Avaliação de aspectos inatos e adaptativos do sistema imune na psoríase: análise fenotípica e... por Mariana Dias Batista - Versão HTML
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Ag a cél. T
Fumo
Células
dendríticas
naive
plasmocitóides
Migração de
liberam IFN-α
células
IL-12
dendríticas
CXCR3
Migração de
plasmocitóides
Diferenciação
outras células T
em resposta a
Th-1/Tc-1
IFN-γ
Processo
quemerina
Células
inflamatório
CLA
dendríticas
Células NK e
mielóides
NKT liberam
ativadas
IFN-γ
Imunidade Inata
Imunidade Adaptativa
Figura 1.1 Mecanismos patogenéticos na psoríase. Indivíduos geneticamente
predispostos, quando expostos a fatores desencadeantes, iniciam processo de ativação
dos queratinócitos, que culmina com a liberação de IFN-" por células dendríticas
plasmocitóides, que por sua vez estimulam células dendríticas mielóides a apresentar
antígeno ainda não determinado para células T naïve. Estas por sua vez, sob estímulo
de IL-12, se diferenciam para a via Th-1, com expressão de CXCR3 e CLA, e migram
para a pele. Por outro lado, células T naïve sob o estímulo de IL-23 se diferenciam em
células Th-17, que expressam CLA, CCR6 e CCR4 e também contribuem para a
patogênese da doença. Adaptado de: Nestle, 2009 e Nograles, 2008.
16
2 OBJETIVOS
2.1 Objetivos gerais
2.1.1 Investigar se o fenótipo de células NK e células T na psoríase é compatível com
imuno-senescência e maior capacidade efetora
2.1.2 Avaliar características funcionais de células T na psoríase
2.2 Objetivos específicos
2.2.1 Avaliar a expressão do marcador de imunosenescência CD57, do marcador
inibitório de células NK NKG2A e do marcador ativador de células NK NKG2C em
células T e células NK isoladas de pele lesional, não afetada e células mononucleares
de sangue periférico (CMSP) de pacientes com psoríase, comparadas àquelas isoladas
de pele e CMSP de controles sadios
2.2.2 Avaliar a produção das citocinas IL-17A, IL-22, IL-2, TNF-", IFN-#, IL-21, IL-
27 e IL-33 por células T CD4+ e CD8+ isoladas da pele lesional e não afetada de
pacientes com psoríase, após expansão em cultura e estímulo com mitógenos.
17
3 MÉTODOS
3.1 Casuística
Os pacientes foram recrutados no Serviço de Dermatologia do San Francisco General
Hospital, na Universidade da California, São Francisco, após aprovação pelo Comitê de
Ética em Pesquisa daquela instituição e da Universidade de São Paulo (anexos A e B).
Todos os pacientes participantes do estudo preencheram o Termo de Consentimento
Livre e Esclarecido (anexo C). Na primeira fase de recrutamento, foram incluídos 12
pacientes (P1 a P12), que participaram da porção fenotípica do estudo. Em uma fase
subsequente, foram incluídos outros 13 pacientes (F1 a F13), que participaram da
porção de ensaios funcionais de células T, conforme demonstra a tabela 3.1.
Tabela 3.1- Características amostrais e demográficas dos pacientes
Tempo desde o
Subtipo de
diagnóstico
Paciente
Sexo
Idade
psoríase
Gravidade
Amostras Coletadas
Fatores Predisponentes
(anos)
P1
M
22
vulgar
moderada
pele lesional/ pele não afetada/ sangue
estresse/ trauma cutâneo
10
P2
F
35
gutata
moderada
pele lesional/ pele não afetada/ sangue
nenhum
27
P3
M
61
vulgar
moderada
sangue
trauma cutâneo/ inverno
3
P4
F
84
gutata
grave
sangue
estresse/ inverno
20
P5
M
67
gutata
leve
pele lesional/ pele não afetada/ sangue
nenhum
7
P6
M
44
gutata
leve
pele lesional/ pele não afetada
estresse
24
P7
M
21
gutata
leve
pele lesional/ pele não afetada/ sangue
inverno
1
P8
F
41
vulgar
leve
pele lesional/ pele não afetada/ sangue
estresse/ consumo de álcool/ inverno
11
P9
M
37
gutata
leve
pele lesional/ pele não afetada/ sangue
inverno
1
P10
M
67
vulgar
leve
pele lesional/ pele não afetada/ sangue
nenhum
9
P11
M
52
gutata
leve
pele lesional/ pele não afetada/ sangue
nenhum
40
P12
M
61
vulgar
leve
pele lesional/ pele não afetada/ sangue
estresse
10
F1
M
50
vulgar
grave
pele lesional/ pele não afetada
estresse
5
F2
F
53
vulgar
leve
pele lesional/ pele não afetada
estresse/ inverno
38
F3
M
68
vulgar
leve
pele lesional/ pele não afetada
estresse
3
F4
F
53
vulgar
leve
pele lesional/ pele não afetada
trauma cutâneo/ inverno
50
F5
M
40
vulgar
moderada
pele lesional/ pele não afetada
estresse/ inverno/ trauma cutâneo
12
F6
M
46
vulgar
moderada
pele lesional/ pele não afetada
nenhum
14
F7*
F
54
vulgar
moderada
pele lesional/ pele não afetada
estresse/ inverno
12
F8
F
27
vulgar
grave
pele lesional/ pele não afetada/ sangue
estresse/ inverno/ gravidez
8
F9
M
60
vulgar
moderada
pele lesional/ pele não afetada/ sangue
nenhum
42
F10
F
58
vulgar
moderada
pele lesional/ pele não afetada/ sangue
