance (NMR) signals [157] using an
Avance III 400 MHz spectrometer (Brucker, Billerica, MA). Aliquots were prepared for
each experimental week (7.5 mg), freeze-dried and kept at -20ºC until use.
Study Design
Thirty transgenic (HPV16+/-) and 15 wild-type (WT, HPV16-/-) female 18-20 weeks-
old mice, showing diffuse cutaneous crusting and papillomatosis were employed. The
animals were separated into three experimental groups: group 1 (n = 15, HPV16-/- mice),
group 2 (n = 15, HPV+/- untreated mice) and group 3 (n = 15, HPV16+/- mice treated orally
with 0.5 mg ptaquiloside per week, for 10 consecutive weeks). The experimental animals
were monitored daily for signs of stress or disease. All surviving mice were euthanized at
28-30 weeks of age by an intraperitoneal pentobarbital overdose, followed by cardiac
puncture and exsanguination. Chest skin samples (approximately 4 cm2) were collected
for flow cytometry analysis. Matched samples were collected for histological examination.
Ptaquiloside administration and toxicity
Each week, a 7.5 mg ptaquiloside aliquot was dissolved in 300 µl ethanol (25
mg/mL) and the individual 0.5 mg dose (20 µl ethanol) was added to a standard wheat
cookie (Vieira, Vila Nova de Famalicão, Portugal) fragment weighting ca. 100 mg. Each
dosed fragment was allowed to dry at room temperature for 10 minutes and individually
administered to a group 3 animal, in an individual empty cage; the ingestion was visually
monitored. In order to confirm that ptaquiloside was indeed active at the administered
dosage, we looked for a lymphoid malignancy [128]. Thus, histological analysis of kidney
samples was used to confirm neoplastic lymphoid cell infiltration in this organ.
34
Skin histology
Skin samples were fixated in 10% neutral buffered formalin. Samples were
dehydrated through graded alcohols and xylene and, paraffin embedded in an automatic
STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with
haematoxylin-eosin (H&E) for histological examination on light microscopy. Skin samples
were classified as normal skin, epidermal hyperplasia, multifocal epidermal dysplasia in a
hyperplastic background and diffuse epidermal dysplasia.
Isolation of a Single-Cell Suspension
Chest skin samples were cleaned from excessive blood vessels and fat tissue and
cut into small pieces. This fragments were incubated for 2 hours with 125 U/ml of type I
collagenase (Gibco, Life Technologies, Paisley, UK) in RPMI-1640 medium
complemented with 1% glutamine, 1% penicillin-streptomycin-amphotericin B, 1% HEPES
buffer (all from Sigma) and 10% foetal bovine serum (BioWest, Nuaillé, France) at 37 ºC
and 150 rpm in a 3031 shaking incubator (GFL, Burgwedel, Germany). The resulting cell
suspension was then filtered and centrifuged at 300 � for 10 min at 4 ºC. Cells were then
ressuspended in phosphate buffered saline containing 1% bovine serum albumin and 20
mM sodium azide followed by extracellular staining.
Flow Cytometry Analysis
Following cell isolation, the cellular immune phenotype was assessed by flow
cytometry using monoclonal antibodies (mAb). Non-specific antibody binding was
prevented by incubating cells with anti-mouse CD16/CD32 mAb for FcγR blocking. This
was followed by incubation with anti-CD8 phycoerythrin-cychrome 5-conjugate (clone 53-
6.7, BD Biosciences, San Diego, CA), anti-CD107a (LAMP1) phycoerythrin-conjugate
(clone eBio1d4b) and anti-CD44 phycoerythrin-cychrome 7-conjugate (clone IM7) mAb
(both from eBioscience, San Diego, CA). Following extracellular staining, the cells were
washed, fixed in 2% formaldehyde and washed with phosphate buffered saline containing
1% bovine serum albumin and 20 mM sodium azide. Antibody-labelled cells were
analysed in an EPICS XL flow cytometer using the EXPO32ADC software (Beckman
Coulter, Miami, FL). The collected data files were analysed using the FlowJo software
v10.0.7 (FLOWJO, LLC, Ashland, OR).
Statistical Analysis
Flow cytometry statistical analyses were executed in the GraphPad software
(version 6.0, GraphPad Software, Inc. La Jolla, CA). In column and dot graphs, each point
is representative of an individual mouse and bars represent the mean value for the
35
respective group. Analysis between group pairs was performed using the Mann-Whitney
test. Kaplan-Meier survival analysis coupled with a log rank test was performed using the
PASW Statistics software (version 18, IBM® SPSS®, Quarry Bay, Hong Kong).
Results
General findings
Ptaquiloside was isolated at a 0.01% yield from bracken, as previously reported
[128]. The compound's structure (Fig. 4a) was confirmed using NMR analysis and no
other illudane glycoside was detected (Fig. 4b, c). Transgenic animals showed
characteristic diffuse cutaneous hyperkeratosis and erythema, together with variably
intense pruritus, while WT mice showed normal skin. All mice in groups 1 and 2 survived
the 10 weeks experimental period. Ten mice (66.7%) from group 3 succumbed before the
end of the study (Fig. 4d). Histological analysis of kidney samples showed normal
histology in WT and untreated HPV+/- animals (Fig. 4e) and, moderate to severe,
multifocal, perivascular infiltration of lymphoblastic cells, showing highly pleomorphic
nuclei and up to 3 mitotic figures per high-power field in 100% of ptaquiloside-treated
animals (group 3) (Fig. 4f).
