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UNIVERSIDADE PAULISTA
Artigo1: CULTURE AND PROPAGATION OF
MICROSPORIDIA OF VETERINARY
INTEREST-CULTURE OF MICROSPORIDIA
Artigo 2: B-1 CELLS AS A COMPONENT OF THE IMMUNE
RESPONSE AGAINST MURINE ENCEPHALITOZOONOSIS
Tese apresentada ao Programa de Pós-
Graduação em Patologia Ambiental e
Experimental da Universidade Paulista –
UNIP, para obtenção do título de Doutor em
Patologia Ambiental e Experimental.
LIDIANA FLORA VIDÔTO DA COSTA
SÃO PAULO
2015
UNIVERSIDADE PAULISTA
Artigo1: CULTURE AND PROPAGATION OF
MICROSPORIDIA OF VETERINARY
INTEREST-CULTURE OF MICROSPORIDIA
Artigo 2: B-1 CELLS AS A COMPONENT OF THE IMMUNE
RESPONSE AGAINST MURINE ENCEPHALITOZOONOSIS
Tese apresentada ao Programa de Pós-
Graduação em Patologia Ambiental e
Experimental da Universidade Paulista –
UNIP, para obtenção do título de Doutor em
Patologia Ambiental e Experimental, sob a
orientação da Profª Drª Maria Anete Lallo e
Co-orientação da Profª Drª Anuska Marcelino
Alvares Saraiva
LIDIANA FLORA VIDÔTO DA COSTA
SÃO PAULO
2015
Costa, Lidiana Flora Vidôto da. Culture and propagation of microsporidia of veterinary interest - Culture
of microsporidia; B-1 cells as a component of the immune response against murine encephalitozoonosis / Lidiana Flora Vidôto da Costa. – 2015. 59 f. : il. color. + CD-ROM. Tese de Doutorado Apresentada ao Programa de Pós Graduação em
Patologia Ambiental e Experimental da Universidade Paulista, São Paulo,
2015. Área de Concentração: Patogenia das Enfermidades Infecciosas e
Parasitárias. Orientadora: Prof.ª Dra. Maria Anete Lallo. Coorientadora: Prof.ª Dra. Anuska Marcelino Alvares Saraiva.
1. Encephalitozoon cuniculi infection. 2. Microsporidia culture. 3. B-cell.
4. B-1 cell. 5. B-2 cell. 6. XID mice. 7. Animals. I. Lallo, Maria Anete (orientadora). II. Alvares-Saraiva, Anuska Marcelino
(coorientadora). III. Título.
LIDIANA FLORA VIDÔTO DA COSTA
Artigo1: CULTURE AND PROPAGATION OF
MICROSPORIDIA OF VETERINARY
INTEREST-CULTURE OF MICROSPORIDIA
Artigo 2: B-1 CELLS AS A COMPONENT OF THE IMMUNE
RESPONSE AGAINST MURINE ENCEPHALITOZOONOSIS
Tese apresentada ao Programa de Pós-
Graduação em Patologia Ambiental e
Experimental da Universidade Paulista –
UNIP, para obtenção do título de Doutor em
Patologia Ambiental e Experimental.
Aprovado em:
BANCA EXAMINADORA
________________________________/_/___
Prof. Dr. Mário Mariano
Universidade Paulista – UNIP
_________________________________/_/___
Prof. Drª Maria Anete Lallo.
Universidade Paulista – UNIP
_________________________________/_/___
Prof. Drª Anuska Marcelino Alvares Saraiva
Universidade Paulista – UNIP
_________________________________/_/___
Prof. Drª Diva Denelle Spadacci Morena
Universidade de São Paulo- USP
_________________________________/_/___
Profª Drª Sílvia Regina Kleeb
Universidade Metodista de São Paulo- UMESP
DEDICATÓRIA
Dedico esse trabalho à minha família...