inverno
37
F11
F
48
vulgar
leve
pele lesional/ pele não afetada
estresse/ inverno/ período pré-menstrual
32
F12
F
55
vulgar
grave
pele lesional/ pele não afetada
estresse/ trauma cutâneo
50
F13
F
32
vulgar
grave
pele lesional/ pele não afetada
estresse
19
Os pacientes apresentavam diagnóstico de psoríase em placas ou gutata, com
gravidade classificada com base na área de superfície corporal afetada ( body surface
affected area- BSA), da seguinte maneira: leve- menos de 5%, moderada- 5 a 30%,
grave- mais de 30%. Os pacientes tinham o diagnóstico de psoríase há pelo menos 1
ano, estavam sem tratamento tópico ou sistêmico há pelo menos 6 meses, e foram
recrutados durante sua primeira consulta no Serviço de Dermatologia. Foram
excluídos pacientes que apresentassem outras doenças auto-imunes e infecção pelo
18
HIV, ou que estivessem em qualquer tipo de tratamento tópico ou sistêmico para a
psoríase. Foram coletadas amostras de sangue, pele lesional e pele não afetada dos
pacientes. Para a porção fenotípica do estudo, foi possível coletar amostras pareadas
de pele lesional, não afetada e sangue de 9 dos 12 pacientes. Para os 3 outros
pacientes, coletou-se apenas sangue ou pele, conforme indica a tabela 1. Quando na
citometria os números de células identificados após o gating eram muito pequenos
(abaixo de 30 eventos) os pacientes foram excluídos das análises, conforme
demonstram as figuras nos resultados. Na porção funcional do estudo, foram
coletadas amostras de pele lesional e não afetada de 13 pacientes, e amostras pareadas
de sangue de 3 (tabela 3.1). Foi excluído um paciente da porção funcional do estudo
(F7) devido a baixa pureza após o sorting celular.
As amostras de sangue de 10 controles sadios foram obtidas após consentimento no
Banco de Sangue de Stanford. A mediana de idade dos controles foi de 45 anos,
conforme demonstra a tabela 3.2. As amostras de pele normal de 3 controles sadios
foram obtidas de tecido descartado após abdominoplastia, em pacientes sem histórico
de psoríase ou outras afecções inflamatórias.
3.2 Processamento de amostras de pele
As amostras de pele foram coletadas através de biópsia com punch de 4mm, uma
realizada na borda de lesão ativa de psoríase, e outra em pele não afetada localizada a
pelo menos 5 cm de distância das lesões ativas. As amostras foram incubadas em
RPMI-1640 com colagenase/dispase na concentração de 1 mg/ml (Roche Diagnostics,
Indianapolis, IN, USA), associadas a 10 unidades/ml de DNAse I recombinante
19
(Roche Diagnostics, Indianapolis, IN, USA), a 37°C por 15 minutos, e posteriormente
a 4°C durante 8 horas. Após este período, foi realizada a separação mecânica entre a
epiderme e a derme, e a epiderme foi tratada com tripsina e EDTA (GIBCO
Invitrogen, Carlsbad, CA, USA) em tampão fosfato (PBS), a 37°C por 15 minutos.
Para determinar se havia necessidade de cultura para isolar as células linfo-
monocitárias da pele, após a separação entre epiderme e derme, foi realizado
experimento com amostras de pele normal proveniente de abdominoplastia, no qual se
comparou células provenientes da etapa de digestão com colagenase sem tempo
nenhum de cultura com outros dois grupos: células cultivadas por 48 horas e por 7
dias. Conforme demonstra a figura 3.1, determinou-se que a melhor viabilidade e
separação de células linfo-monocitárias se deu no período de 48 horas de cultura.
250K
250K
250K
200K
200K
200K
150K 83.8
150K
150K
2.81
43.2
SSC-A
SSC-A
SSC-A
100K
100K
100K
50K
50K
50K
0
0
0
0 102
103
104
105
0
103
104
105
0 102
103
104
105
<AARD-A>: LIVE/DEAD
<AARD-A>
<AARD-A>: live/dead
Figura 3.1 – Determinação do tempo de cultura das células isoladas da pele. A-
Citometria realizada a fresco sem cultura. B- 48 horas de cultura. C- 7 dias de cultura
Assim, nas amostras dos pacientes, após a digestão enzimática com colagenase, a
epiderme e a derme foram subsequentemente cultivadas separadamente a 37°C por 48
horas, em RPMI-1640 suplementado com soro humano a 10% (Gemini Bio Products,
Sacramento, CA, USA), penicilina e estreptomicina a 1%, e tampão HEPES a 1 mol/L
(Invitrogen, Carlsbad, CA, USA), em placas de cultura de 6 poços. Após esse período,
as suspensões celulares foram obtidas filtrando o meio de cultura em filtro de 70 µm
(BD, San Jose, CA, USA). Devido aos pequenos números de células obtidos em
contagem no hemocitômetro, as células isoladas da epiderme e da derme foram
combinadas para produzirem números suficientes para a realização de citometria de
20
fluxo na porção fenotípica do estudo, e para sorting de células T CD4+ e CD8+ na
porção funcional do estudo.