36
Fig. 4: Ptaquiloside and its leukaemogenic effect in K14-HP V16 mice. a – ptaquiloside's structural formula. b and c – partial 2D NMR-HSQC spectra (obtained in CD3OD) for ptaquiloside. Note the correlations between ca rbons and their corresponding hydrogens in the glucose residu e (b). Note in particular the correlations between carbons 12 and 13 at 5.87 ppm and 10.86 ppm respectively, and their corresponding hydrogens in the cyclopropylide ne ring (c). d – Kaplan-Meier survival analysis. HPV +/- mice treated with ptaquiloside show significantly reduced survival ( P � 0.001) compared with untreated HPV -/- or HPV+/- animals. e – Untreated HPV +/- animal (group 2), showing normal kidney histology; H&E 200 �. f – Ptaquiloside-exposed HPV +/- animal (group 3) showing typical ptaquiloside-associated perivascular infiltration by leukaemic l ymphocytes. Note marked nuclear pleomorphism and mi totic figures; H&E 200 �.
37
Cutaneous lesions
The histological analysis showed that all group 1 mice (100.0%) presented normal
skin histology (Fig. 5a) whilst all transgenic mice (groups 2 and 3) presented skin lesions.
In group 2, 3 animals (50.0%) presented diffuse epidermal dysplasia (Fig. 5b); other 3
(50.0%) showed epidermal hyperplasia with multifocal dysplasia. Sub-epidermal
angiogenesis and dermal inflammatory infiltrates were prominent in dysplastic lesions,
showing numerous mixed mononuclear leukocytes and neutrophils. In group 3, the totality
of mice (100.0%) showed diffuse epidermal dysplasia (Fig. 5c).
Fig. 5: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – group 1 animal. Normal skin histology, 400 �. b – group 2 animal. Epidermal hyperplasia extendi ng to the follicular infundibulum, 400 �. c – group 3 animal. Epidermal dysplasia, 400 �. Note loss of keratinocytic polarity and different iation.
CD8+ T lymphocytes are present in ptaquiloside-treated and untreated
animals
In order to determine the proportions of CD8+ T cells present in chest skin with or
without HPV-associated lesions, samples were collected and cells isolated, followed by
flow cytometry analysis (Fig. 6a). As shown in Fig. 6b, group 2 and 3 (HPV+/- and
ptaquiloside-treated HPV+/-, respectively) mice showed significantly higher percentages of
CD8+ T cells compared with HPV-/- mice (P � 0.05). The percentage of CD8+ T cells in
chest skin was not significantly different between ptaquiloside-treated and untreated
HPV16+/- mice (groups 3 and 2, respectively).
38
Fig. 6: Percentage of CD8 + T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. The number wit hin the graph corresponds to the percentage of the gated po pulation. b – Percentages of CD8 + T cells were determined by flow cytometry after skin tissue collection and enz ymatic digestion. Chest skin samples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Group 3, n = 5. Each dot represe nts an individual animal. Bars represent the mean v alue in each group. * P � 0.05.
Ptaquiloside decreases the number of CD8 +CD107a+ T cells in HPV16 +/- mice
Next, CD8+ T cells found in skin samples were analysed with the objective to
determine if they were actively degranulating, which indicates ongoing cytotoxic activity.
For that purpose, flow cytometry analysis was performed to assess the expression of
surface CD107a in these cells (Fig. 7a). A significantly higher percentage of
CD8+CD107a+ T lymphocytes was observed in HPV+/- mice as compared with HPV-/-
animals (P � 0.01) (Fig. 7b). Additionally, HPV+/- mice exposed to ptaquiloside also
presented a higher percentage of CD8+CD107a+ T lymphocytes when compared to WT
animals, but this was significantly reduced when compared to untreated HPV+/- mice (P �
0.05).
39
Fig. 7: Percentage of CD8 +CD107a+ T cells within total CD8 + T cells obtained from chest skin samples from WT a nd K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. Numbers within graphs correspond to the percentage of the gated populatio n. b – Percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after skin tissue collection and enzymatic digestion. Chest sk in samples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Gro up 3, n = 5. Each dot represents an individual anim al. Bars represent the mean value in each group. * P �0.05; ** P � 0.01.
Reduced number of CD8 +CD44+ T cells in ptaquiloside-treated transgenic
animals Having determined that CD8+ T cells presented a cell surface phenotype
associated with degranulation, expression of CD44, a marker indicating a memory cell
phenotype [158], was also assessed. As shown in Fig. 8 (a and b), a significantly higher
percentage of CD8+CD44+ T lymphocytes was found in HPV+/- mice as compared with
HPV-/- animals (P � 0.01). Ptaquiloside-treated HPV+/- mice presented a significantly
reduced percentage of CD8+CD44+ T lymphocytes compared with untreated HPV+/- mice
(P � 0.05). In fact, group 3 did not show statistically different values to group 1 (P � 0.05).