...ao meu marido e amado companheiro Alberto Luiz, pelo amor e paciência em todos esses
anos de convivência. Por ter me acompanhado nesta jornada e nunca ter desacreditado em
nossos sonhos.
...aos meus filhos amados e queridos Guilherme e Isabella, razão de todos os meus esforços e
esperanças!
AGRADECIMENTOS
Ao nosso Deus, por ter me dado a oportunidade de encontrar pessoas maravilhosas
nessa jornada desafiadora, por tornar meu caminho seguro e ordenado, por me dar as
ferramentas necessárias para que pudesse construir essa etapa e por me amparar nas horas que
necessitei.
À Professora Drª Maria Anete Lallo, minha orientadora, por me colocar o desafio de
fazer a tese de doutoramento, pela paciência, competência, disponibilidade e generosidade
dedicadas à mim durante o desenvolvimento desse trabalho. Seu entusiasmo e autruísmo
estarão sempres presentes.
Á Professora Drª Anuska Marcelino Alvares, pela competência ensinamentos e
orientações dadas. Pela dedicação, bem como pela disponibilidade e amizade então
demonstradas.
À Professora Drª Elizabeth Cristina Perez Hurtado, pela competência, pelas inúmeras
orientações, amizade e pelo inestimável apoio sempre manifestados.
À Professora Drª Diva Denelle Spadacci Morena, pelas orientações e competência,
disponibilidade e otimismo como que sempre nos recebeu no Instituto Buntantan,
imprescindível para que esse trabalho fosse realizado.
Ao Prof. Dr. Mário Mariano, pelas sugestões inovadoras sempre presentes e pelo
magnetimo que atrai seus alunos à imaginar possibilidades e conquistas científicas.
À Drª Fabiana Toshie de Camargo Konno, responsável pelo Laboratório de Biologia
Molecular e Celular do Instituto de Ciências da Saúde- UNIP pelo incansável apoio,
orientação e disponibilidade, que em muito contribuíram para a conclusão desta tese.
Ao corpo docente e discente do Programa de Mestrado e Doutorado em
Patologia Ambiental e Experimental na UNIP, pelos valiosos ensinamentos e convivência.
À minha amiga Profª Drª Raquel Machado Cavalca Coutinho, pelo incentivo e pelo
apoio que me fortaleceu na escolha de fazer a tese de doutoramento, pelas palavras amigas de
sempre, pela paciência, caronas e principalmente pelos exemplos de perseverança, trabalho, e
determinação com que sempre me motivaram a nunca desistir do que acredito.
À minha amiga Profª Drª Eliana Maria Scarelli Amaral, pelo companheirismo nesta
jornada, pelos auxílios no trabalho e nos desabafos. Pelo cuidado com meu dia a dia, parceira
e companheira de todas as horas.
À todos os funcionários do Programa de Mestrado e Doutorado em
Patologia Ambiental e Experimental na UNIP, pela ajuda em especial à Christina, Cleonice,
Wilton, Rodolfo pelo companheirismo e dedicação em suas atividades.
Ao meu companheiro e amado, presente em todos os momentos de minha vida, razão
de minha alegria, Alberto Luiz, pelo inestimável apoio familiar, carinho, paciência e
compreensão revelados ao longo destes anos.
Aos meus queridos filhos, Guilherme e Isabella pela compreensão e ternura sempre
manifestadas. Espero que todo o entusiasmo, seriedade e empenho que me dediquei a este
trabalho ao longo dos anos lhes possam servir de estímulo para o bem e espelho para a vida.
Agradeço as instituições abaixo descritas pelo apoio, excelentes condições de trabalho
que me proporcionaram e colaboração prestados, sem os quais não seria possível a
concretização do trabalho de doutoramento:
Universidade Paulista
Escola Paulista de Medicina
Instituto Adolfo Lutz
Instituto Butantan
Mais uma vez, a todos os meus sinceros agradecimentos.
EPÍGRAFE
“Nada lhe posso dar que já não exista em você mesmo.