Com o objetivo de determinar se o tratamento com colagenase/dispase poderia alterar
o processo de coloração com os fluorocromos para citometria de fluxo, foi realizado
experimento no qual se comparou CMSP tratadas com colagenase/dispase com outras
que não receberam este tratamento, e após citometria de fluxo não houve diferença
entre os grupos.
Foi também realizado experimento piloto com amostra proveniente de
abdominoplastia para determinar se a expressão de receptores observada em células
isoladas a partir de punch de 4mm era representativa da expressão observada após
digestão enzimática de amostra maior, de 3x3 cm de pele. Não foi observada
diferença entre as amostras, sugerindo que o punch de 4mm foi representativo do
ambiente inflamatório local.
3.3 Processamento de amostras de sangue
As amostras de sangue foram coletadas em tubos vacutainer com EDTA, e as CMSP
foram isoladas através do gradiente de Ficoll-Hypaque, no período de até 6 horas após
a coleta. Após a separação, as CMSP foram lavadas 2 vezes com PBS, e re-suspensas
em tampão FACS para citometria de fluxo (PBS com albumina bovina 0.5% e 2mM
EDTA).
3.4 Citometria de fluxo
Para a porção fenotípica do estudo, após o isolamento das suspensões celulares, foi
realizada fenotipagem para citometria de fluxo. Primeiramente, os receptores Fc nas
células foram bloqueados com IgG humana 10 µg/ml (Sigma Aldrich, S. Louis, MI,
USA), durante 20 minutos no gelo. Posteriormente, as células foram marcadas com
anticorpos monoclonais, descritos na tabela 3.3, por 30 minutos no gelo, depois
lavadas duas vezes com tampão FACS, e fixadas com paraformaldeído a 2%
(Touisimis, Rockville, MD) em PBS. As amostras foram adquiridas no citômetro de
fluxo LSR II (BD Biosciences). Por fim, os dados gerados foram analisados através
do software FlowJo versão 8.8.6 (Tree Star, San Carlos, CA, USA).
21
As janelas de aquisição ( gating) utilizadas estão representadas na figura 3.2.
Primeiramente, foram selecionadas as células únicas (área e altura do tamanho
celular), em seguida foi utilizada a janela de aquisição baseada na granularidade e
tamanho celular. Posteriormente, foram excluídas células mortas através de seleção de
células Amine-Acqua negativas, e novamente por tamanho e granularidade
identificou-se a população de linfócitos. Para identificação mais fidedigna dos
linfócitos, foram selecionadas as células CD45+. Posteriormente, em gráfico de CD3
X CD14/CD19, foram selecionadas 2 subpopulações: CD3+CD14-CD19-, para
células T, que foram então subdivididas em CD4+ e CD8+, e CD3-CD14-CD19-, para
células NK, que foram então subdivididas de acordo com a expressão de CD56 e
CD16. Os marcadores CD14 e CD19 foram utilizados para excluir monócitos e
linfócitos B, respectivamente.
22
Figura 3.2- Estratégia de gating para citometria de fluxo
3.5 Sorting de células T CD4+ e CD8+
Após o isolamento a fresco de CMSP e de células provenientes das amostras de pele,
foi realizada marcação com anticorpos monoclonais, conforme indica a tabela 3.4, por
30 minutos no gelo. As células foram lavadas duas vezes com tampão FACS, e foi
realizado sorting celular no citômetro FACS Aria (BD Biosciences, San Jose, CA,
USA). A estratégia de aquisição realizada envolveu seleção de células únicas, células
CD45+, CD3+CD4+ ou CD3+CD8+. No primeiro experimento (n=6), não houve
viabilidade das células T CD3+CD4+ após o sorting, e as etapas subsequentes foram
realizadas apenas com as células T CD3+CD8+. No segundo experimento (n=7),
células T CD3+CD4+ e CD3+CD8+ foram isoladas e utilizadas nas etapas
subsequentes. A pureza de todos os procedimentos de sorting foi superior a 95%,
23
exceto para o paciente F7, cujas amostras obtiveram pureza de 75% e foram excluídas
das etapas subsequentes.