40
Fig. 8: Percentage of CD8 +CD44+ T cells within total CD8 + T cells obtained from chest skin samples from WT a nd K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. Numbers within graphs correspond to the percentage of the gated populatio n. b – Percentages of CD8 +CD44+ T cells in gated CD8 + T cells were determined by flow cytometry after skin tissue collection and enzymatic digestion. Chest skin sam ples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Group 3, n = 5. Each dot represents an individual animal. B ars represent the mean value in each group. * P � 0.05;** P � 0.01.
Discussion
CD8+ T lymphocytes are important effectors of the cell-mediated immune
response, especially against virus-infected or tumour cells [117]. Upon activation these
cells may degranulate, releasing a set of lytic enzymes capable of destroying target cells
[159]. CD107a is a lysosome-associated membrane protein which can be used as
surrogate marker of degranulation [145]. In CD8+ T lymphocytes, CD107a reaches the cell
surface when lytic granules are exocytosed, thus exposing its membrane proteins [144].
As CD8+ T cells need to be activated in order to release cytotoxic granules, the presence
of CD107a at the cell surface is useful to determine their activation status [145].
Additionally, CD8+ T cells may also acquire a memory phenotype, allowing a quicker recall
response, as memory T cells can be more easily activated than naive T cells [159]. CD44
is a cell-surface glycoprotein and is currently considered the best marker to identify
memory CD8+ T cells [158]. Furthermore, CD44 is thought to act as a regulator of the
motility of CTL in the tumour microenvironment [160].
41
Although many papillomavirus (including some oncogenic HPV and BPV types)
down-regulate MHC I expression as a strategy to evade immune surveillance mediated by
CTL [154], these cells still play a major role against papillomavirus-induced lesions,
namely in HPV-induced oropharyngeal cancers [155]. Evading the host immune response
is critical for maintaining a long-term infection, allowing tumour progression. In this
context, environmental (e.g. dietary) immunosuppressant products such as bracken and
its toxin, ptaquiloside, may play an important role.
In this work, as expected, WT mice had no HPV-associated lesions, whilst age-
matched K14-HPV16 transgenic mice showed hyperplastic lesions with dysplastic foci or
diffuse dysplasia. It is possible that the diffuse dysplastic lesions observed in ptaquiloside-
treated animals represent a more aggressive stage in multi-step carcinogenesis compared
with the often focal dysplasia observed in untreated animals. However, the small number
of animals that survived the experimental period and the absence of obviously invasive
lesions do not allow us to firmly conclude that ptaquiloside enhanced tumour progression.
The increasing aggressiveness of dysplastic versus hyperplastic lesions was
accompanied by an increased bulk of infiltrating immune cells.
These results indicate that HPV-induced hyperplastic and dysplastic skin lesions in
K14-HPV16 transgenic mice show increased infiltration of CD8+ T cells when compared to
normal skin from age-matched WT mice. Ptaquiloside reduced the CD8+ T cell infiltration,
although only slightly (P > 0.05). Nevertheless, ptaquiloside did induce a significant
reduction in CD107a+ and CD44+ CD8+ T cells.
Altogether, these data show that cytotoxic T cells migrate to the site of HPV-
induced lesions, are activated to degranulate and acquire a memory-phenotype and, that
ptaquiloside is capable of impairing the function of these cells, explicitly by inhibiting
cellular activation and memory phenotype acquisition. The molecular mechanism behind
this impairment should be an alluring topic for future research.
Grazing animals and human populations worldwide are easily exposed to bracken
and its illudane toxins, including ptaquiloside and other related compounds [123]. Bracken
is an abundant weed, especially in poorer pastures, and ptaquiloside accumulates in
bovine and ovine milk and meat [161-163], besides contaminating underground waters
[164]. Moreover, some human populations in different countries consume bracken shoots
(known as croziers, broto de samambaia in Portuguese or warabi in Japanese) as part of
their daily diet [123]. In China, the production of dry Pteridium aquilinum var. latiusculum
for food uses (locally known as juecai) was estimated to involve approximately 1000
companies in a business worth 300 million USD per annum [165]. This is considered the
most widely-consumed fern in China, with an estimated annual production of 1200 tons in
the Zhouzhou county, province of Hunan. Bracken and ptaquiloside are well-known
42
carcinogens [128, 166-169] but also show important immunotoxic properties. The present
ptaquiloside yield is in agreement with other previous studies using samples from the
same area [128]. Ptaquiloside extreme instability makes its isolation, manipulation and
administration very challenging, as it easily originates the inactive pterosin B [157].
However, the typical toxicities and dramatic mortality rate now induced leave no doubts as
to its activity. The dosage now employed was similar to the one used intraperitoneally on
CD-1 mice [128]. However, the transgenic K14-HPV16 mouse strain shows added frailty
due to its characteristic lesions, and this is likely to explain the higher mortality now
observed.
Bracken induces other immunosuppressive effects involving innate immune cells.
These include neutropenia and the down-regulation of NK cell activity [127]. This second
effect was recently proposed to contribute to urethane-induced lung carcinogenesis [170].
However, due to ptaquiloside scarcity and instability, studies on bracken toxicology
frequently resort to complex and poorly characterized plant extracts, thereby
compromising the study's ability to draw significant conclusions. The present study is the
first to address the immunosuppressive effects of ptaquiloside in a relevant model of
papillomavirus-induced cancer.