Não posso abrir-lhe outro mundo de imagens, além daquela que há em sua própria alma.
Nada lhe posso dar a não ser a oportunidade, o impulso, a chave.
Eu o ajudarei a tornar visível seu próprio mundo, e isso é tudo...”
Hermann Hesse
SUMÁRIO
1 INTRODUÇÃO ................................................................................................................. 8
2 ARTIGOS ORIGINAIS DE PESQUISA ...................................................................... 10
2.1 Artigo 1 - Culture and propagation of microsporidia of veterinary interest ............ 10
2.2 Artigo 2 - B-1 cells as a component of the immune response against murine
encephalitozoonosis .............................................................................................................. 30
3 CONSIDERAÇÕES FINAIS ......................................................................................... 57
4 REFERÊNCIAS BIBLIOGRÁFICAS .......................................................................... 58
8
1 INTRODUÇÃO
Os microsporídios são parasitas intracelulares obrigatórios que são encontrados tanto
em hospedeiros invertebrados como em vertebrados (Anane; Attouchi, 2010). Estes
organismos foram descritos como protozoários, no entanto, atualmente são reconhecidos
como fungos atípicos sem mitocôndrias (Frazen, 2008). De mais de 1200 espécies de
microsporídios, 17 são conhecidas por infectarem seres humanos (Fayer; Santin-Duran,
2014). Os estudos indicam que as espécies que infectam o homem também infectam animais e
a diferença entre as mesmas está ligada a variações genotípicas (Frazen, 2008).
Os conhecimentos sobre microsporidiose humana e animal ainda são limitados devido
às dificuldades na identificação dos esporos, sua forma infectante, pela microscopia de luz,
por serem pequenos, mesmo utilizando-se a maior aumento de 1000x (Weber et al., 1994).
Em meados da década de 80, verificou-se que a microsporidiose quando associada à
imunossupressão causada pela infecção com o Vírus da Imunodeficiência Humana - HIV,
determinava um quadro de diarréia aguda importante (Desportes et al., 1985), e o número
desses casos descritos aumentaram após 1990 (Didier, 2005).
A imunossupressão causada por anticorpos anti-TNF , pelo uso de drogas
quimioterápicas e/ou imunomoduladoras usadas em transplante de órgãos tem sido
reconhecidos como importantes fatores de risco para o desenvolvimento da infecção por
microsporídios (Khan et al., 2001; Lallo et al., 2012). No entanto, a microsporidiose humana
não é restrita a pessoas imunossuprimidas, mas também pode ocorrer em indivíduos
imunocompetentes. Até agora, os microsporídios descritos em seres humanos incluem:
Encephalitozoon cuniculi, E. intestinalis, E. hellem, Enterocytozoon bieneusi,
Microsporidium africanum, M. ceylonensis, Microsporidium sp, Pleistophora ronneafiei,
Trachipleistophora hominis, T. anthropopthera, Tubulinosema sp, Anncalia algerae, A.
connori, A. vesicularum, Nosema ocularum, Nosema sp. e Vittaforma corneas (Didier, 2005).
O cultivo in vitro de microsporídios permite o estudo do ciclo de vida, do
metabolismo, da patogênese, do diagnóstico e também funciona como repositório desses
patógenos o que possibilita pesquisas biomoleculares, genéticas e epidemiológicas (Molestina
et al., 2014). Além disso, as culturas também têm sido usadas para determinar a eficácia dos
agentes antimicrobianos contra vários microsporídios, incluindo Encephalitozoon cuniculi, E.
hellem, e E. intestinalis (Wolk et al., 2000). Embora o cultivo de microsporídios não seja
9
fundamental para os laboratórios de diagnóstico de rotina, o cultivo in vitro continua a
fornecer o diagnóstico de confirmação de microsporidiose em seres humanos e animais, bem
como na utilização desses esporos para infecções experimentais, que forneceram dados
relevantes sobre a resposta imune contra esses patógenos (Meng et al., 2014). Como o estudo
de microsporidiose em medicina veterinária é incipiente e que a cultura celular é uma
ferramenta indispensável para o seu estudo, o primeiro artigo aborda uma revisão
bibliográfica que tem como objetivo (i) descrever os microsporídios relevante para a área
veterinária e (ii) descrever cultivo in vitro destes patógenos.