3.6 Cultura de células T
Células T CD4+ e CD8+ isoladas de pele lesional, não afetada e sangue de pacientes
com psoríase foram cultivadas por 8 horas a 37°C e 5% CO2, numa placa de cultura
de 96 poços, fundo em U. Cada amostra foi dividida em dois, e a metade das células
foi estimulada com acetato de forbol miristato (PMA) (50ng/ml) e ionomicina
(500ng/ml) (Sigma-Aldrich, St. Louis, MO, USA), e a outra metade das células não
recebeu a estimulação. Após o período de 8 horas, a placa foi centrifugada e os
sobrenadantes foram retirados. As células foram lavadas com PBS, e meio de cultura
contendo IL-2 400 UI/ml foi adicionado. As células foram então para cultura por 7
dias a 37°C e 5% CO2. No sétimo dia as placas foram centrifugadas e os
sobrenadantes foram congelados a -20°C para uso subsequente nos ensaios de
multiplex.
3.7 Ensaios de Multiplex
A produção de IFN%#, TNF%", IL-2, IL-17A, IL-22, IL-21, IL-27 e IL-33 foi avaliada
através do ensaio de multiplex para citocinas denominado Legendplex (Biolegend,
San Diego, CA, USA). Foi montada placa sob encomenda para o ensaio, que continha
todas as citocinas simultaneamente. A presença das citocinas foi avaliada nos
sobrenadantes coletados após a cultura de células T CD4+ e CD8+ previamente
isoladas por sorting. As amostras foram adquiridas no analizador Labscan 200
(Luminex, Austin, TX, USA), utilizando o software Bio-Plex manager versão 6.0
(Bio-Rad, Hercules, CA, USA).
24
3.8 Análise Estatística
As análises estatísticas foram realizadas no software Prism versão 4.0a (GraphPad, La
Jolla, CA, USA). As análises foram realizadas através do teste não-paramétrico de
Mann-Whitney quando se comparavam 2 grupos, ou do teste não paramétrico de
Kruskal-Wallis quando se comparavam 3 grupos. O teste não-paramétrico de
Wilcoxon foi utilizado nas análises de amostras pareadas. Foi utilizado valor mínimo
de significância estatística de p=0.05. O software SPICE versão 5.2 (Exon NIAID,
Bethesda, MD, USA) foi utilizado para as análises de co-expressão realizadas na
porção fenotípica do estudo.
25
4 RESULTADOS
4.1 Manuscrito de artigo científico sobre células NK
4.1.1 Folha de Rosto
Title: Skewed Distribution of Natural Killer Cells in Psoriatic Skin Lesions
Running Head: NK Cells in Psoriatic Skin Lesions
Authors: M. D. Batista, MD1,2, E. L. Ho, MD, PhD1#, P. J. Kuebler, PharmD1, J. M.
Milush, PhD1, L. L. Lanier, PhD3, E. G. Kallas, MD, PhD2,4, V. A. York, BS1, D.
Chang, MD5, W. Liao, MD6, P. Unemori, MD6, K. S. Leslie, M.B.,B.S., FRCP6, T.
Maurer, MD6, D. F. Nixon, MD, PhD1*
Affiliations: 1Division of Experimental Medicine, Department of Medicine,
University of California San Francisco, San Francisco, California, USA, 2Division of
Clinical Immunology and Allergy, School of Medicine, University of São Paulo, São
Paulo, Brazil, 3Department of Microbiology and Immunology and the Cancer
Research Institute, University of California, San Francisco, San Francisco, USA,
4Instituto de Investigação em Imunologia, University of São Paulo, São Paulo, Brazil,
5California Pacific Medical Center - Davies Campus, San Francisco, CA, USA,
6Department of Dermatology, University of California, San Francisco, USA. #
Current Address: Department of Neurology, University of Washington, Seattle, WA.
O presente manuscrito foi submetido e encontra-se em revisão no periódico
Experimental Dermatology (Anexo D).
26
4.1.2 Abstract
Background: Psoriasis is a hyper-proliferative inflammatory disease of the skin in
which immunological mechanisms play a direct role in disease pathogenesis. There
have been limited studies of natural killer (NK) cells in psoriasis.
Objectives: To examine the phenotype and functional role of NK cells in skin biopsies
and peripheral blood mononuclear cells from patients with psoriasis and healthy
controls.
Methods: CD56+CD16- and CD56+CD16+ NK cells were isolated from lesional skin,
unaffected skin and PBMC of psoriasis patients, and normal skin and PBMC from
healthy controls. The expression of CD57, NKG2A, and NKG2C was assessed by
flow cytometry.
Results: NK cells in psoriatic skin lesions were skewed in their expression of CD57, a
marker of NK cell maturity, with CD57 expression significantly reduced and NKG2A
expression increased on NK cells in lesional and unaffected skin compared to
controls.
Conclusions: These data suggest that NK cells in psoriasis lesions exhibit an
immature phenotype, but further studies are needed to determine the functional role of
these cells in psoriasis.
Key Words: psoriasis, natural killer cells, CD57, immunosenescence
27
4.1.3 Introduction
Psoriasis is a chronic disease of the skin with significant morbidity, where clinical
improvements can be observed with the use of different therapeutic modalities
including topical and systemic immunosuppressive agents, anti-cytokine biological
therapies, and ultraviolet light therapies. Histologically, psoriasis is characterized by
psoriasiform epidermal hyperplasia, parakeratosis, loss of the granular cell layer, and
the formation of spongiform pustules (1).