Over the years, the K14-HPV16 transgenic mouse model emerged as a valuable in
vivo mock-up for the study of HPV-induced multi-stage carcinogenesis [73, 74].
Importantly, the expression of viral genes is targeted to basal keratinocytes, the cellular
type affected by natural papillomavirus infections. The high histological resemblance
between the lesions taking place in transgenic mice and the ones occurring in human
patients support the utility of this animal model [76]. Furthermore, K14-HPV16 mice are
useful to study the HPV-associated immune response that accompanies carcinogenesis
[93, 113, 114].
The knowledge on the immune response surrounding papillomavirus-induced
carcinogenesis is in constant growth but the cell-mediated immune response against
neoplastic and pre-neoplastic lesions remains poorly understood. This lack of knowledge
may be holding back the development of more effective therapeutic strategies, including
therapeutic vaccines.
Conclusion
The results confirm the role of ptaquiloside as an immunosuppressive toxin,
capable of enhancing the immune evasion strategies characteristic of papillomavirus-
transformed cells. Ptaquiloside reduces CD8+ T cell infiltration of papillomavirus-induced
lesions, as well as the effector and memory functions of these cells, as shown by a
43
reduced expression of CD107a and CD44. These findings are of particular concern in the
context of oropharyngeal high-risk papillomavirus infections, where dietary toxicants like
ptaquiloside may promote tumour progression and aggressiveness.
44
45
Chapter 5: Celecoxib promotes degranulation of CD8 + T
cells in HPV-induced lesions of transgenic mice 2
2 The contents of this chapter were adapted from:
- Santos C., Neto T., Ferrerinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Celecoxib promotes degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice. Submitted to the journal Antiviral Research with the number AVR-D-16-00002.
46
47
Introduction
Human papillomavirus (HPV) has been described as an etiologic factor of several
diseases, such as cervical and other anogenital cancers, and a subset of HPV-positive
head and neck cancers [4, 6, 141, 142]. Oropharyngeal cancers have lately drawn
considerable attention, given their growing incidence [152]. In order for a HPV-associated
neoplasm to develop, a persistent infection by an oncogenic HPV type (mostly types 16
and 18) is mandatory [3]. Only in a minority of cases, in which the immune system fails to
eradicate the virus, will the infection progress to cancer [2].
Cytotoxic T lymphocytes (CTL), differentiated from CD8+ T cells, play an important
role in the complex immune response which accompanies HPV-induced carcinogenesis
[11, 148]. CD8+ T cells are recruited to the lesions microenvironment by chemokines
released by stromal and/or innate immune cells on site [149]. In the presence of activator
stimuli, differentiated CTL can specifically target virus-infected and tumour cells, which are
eliminated either through the release of lytic granules, engagement of death receptors
(e.g. FAS-FASL or TRAIL) or following interferon-γ-dependent mechanisms [116-118]. In
particular, CTL are critical to control cervical lesions and were recently demonstrated to
drive the immune response in patients following the administration of an experimental
therapeutic vaccine [171]. Recent studies have also shown the number of CD8+ T cells to
be an important independent prognostic marker in HPV-positive head and neck cancer
patients, while high CD8+ T cell levels correlate with a better prognosis, enhancing overall
survival as well as progression-free survival [155, 172, 173]. Understanding and
enhancing CTL function in patients with HPV-induced malignancies seems, therefore, a
priority for cancer therapy.
It has recently been suggested that cyclooxygenase-2 (COX-2) and its product
prostaglandin E2 down-regulate the function of activated CD8+ T cells and induce their
senescence [174]. This effect may explain how the abrogation of COX-2 signalling
reduces tumour growth in mouse models of glioma [175] and mammary cancer [176]. In
light of these findings, it is tempting to study whether CD8+ T cells present in HPV-induced
lesions are affected by COX-2 inhibition.
In this study, the aim was to examine the effect of a selective COX-2 inhibitor,
celecoxib, on the infiltration and activation of CD8+ T lymphocytes in HPV-induced lesions.
For this purpose, chest skin lesions of K14-HPV16 transgenic mice were employed [73].
These mice have been designed to target the expression of HPV16 oncogenes to skin
and keratinized mucosal keratinocytes. The multi-stage disease progression in these mice
greatly resembles HPV-associated disease in cancer patients, both morphologically and
48
molecularly [76], making K14-HPV16 mice a particularly useful model for HPV research.
Material & Methods
Animals
Generation of K14/HPV16 mice on a FVB/n background has been previously
reported [73]. K14-HPV16 transgenic mice were kindly donated by Drs. Jeffrey Arbeit and
Douglas Hanahan (University of California) through the USA National Cancer Institute
Mouse Repository. The animal experiments were approved by the Universidade de Trás-
os-Montes e Alto Douro Ethics Committee (10/2013) and the Portuguese General
Veterinary Directorate (approval no. 0421/000/000/2014). Animals were maintained and
bred according to Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive
2010/63/EU) legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark
cycle (12h light/12h dark) and relative humidity (50 ± 10 %), using corncob bedding. Food
and water were provided ad libitum.