The etiologic cause of psoriasis is unknown, although certain environmental triggers
(e.g. infection, skin injury, stress, weather, and medications) are thought to contribute
to its onset. The development of psoriatic lesions is thought to be due to the
interaction of these triggers in persons with genetic predisposition (1). The major
genetic determinant of psoriasis is located within the MHC region on chromosome 6,
in a locus known as PSORS1 (2). From this region, HLA-C*06:02 allele is most
strongly associated with psoriasis (3). Considerable genetic variability can be
observed among different subtypes of psoriasis, with guttate psoriasis being most
associated with PSORS1 (4). However, the underlying cellular and molecular
mechanisms leading to the hyper-proliferative inflammatory nature of psoriasis
remain incompletely understood.
Several studies have characterized the cellular and cytokine immune composition of
healthy and psoriatic skin. These studies suggest contributions from both the innate
and adaptive immune systems and their interaction with keratinocytes and other skin
cells in the pathogenesis of psoriasis (5-7). Increasingly, evidence points to a pro-
inflammatory environment with local expansion of activated lymphocytes within
psoriatic lesions, elevated IFN-#, TNF-", IL-17, IL-22, and IL-23, as well as secretion
of antimicrobial peptides such as cathelicidin from keratinocytes (8-10). Although IL-
23 may contribute to disease, a role for IL-12 in psoriasis is also described (11). IL-17
and IL-22 co-producing T cells are also considered important contributors to the pro-
inflammatory milieu associated with psoriasis pathology (8, 12, 13).
28
Natural Killer (NK) cells are lymphocytes at the interface of the innate and adaptive
immune systems. They migrate into sites of infected and damaged tissue in response
to elevated levels of pro-inflammatory chemokines and can further produce
significant amounts of pro-inflammatory cytokines, such as IFN-#. While innate
immune mechanisms may also play a role in the immunopathogenesis of psoriasis, the
role of NK cells in psoriasis is less studied (14-16). The interaction between HLA-C
and Killer cell Immunoglobulin-like Receptors (KIRs) on NK cells has been
investigated in psoriasis, and KIR2DS1 is associated with psoriasis vulgaris and
psoriatic arthritis (17-19). Furthermore, greater expression of HLA-G, a known
inhibitor of NK cell cytolysis, is observed in the skin of psoriasis patients compared to
normal skin (20, 21).
NK cells express a heterogeneous repertoire of germline-encoded receptors that serve
to educate them through an appropriate balance of activating and inhibitory signals as
they mature (22). These receptors also regulate NK cell effector function (23).
Myeloid dendritic cells can influence NK cell function through cytokine secretion
(24), particularly IL-12, which is a potent inducer of IFN-# production by NK cells.
We investigated the expression of the inhibitory receptor NKG2A and the activating
receptor NKG2C, which have a role in immunosurveillance by binding to HLA-E
(25), on NK cells in the skin and blood of patients with psoriasis and healthy controls.
Although both NKG2A and NKG2C bind HLA-E, NKG2C binds with a lower
affinity than NKG2A (25, 26). However, as NK cells mature, they tend to express
more NKG2C and lose NKG2A expression (27).
Although NK cell function (cytotoxicity and cytokine secretion) is related to their
stage of development, both immature CD56brightCD16neg and mature
CD56dimCD16+ NK cell subpopulations secrete IFN-#, which is critical in the
pathogenesis of psoriasis (28). Indeed, one study demonstrated how specific target
cell ligands dictate the qualitative and temporal aspects of NK cell cytokine responses
resulting in graded responses, depending on the multiplicity of activating receptors
engaged. That study suggested the CD56dimCD16+ NK cell subpopulation may be
the most important source of cytokines upon recognition of aberrant target cells (29).
Expression of CD57 identifies terminally differentiated T cells (30). Recently, we,
29
and others, have shown that CD57 is also expressed on highly mature cells within the
CD56dimCD16+ NK cell compartment, became NKG2Chi, and finally acquired
CD57 (31, 32). This suggests that CD57 might provide a marker of "memory" NK
cells (27). These CD57+ NK cells have a poor replication capacity in vitro, but a high
capacity to produce IFN-# following stimulation through the activating receptor CD16
(31, 32). More importantly, CD57+CD56dimCD16+ NK cells are less responsive to
stimulation
by
IL-12
and
IL-18
compared
to
their
less
mature
CD57negCD56dimCD16+ counterparts (32). These data suggest that infiltration by
less mature CD57negCD56dimCD16+ NK cells may fuel the pro-inflammatory
environment as they are more capable of responding to activating cytokines.
In view of our finding that CD57 expression can identify NK cell subsets capable of
producing high amounts of IFN-# in response to pro-inflammatory cytokines (32), and
our observation that a significant amount of the total IFN-# produced in response to
antigenic stimulation in a polyclonal PBMC population can be attributed to NK cells
rather than T cells (33), we examined the phenotype of NK cells in psoriasis by
investigating the distribution of CD57+ NK cells in lesional and non-lesional skin, as
well as peripheral blood of patients with psoriasis compared to controls.
30
4.1.4 Methods
Study design and skin samples
Peripheral blood mononuclear cells (PBMCs) and skin biopsies were collected from
patients with psoriasis (lesional and non-lesional skin) (n=12) and PBMC alone were
collected from healthy controls (n=10). Normal skin from healthy donors was
obtained from discarded tissue following plastic surgery procedures (n=3). All
patients gave informed consent and the study was approved by the UCSF Institutional
Review Board. Patients with mild to severe psoriasis of both guttate or plaque subtype
were included. Severity was assessed based on affected body surface area (BSA), as
follows: mild - less than 5% BSA, moderate 5 - 30% BSA, severe - more than 30%
BSA. Additionally, patients had a history of psoriasis for at least 1 year and were
treatment-free in the last 6 months. Patients undergoing any topical or systemic
therapy were excluded.
Skin sample preparation
Tissue samples from psoriasis patients were collected as a 4-mm punch biopsy. One
punch biopsy was collected from an active psoriasis lesion (close to the lesion
margin) and another from non-lesional skin, at least 5 cm away from an active lesion.
Tissue samples were incubated overnight at 4°C in 1 mg/ml collagenase and dispase
(Roche Diagnostics, Indianapolis, IN, USA) and 10 units/ml recombinant DNAse I
(Roche Diagnostics, Indianapolis, IN, USA) in RPMI-1640. The epidermis was
subsequently treated with 0.25% trypsin and EDTA (GIBCO Invitrogen, Carlsbad,
CA, USA) in PBS at 37°C for 15 minutes. Epidermis and dermis were both cultured
separately for 48 hours at 37°C in RPMI-1640 supplemented with 10% pooled human
serum (Gemini Bio Products, Sacramento, CA, USA), 1% penicillin and
streptomycin, and 1 mol/L HEPES buffer (Invitrogen, Carlsbad, CA, USA) in 6-well
culture plates. Single cell suspensions were obtained after rinsing through a 70 µm
cell strainer (BD, San Jose, CA, USA), and cells isolated from epidermis and dermis
were subsequently combined to produce sufficient cell numbers for flow cytometry.
We also treated PBMC with collagenase and dispase to determine whether NK cell
staining within PBMC was perturbed by this treatment. No differences were observed
(data not shown).
31
Flow cytometry
Twelve-parameter flow cytometry was performed using a LSR II flow cytometer (BD
Biosciences). Fc receptors on cells were first blocked using 10 µg/ml human IgG
(Sigma Aldrich, S. Louis, MI, USA) for 20 minutes on ice. Cells were then stained for
30 minutes on ice with fluorophore-labeled antibodies, washed with FACS buffer
containing PBS, 0.5% bovine serum albumin, and 2mm EDTA, and fixed with 2%
paraformaldehyde in PBS. The data files were analyzed using FlowJo Software
version 8.8.6 (Tree Star, San Carlos, CA, USA).
Statistical Analyses
The statistical analyses were performed using Prism Software version 4.0a
(GraphPad, La Jolla, CA, USA). Groups were compared using the Mann-Whitney
non-parametric test with a minimum significance value of p = 0.05. SPICE software
version 5.2 was used for the analyses presented in Figure 2.
32
4.1.5 Results
Patient Demographics
Twelve subjects with psoriasis were studied, including 9 males and 3 females. The
median age was 48 years (range 21 to 84). With respect to psoriasis subtype, 5
subjects had plaque psoriasis and 7 subjects had guttate psoriasis. The majority of
subjects had mild disease (n = 8), 3 subjects had moderate disease, and 1 subject had
severe disease. Matched skin and PBMC samples were available from 9 out of 12
psoriasis subjects. Healthy control PBMC samples were available for 10 individuals,
with a median age of 45 years. In patients for whom serum was available, CMV
serology was positive. This positivity is representative of the general demographic of
the local population.
Distribution and phenotype of NK cells in lesional and non-lesional skin and
peripheral blood of psoriasis patients compared to healthy controls
We examined the distribution and phenotype of NK cells in skin and peripheral blood
of psoriasis patients and healthy controls. Lymphocytes were identified by forward
and side scatter followed by staining with CD45. Within the CD45+ lymphocyte
subset, both lesional and non-lesional skin of psoriatic patients had similar
frequencies of CD56+CD16+ NK cells; however, in the blood there was a trend toward
an increased frequency of CD56+CD16+ NK cells in patients compared to healthy
controls (Fig. 1a). To assess the maturity of NK cells in the skin and peripheral blood,
we measured CD57 expression on CD56+CD16+ NK cells. The frequency of CD57+
NK cells in lesional skin (less than 10%) was significantly lower than in non-lesional
skin (~ 40%) (Fig. 1b). Interestingly, the non-lesional skin in psoriasis subjects had
significantly fewer CD57+ NK cells than in skin of healthy controls. This difference
was specific to the skin, as no significant difference in CD57+CD56+CD16+ NK cells
was observed in the blood of patients compared to healthy controls (Fig. 1b).