Mice genotyping
Animals were genotyped at weaning, using tail tip samples as described previously
[113, 114]. Briefly, nucleic acids were extracted and DNA quality and purity were
assessed. HPV16-E6 and -E2 genes were amplified to confirm the presence of HPV DNA
and a fragment of mouse β-globin was also amplified as endogenous control. Lengths of
the amplicons were confirmed by agarose gel electrophoresis. Only hemizygous females
were used for the transgenic mouse groups in the experiment.
Experimental Design
Fifty-four 18 to 20 weeks-old female mice were divided into four experimental
groups, according to their genotype and taking into consideration that HPV transgenes
and celecoxib (CXB) administration could induce some mortality: group 1 (HPV16-/-
untreated animals, n = 12), group 2 (HPV16+/- untreated animals, n = 12), group 3
(HPV16-/- CXB-treated animals, n = 15) and group 4 (HPV16+/- CXB-treated animals, n =
15). All surviving mice were humanely euthanized at 24-26 weeks of age by
intraperitoneal pentobarbital overdose, followed by exsanguination by cardiac puncture.
Chest skin samples (approximately 4 cm2) were collected for flow cytometry analysis and
matched skin samples were collected for histological analysis.
49
Celecoxib administration
Celecoxib (Pfizer, New York, NY) was dissolved in drinking water at a
concentration of 0.5 mg/ml, estimating an average daily water intake of 5.0ml per mouse,
and a dose of 46.7 mg/kg/day and 2.5 mg/animal/day in an average mouse weighting 30
g. This is a well-tolerated moderate dose, as shown in previous assays [177]. However,
K14-HPV16 animals dramatically increased their water intake when CXB was added
(possibly due to the highly palatable lactose present in the vehicle) reaching up to 15 ml
per animal. The CXB concentration was thus reduced from the start of the third week
onwards down to 0.2 mg/ml, resulting in a decrease in consumption and an effective dose
of 93 mg/kg/day and 2.8 mg/animal/day. The average dose during the overall
experimental period was thus 124 mg/kg/day and 3.72 mg/animal/day.
Histological Analysis
Skin samples were fixated in 10% neutral buffered formalin for 48 hours. Samples
were dehydrated through graded alcohols and xylene and paraffin embedded in an
automatic STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with
haematoxylin-eosin (H&E) for histological evaluation on light microscopy. Skin samples
were classified as normal skin, epidermal hyperplasia and epidermal dysplasia.
Preparation of Single-Cell Suspensions
Chest skin samples were cut into small pieces after excessive blood vessels and
fat tissue removal. Skin fragments were incubated for 2 hours with 125 U/ml type I
collagenase (Gibco, Life Technologies, Paisley, UK) in RPMI-1640 medium
complemented with 1% glutamine, 1% penicillin-streptomycin-amphotericin B, 1% HEPES
buffer (all from Sigma, St. Louis, MO) and 10% foetal bovine serum (BioWest, Nuaillé,
France) at 37 ºC and 150 rpm in a 3031 orbital incubator (GFL, Burgwedel, Germany).
Subsequently, the resulting cell suspension was filtered and centrifuged at 300 � for 10
min at 4 ºC. Cells were ressuspended in phosphate buffered saline containing 1% bovine
serum albumin and 20 mM sodium azide followed by flow cytometry analysis.
Immunophenotyping
Following cell isolation, the surface phenotype of the collected cells was assessed
by flow cytometry using specific monoclonal antibodies (mAb). Cells were incubated with
anti-mouse CD16/CD32 mAb for FcγR blocking, to prevent non-specific antibody binding.
Next, cells were incubated with anti-CD8 mAb phycoerythrin-cychrome 5-conjugate (clone
53-6.7, BD Biosciences, San Diego, CA) and anti-CD107a (LAMP1) mAb phycoerythrin-
50
conjugate (clone eBio1d4b, eBioscience, San Diego, CA). Following extracellular staining,
the cells were washed, fixed in 2% formaldehyde and washed with phosphate buffered
saline containing 1% bovine serum albumin and 20 mM sodium azide. Antibody-labelled
cells were analysed in an EPICS XL flow cytometer using the EXPO32ADC software
(Beckman Coulter, Miami, FL). The assembled data files were analysed using the FlowJo
software v10.0.7 (FLOWJO, LLC, Ashland, OR).
Statistical Analysis
Statistical analyses were performed using the GraphPad software (version 6.0,
GraphPad Software, Inc. La Jolla, CA). In column and dot graphs each point represents
individual mice, while bars represent the mean for the respective group. Statistical
analysis between group pairs was done using the Mann-Whitney test.
Results
General findings
All K14-HPV16 mice showed typical cutaneous changes, including diffuse
hyperkeratosis and erythema. Celecoxib-treated HPV+/- mice (group 4) showed significant
mortality: 10 out of 15 mice (66.7%) succumbed before the end of the study. All mice in
groups 1, 2 and 3 survived until the end of the study.