An assessment of inhibitory, NKG2A, and activating, NKG2C, receptor expression
on skin and peripheral blood CD56+CD16+ NK cells revealed a significant expansion
of NKG2A+ NK cells in lesional and non-lesional skin of patients compared to
healthy controls (Fig. 1c). However, the frequency of NKG2A+ NK cells in the
peripheral blood was similar between psoriasis patients and healthy controls. In
contrast, the proportion of NKG2C+ NK cells was similar in both lesional and non-
lesional skin to healthy controls. In the blood, psoriasis patients had a significantly
33
greater frequency of NKG2C+ NK cells compared to healthy controls of similar
median age (Fig. 1d).
When we examined co-expression of CD57, NKG2A, and NKG2C on skin NK cells
we observed a marked difference between normal skin from healthy donors and
lesional and non-lesional skin from psoriasis patients (Fig. 2). The majority of NK
cells from the skin of psoriasis patients only expressed NKG2A, particularly in
lesional skin. In contrast, the majority of NK cells in the skin of healthy donors
contained CD57+ NK cells that were NKG2A- and NKG2C-. These data suggest NK
cells in the skin of psoriasis patients, particularly in the lesion itself, are less mature
NK cells. When we compared plaque and guttate psoriasis for all the measured
parameters, no difference was found (Supplemental Fig. 1).
34
Figure 1: (a) Frequency of CD56+CD16+ NK cells in lesional skin (n=7) and
unaffected skin (n=8) of psoriasis patients and skin from healthy controls (n=3), and
in the blood of psoriasis patients (n=11) and healthy controls (n=10). (b)
CD57+CD56+CD16+ NK cells in skin and the blood. (c) NKG2A expression on
CD56+CD16+ NK cells in skin and the blood. (d) NKG2C expression on
CD56+CD16+ NK cells from skin and PBMC. * = p<0.05, *** = p<0.001. Missing
values correspond to samples where number of events was too low for analysis.
35
Figure 2: Co-expression of CD57, NKG2A, and NKG2C on CD56+CD16+ NK cells
in lesional and unaffected skin of psoriasis patients and normal skin of healthy
controls. Both pie charts and bar graphs are shown. Comparisons are made between
psoriasis groups (lesional and unaffected skin) in relation to the control group (normal
skin). Significant differences (p<0.05) are represented by a (+). A significant
difference between groups was noted in CD57 and NKG2A expression alone and in
NKG2A and NKG2C co-expression. + = p<0.05.
36
Supplemental Figure 1: Lack of difference between plaque and guttate psoriasis for
the expression of markers on CD56+CD16+ NK cells. (a) CD57. (b) NKG2A. (c)
NKG2C.
37
We also examined the distribution and phenotype of CD56+CD16- NK cells. Within
the CD45+ subset, there was a trend toward an increased frequency of CD56+CD16-
NK cells in psoriasis lesional and unaffected skin compared to the skin of healthy
controls (Fig. 3a). No difference was observed in the blood. Similar to what was
observed for the CD56+CD16+ NK cell subset, CD57 expression on CD56+CD16- NK
cells was significantly reduced in lesional and non-lesional skin of patients compared
to normal skin of healthy controls (Fig. 3b). NKG2A expression was more prominent
in lesional skin compared to unaffected skin and normal skin of healthy controls,
although this trend did not meet statistical significance (Fig. 3c). No difference was
observed for NKG2C expression in the skin or blood of psoriasis patients compared to
healthy controls (Fig. 3d). No difference was observed between plaque and guttate
psoriasis (Supplemental Fig. 2).
38
Figure 3: (a) Frequency of CD56+CD16- NK cells in lesional skin (n=10) and
unaffected skin (n=10) of psoriasis patients and skin from healthy controls (n=3) and
in the blood of psoriasis patients and healthy controls. (b) CD57+CD56+CD16- NK
cells in skin and the blood. (c) NKG2A expression on CD56+CD16- NK cells in skin
and the blood. (d) NKG2C expression on CD56+CD16- NK cells from skin and
PBMC. * = p<0.05. Missing values correspond to samples where number of events
was too low for analysis.
39
Supplemental Figure 2: Lack of difference between plaque and guttate psoriasis for
the expression of markers on CD56+CD16- NK cells: (a) CD57. (b) NKG2A. (c)
NKG2C.
40
4.1.6 Discussion
Psoriasis has been largely considered a disease driven by inflammatory processes.
Much recent attention has been given to the role of Th17 and Th22 cell subsets in the
immunopathogenic process (12). Genomic studies have reinforced the importance of
aberrant inflammation by identifying relationships between inflammatory genes and
psoriasis (34, 35). Moreover, the efficacy observed with biologics targeting pro-
inflammatory elements further elucidates the contribution of cytokines such as
TNF%", IL-12, and IL-23 (7). Nevertheless, questions remain about what triggers the
onset of psoriasis that initiates the cascade of inflammation, as well as the persistence
and localization of inflammation to the involved area.
Whether NK cells are involved in the immunopathogenesis of psoriasis has been
much less studied relative to other cell types and inflammatory pathways, although
genetic and immunological analyses support a role for NK cells in psoriasis. Genetic
analyses revealed a susceptibility locus for psoriasis, the A5.1 allele of MICA, a
ligand for NKG2D (36). Others have found a psoriasis SNP between HLA-B and
MICA (37), and more recently MICA*016 was associated with psoriasis (38).
KIR2DS1 is associated with psoriasis vulgaris, and is the only activating KIR receptor
that associates to HLA-Cw6 (16-18). NK cells appear to be concentrated at the
dermal/epidermal junction (14, 28) and may have a clinically relevant role in
psoriasis, as they can function without prior sensitization and possibly act in concert
with myeloid and plasmacytoid dendritic cells (DC) to initiate plaque development.
Activated NK cells are a source of Th1 cytokines (IFN-#) and might initiate events in
psoriasis with subsequent T cell infiltration (14).
In this study, we found variations in NK cell distribution in blood and tissue of
psoriasis patients when compared to healthy subjects without psoriasis. Overall, a
trend toward higher CD56+CD16- NK cell proportions was observed in skin of
psoriasis patients compared to non-psoriasis subjects (Fig. 3a). This could be
explained as a consequence of the ongoing inflammatory process in psoriatic plaques
resulting in upregulation of integrins and chemokines that lead to increased trafficking
to the site of inflammation. CD56brightCD16- NK cells express high levels of the
41
chemokine receptors CXCR3 and CCR5, which can account for preferential homing
to the skin (28). Taken together, the skin of psoriasis patients appears to be primed for
a heightened degree of immune stimulation, at least for those in whom active
psoriasis plaques exist. The similar distribution of NK cells in lesional and non-
lesional tissue and phenotypic differences within the NK cell population suggest
underlying functional differences in NK cell maturation in psoriasis.
We, and others, recently described NK cell differentiation as a progression from a less
mature phenotype displaying high CD56 expression together with NKG2A but
lacking CD16, to a more differentiated phenotype expressing CD16 and CD57 with a
decreasing ability to replicate in vitro (27, 31, 32). Although we observed no
difference between the proportion of CD57+CD56+CD16+ NK cells in the blood of
patients and healthy controls, we observed a substantially lower percentage of
CD57+CD56+CD16+ NK cells in skin from psoriasis plaques compared to control
skin. Interestingly, non-involved skin from patients presented as an intermediate
between the two. CD57-CD56+CD16+ NK cells exhibit increased HLA-DR and
CD27 expression, as well as Ki67 and CD107a, and correspond to activated cells with
a higher turnover and degranulation ability (39). Another possible mechanism that
could explain the preferential localization of CD57- NK cells to lesions is their high
responsiveness to IL-2 stimulation, as psoriasis lesions contain high levels of IL-2
(40). Importantly, CD57-CD56+CD16+ NK cells are also more sensitive and produce
higher amounts of IFN-gamma following cytokine stimulation compared to
CD57+CD56+CD16+ NK cells. Furthermore, CD57-CD56+CD16+ NK cells may
help perpetuate the inflammation by producing high levels of IFN-gamma that
activate surrounding T cells and myeloid cells. In contrast, CD57+CD56+CD16+ NK
cells have increased KIR and granzyme B expression and correspond to more mature
cells, with lower replicative capacity, but are nonetheless functionally active (32, 39).
One previous study has analyzed CD57 expression by immunohistochemistry in
psoriasis skin, and found an increased expression of this marker in lesional skin (41).
However, that study did not identify which cells expressed CD57, and it is possible
that other cell types, such as T cells, which are more abundant in the psoriatic
inflammatory infiltrate, were CD57+.
We observed no differences in the proportion of NK cells positive for NKG2A in the
42
blood; however, there was a significant difference in the proportion of NKG2A+ cells
in lesional skin when compared to non-lesional and normal skin. NKG2A is inducible
by IL-12, which is likely to be highly expressed in the lesions, and might explain why
NKG2A is highly expressed. Alternatively, some groups have proposed that the
acquisition of functional capacities by NK cells, specifically IFN-gamma production,
is correlated with NKG2A expression on CD56+CD16+ NK cells (42). Our results are
in accordance with this observation, as lesional skin NK cells express more NKG2A.
Of interest, the expression of NKG2A was increased on circulating CD8+ T cells
from psoriasis patients, and correlated with PASI scores (43). The proportion of
NKG2C+ cells in blood showed a modest, but statistically significant difference,
whereas skin showed no such difference. This could be due to differences in CMV
serostatus because NK cells expressing NKG2C have been shown to be preferentially
expanded in CMV seropositive individuals. However, we were unable to determine
CMV serostatus on all patients in the study.
Together, these data suggest that psoriasis patients harbor a less differentiated NK cell
population in the skin. This appears to be localized to the skin itself, further