Histological analysis
Histological analysis of skin samples (Table 2) showed that all WT mice, groups 1
and 3, presented normal skin histology (Fig. 9a). In groups 2 and 4 (untreated and CXB-
treated HPV+/- mice, respectively), the totality of mice showed simple to papillary, diffuse,
variably severe epidermal hyperplasia extending to the follicular infundibula and
papillomatosis with orthokeratotic hyperkeratosis (Fig. 9b). Additionally, 2 animals (16.7%)
from group 2 presented multifocal epidermal dysplastic foci with parakeratotic
hyperkeratosis within the hyperplastic background (Fig. 9c). There were signs of mild
inflammation, with a small amount of macrophages, mast cells and lymphocytes in the
superficial dermis. Dysplastic foci were associated with increased numbers of leukocytes
infiltrating the dermo-epidermic junction and with intense angiogenesis.
51
Table 2: Histological classification of HPV16-induced skin l esions from 24-26 weeks-old female mice. Celecoxib –
CXB.
Group Cutaneous lesions
Incidence (%)
Normal skin Epidermal hyperplasia Epidermal dysplasia
1 (HPV-/-, n = 12) 12/12 (100%) 0/0 (0%) 0/0 (0%)
2 (HPV+/-, n = 12) 0/0 (0%) 12/12 (100%) 2/12 (16.7%)
3 (HPV-/- + CXB, n = 15) 15/15 (100%) 0/0 (0%) 0/0 (0%)
4 (HPV+/-+ CXB, n = 5) 0/0 (0%) 5/5 (100%) 0/0 (0%)
Fig. 9: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – WT animal; Normal skin histology, 100 �. b – CXB-treated HPV16 +/- animal; Epidermal hyperplasia extending to the fol licular infundibulum and isthmus, 100 �. c – Untreated HPV16 +/- mouse; Epidermal dysplasia, 200 �. Note marked parakeratotic hyperkeratosis, loss of cell polarity, enhanced ani sokaryosis and mitotic activity. The dermal-epiderm al junction is obscured by severe inflammatory cell infiltration.
CTL infiltration and activation in celecoxib-treate d mice
With the purpose to study CD8+ T cell infiltration in HPV-induced lesions, lymphoid
cells from mice chest skin tissue were isolated and a flow cytometry analysis of the
recovered cells was performed (Fig. 10a). Skin samples from untreated HPV+/- mice
showed a significantly higher percentage of CD8+ T cells compared with untreated WT
animals (P � 0.01) (Fig. 10b). Also, although statistical significance was not achieved,
CXB-treated WT mice showed decreased CD8+ T cell infiltration when compared to CXB-
treated HPV+/- animals and the same was observed between samples from WT mice
treated with CXB (group 3) and untreated WT animals (group 1) (Fig. 10b). Moreover,
HPV+/- mice treated with CXB (group 4) showed less CD8+ T lymphocytes compared with
untreated transgenic animals (group 2) (P � 0.01) (Fig. 10b).
Next, the objective was to study whether the CD8+ T lymphocytes found in mouse
skin samples showed signs of cytotoxic activity (Fig. 10a). Thus, expression of CD107a
(LAMP1) at the cell surface was assessed by flow cytometry. CD107a reaches the surface
of CTL when lytic granules are released, exposing their membrane proteins during
52
exocytosis [144]. Thus, this lysosome-associated membrane protein is frequently used as
an immunological marker of CTL degranulation [145]. Fig. 10c shows a significantly higher
percentage of CD8+CD107a+ cells in samples from HPV+/- mice treated with CXB (group
4) when compared with untreated HPV+/- (group 2) (P � 0.01).
Fig. 10: Percentage of CD8 + and CD8+CD107a+ T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analysis of the gating strategy empl oyed. Numbers within graphs correspond to the percentage of the gated population. b – Percentages of CD8 + T cells and c – percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after sk in tissue collection and digestion with collagenase. Chest sk in samples were collected from WT and HPV +/- mice (Groups 1 and 2, respectively) and CXB-treated (CXB) WT and HPV+/- mice (Groups 3 and 4, respectively) at 24-26 weeks of age. Group 1, n = 5; Group 2, n = 5; Group 3, n = 5 ; Group 4, n = 5. Each dot represents an individual animal. Bars represent the mean value in each group. ** P � 0.01.
53
Discussion
The development of HPV-associated malignancies depends on a persistent HPV
infection. Ultimately, the ability of the immune system to eliminate the virus is the key
element to decide whether a HPV infection is cleared or evolves to cancer [2]. HPV
presents well-known mechanisms to evade host immunity and delay its elimination, thus
facilitating viral persistence [154]. When the virus is detected, an innate immune response
occurs, and leads to the development of an adaptive immune response. A fundamental
part of this adaptive response is cell-mediated immunity, characterized by the activity of a
vast number of CD8+ and CD4+ T lymphocytes [39]. In fact, infiltration by these cells in
HPV-associated lesions has already been shown to lead to regression [148, 178]. CTL
infiltration drives the response induced by an experimental therapeutic vaccine in patients
with cervical intraepithelial lesions [171] and correlates with a better prognosis in patients
with HPV-positive head and neck cancer [155, 172, 173].
Chronic inflammation is a key feature associated with carcinogenesis, namely in
the case of HPV infection [8]. COX-2 is an enzyme with an imperative role in the
metabolism of arachidonic acid, which leads to the production of prostaglandins, which in
turn promote inflammation [129]. In fact, COX-2 is over-expressed in several
malignancies, including cervical cancer [130-134]. As inflammation is known to contribute
for cancer progression [8], COX-2 inhibition is expected to result in tumour growth
inhibition whilst reducing inflammation. Some non-steroidal anti-inflammatory drugs, like
aspirin and ibuprofen, have already shown promising results as anti-tumour therapy in
both patients and pre-clinical animal models [179-182]. However, these drugs have very
low specificity. Selective COX-2 inhibitors such as CXB and rofecoxib have already been
used to prevent the development of colorectal adenomas [183, 184]. Furthermore, this
COX-2 inhibitor has been showing promising results in a tumour model of human colon
cancer when combined with chemotherapeutic drugs [185].
In this study, the effect of the selective COX-2 inhibitor CXB over the trafficking
and activation of CD8+ T lymphocytes was examined in the K14-HPV16 mouse model, a
proper model to study HPV-induced carcinogenesis due to its great similarities with the
human clinical disease [76]. High mortality was observed among CXB-treated animals,
presumably due to CXB-related toxicity.
The results presented herein show that CXB reduces the number of tumour-
infiltrating CD8+ T cells when compared with untreated mice. Still, despite the decrease in
cell numbers, CXB-treated mice have a higher percentage of activated and degranulating
CTL compared with untreated animals. These findings suggest that CXB reduces the
number of tumour-infiltrating CD8+ T cells while enhancing their effector functions. These
54
data are in agreement with a previous study using glioma-bearing mice where COX2-/-
mice had increasing percentages of tumour-infiltrating CD8+CD107a+ lymphocytes [175].
This may be explained by another study reporting that COX-2 activity leads to CD8+ T cell
senescence and this trajectory may be opposed by COX-2 inhibition, as shown by
increased levels of CD28 and interleukin-2 in CD8+ T cells [174].
Furthermore, hyperplastic epidermal lesions were evident in all surviving
transgenic mice, whilst all WT animals presented normal skin histology, as expected.
Multifocal dysplastic epidermal lesions were restricted to untreated HPV+/- animals. These
findings suggest that CXB blocked tumour progression at the hyperplastic stage, but the
small number of dysplastic lesions observed does not allow for any definitive conclusions.
In fact, COX-2 inhibition boosted the efficacy of a DNA vaccine expressing the HPV E7
oncogene, by enhancing tumour-infiltrating CD8+ T cells and slowing tumour growth [186].
However, this study was performed in mice bearing allografted TC1 lung cells
immortalized by the HPV16 E6 and E7 oncogenes and transformed by the c-Ha-ras
oncogene. Comparisons between this model and K14-HPV16 mice are limited, because
allografts do not reproduce HPV-associated multi-step carcinogenesis, being directly
implanted in the subcutis with a fully malignant phenotype. Also, keratinocytes and not
lung cells are the targets for papillomavirus infection.
It remains unclear whether CTL are activated at regional lymph nodes or at the
lesion location and this would be an interesting point to address in the future. The results
presented herein suggest that CXB induces augmented CTL degranulation in HPV-
induced lesions, possibly contributing to prevent malignant progression in this animal
model.
Conclusion
The present data confirm the potential of CXB to enhance degranulation by
tumour-infiltrating CD8+ T lymphocytes. Moreover, the effect of CXB seems to be more
complex than previously thought, as it also reduced the overall number of tumour-
infiltrating CD8+ T cells. Future studies addressing the impact of COX-2 inhibitors on the
prognosis of patients bearing HPV-induced lesions should take into account both the
number of infiltrating CD8+ T cells and their activation status.
55
Chapter 6: General Discussion and Conclusions
56
57
HPV-associated malignancies are a genuine health problem worldwide, with
emphasis on the increase in the number of HPV-associated oropharyngeal carcinomas
[152]. Prophylactic vaccination remains the most effective method to control HPV
infections. Currently, there are two effective commercialized vaccines: Cervarix™
(GlaxoSmithKline Biologicals, Rixensart, Belgium), a bivalent HPV16/18 vaccine; and
Gardasil™ (Merck Vaccines, West Point, PA), a quadrivalent HPV6/11/16/18 vaccine.
Both are HPV L1 virus-like particles (VLP) vaccines and they act by boosting the
production of neutralising antibodies directed against the L1 capsid protein [187]. Despite
their effectiveness, these vaccines present a few limitations, as the virus-type restriction.
The first is restricted to two HPV types (bivalent) and the second is restricted to four types
(quadrivalent). Looking to increase type-specific protection, Merck managed to create the
nonavalent HPV L1 VLP vaccine by adding the VLPs from five oncogenic HPV types (31,
33, 45, 52 and 58) in addition to the four types used in the quadrivalent vaccine. This
resulted in a 20% increase in protection, in addition to the 70% obtained from the
quadrivalent vaccine, in a total of approximately 90% protection against cervival cancer
[188]. However, despite these advances in disease prevention, an effective therapeutic
vaccine against HPV-induced malignancies remains absent.
In order to develop a therapeutic strategy, it is necessary to gather a large amount
of information on the features of disease progression as well as of the events occurring in
the lesions microenvironment. It is accepted that the carcinogenesis process induced by a
HPV infection is marked by the existence of a prominent immune response [11, 18]. Thus,
a great deal of cellular populations and chemical factors are present and play a specific
role in carcinogenesis, either promoting or impairing it. At the end, it should be the
balance between all pro- and anti-tumour stimuli that will decide whether the viral infection
is cleared or is allowed to progress towards malignancy. Therefore, a therapeutic vaccine
could possible enhance the action of anti-tumour effectors and/or inhibit the function of
known pro-tumour mediators.
Cytotoxic CD8+ T lymphocytes, for instance, are the main cells associated with the
elimination of virus-infected and transformed cells. These cells are part of the adaptive
immune response and depend on antigen presentation by MHC I molecules and other co-
stimulatory signals to become activated and exert their functions [116, 117]. However,
during the course of HPV infection, expression of MHC I molecules is usually down-
regulated. This is one of the most common immune evasion mechanisms employed by
the HPV E5 oncoprotein [154]. Infiltration of CD8+ T cells in HPV-positive lesions has
already been associated with better prognosis when compared with HPV-negative ones
[155, 172, 173, 189]. Thus, boosting the activity of CD8+ T lymphocytes seems a possible
therapeutic strategy to eliminate HPV-associated lesions. Still, the HPV immune evasion
58
mechanisms, namely MHC I molecules down-regulation, should by some means be
reversed in order to increase the efficacy of the CTL boosting.
Nevertheless, modulation of a lesion microenvironment (particularly, the CD8+ T
cells) can be accomplished by a great variety of substances such as toxins or medical
drugs. For instance, humans can become easily exposed to bracken (Pteridium spp.) –
and more importantly to its major toxin, ptaquiloside – through bovine and ovine milk and
meat consumption or ingestion of contaminated water [161-164]. Ptaquiloside can cause
several immunosuppressive events such as neutropenia, diminished NK cell cytotoxicity,
reduced expansion of antigen-specific T lymphocytes and B-cell lymphoproliferative
malignancy [125-128]. However, its effect over CTL or HPV-induced lesions was not
addressed until this moment. In turn, the effect of the selective COX-2 inhibitor celecoxib
over CD8+ T cells has already been examined in a number of studies albeit not in the
context of HPV-induced lesions and with inconsistent results [136-140]. This anti-
inflammatory drug appears as a possible therapeutic drug against inflammation-
associated cancers where COX-2 is over-expressed.
The K14-HPV16 transgenic mouse model stands out as an excellent replica to
study HPV-associated carcinogenesis. Its greatest advantage is the targeted expression
of the entire HPV early region genes to skin and keratinized mucosal keratinocytes by the
cytokeratin-14 promotor. This model expresses the virus proteins in the same cell types
that are infected by HPV in human patients, therefore supporting the use of this model.
Furthermore, the microscopic morphologic similarities between this mouse and the human
disease also validate its appliance in HPV-induced carcinogenesis research [76].
Therefore, the K14-HPV16 mice can be employed in the study of the immune response
against HPV and, more precisely, in the disclosure of the infiltrating cells in the lesions
microenvironment. In addition, this model is appropriate to study the immunomodulatory
effects of multiple substances at the lesions microenvironment. Hence, in this work, these
transgenic mice were utilized to analyse the infiltration and activity of CD8+ T cells in HPV-
induced lesions at different ages and after the treatment with two distinct
immunomodulatory agents.
The results presented herein suggest that CD8+ T lymphocytes heavily infiltrate
HPV-induced lesions. These cells show signs of cytotoxic activity and the number of
degranulating CTL increases in response to more aggressive lesions. However, CTL
infiltration and degranulation were not sufficient to induce lesion regression. This may be
related to the model now employed, in which all keratinocytes express the HPV
oncogenes. In these conditions, even if many neoplastic keratinocytes are cleared by the
immune response, there are no normal cells to replace them, but only an endless
population of transformed cells that will sustain tumour progression.
59
Furthermore, the CD8+ T cell response can be modulated. Ptaquiloside impaired
the cytotoxic activity of these cells and diminished the differentiation of memory CTL.
Apparently, ptaquiloside promoted the development of more aggressive lesions, although
this correlation could not be statistically confirmed. This is likely to constitute another
mechanism through which ptaquiloside contributes to the persistence and the malignant
transformation of viral lesions.
In contrary, celecoxib significantly enhanced the release of lytic granules by CTL,
even though greatly reducing the number of infiltrating lymphocytes. However, increased
cytotoxicity was not perceptibly associated with lesion regression, which may be due to
the model limitations, as outlined above. Nonetheless, the focus of this work was the
characterization of the CD8+ T cell infiltrate. Thus, additional studies are required to
understand why CTL activity is not sufficient to induce the regression of HPV-associated
lesions.
In the near future it will be worth exploring which other immune cellular populations
may also infiltrate HPV-induced lesions and what soluble mediators they could be
producing. This knowledge should provide a better characterization on the pro- and anti-
tumour stimuli in the lesion microenvironment, therefore helping to decide which cells
should be boosted and which should be inhibited in order to attain regression. In
conclusion, this work once more recognizes the value of the K14-HPV16 transgenic
mouse model for HPV-induced carcinogenesis research and it should also be a very good
model for the testing of new therapeutic strategies. Finally, the results presented here are
encouraging in regard to the search for lesion regression, as they show high infiltration
and activity of the most common anti-tumour immune effector cells.
60
61
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