A imunidade celular é crítica para a sobrevivência de hospedeiros infectados por E.
cuniculi, sendo as células T CD8+as principais responsáveis pela resistência à infecção.
Existem linhagens de camundongos mais sensíveis e outras mais resistentes à infecção por E.
cuniculi, o que tem sido evidenciado pela alta porcentagem de macrófagos parasitados após a
inoculação intraperitoneal, sugerindo uma base genética para a resistência inata (Niederkorn
et al., 1981). Por se tratar de um agente oportunista, os camundongos imunodeficientes, tais
como os atímicos e os SCID, são os que desenvolvem a doença letal após a inoculação
experimental de E. cuniculi (Schmidt; Shadduck, 1983; Koudela et al., 1993), geralmente
manifestada na forma dissemida e caracterizada por ascite e a presença de esporos em todos
os sistemas orgânicos.
Em contrapartida, pouco se sabe sobre a participação das células B (B-1 e B-2) no
desenvolvimento de uma resposta imune contra este agente patogênico e sobre as suas
relações com as células efetoras, especialmente macrófagos e células T CD8 + responsáveis
pela imunidade e a eliminação da E. cuniculi.
No segundo estudo, objetivou-se avaliar o papel das células B-1 na infecção
experimental pelo E. cuniculi, verificando se o camundongo BALB/c XID (deficiente em
células B-1) é uma linhagem mais suscetível à infecção por E. cuniculi, e traçar o perfil das
populações celulares e das citocinas Th1/Th2 na infecção experimental pelo E. cuniculi.
10
2 ARTIGOS ORIGINAIS DE PESQUISA
2.1 Artigo 1
The Journal of Veterinary Medical Science
Review article
CULTURE AND PROPAGATION OF MICROSPORIDIA OF VETERINARY
INTEREST
CULTURE OF MICROSPORIDIA
Maria Anete Lallo1*
, Lidiana Flora Vidoto da Costa1, Anuska Marcelino Alvares-Saraiva
1, Paulo
Ricardo Dell’Armelina Rocha1,2
, Diva Denelle Spadacci-Morena3, Fabiana Toshie de Camargo
Konno1, Ivana Barbosa Suffredini
1
1Environmental and Experimental Pathology, Paulista University, São Paulo, Brazil
2Sao Paulo State University, Faculty of Veterinary Medicine, Aracatuba, Brazil
3Physiopathology Laboratory, Butantan Institute, São Paulo, Brazil
*Corresponding author. Address: Environmental and Experimental Pathology, Paulista
University, Rua José Maria Whitaker 290, São Paulo, Brazil. Phone: (55) 11 99869607
E-mail address: [email protected] or [email protected]
11
Abstract
Microsporidia are obligate intracellular mitochrondria-lacking pathogens that rely on host
cells to grow and multiply. Microsporidia, currently classified as fungi, are ubiquitous in
nature and are found worldwide. They infect a large number of mammals and are recognized
as opportunistic infection agents in HIV-AIDS patients. Its importance for veterinary
medicine has been unveiled in recent years through the description of clinical and subclinical
forms of infection in domestic and wild animals. Domestic and wild birds may be infected by
the same human microsporidia, reinforcing their zoonotic potential. Microsporidiosis in fish
are prevalent and cause significant economic losses for fish farming. Some species of
microsporidia have been propagated in cell cultures, which may provide conditions for the
development of diagnostic techniques, understanding of pathogenesis and immune responses
and for the discovery of potential therapies. Unfortunately, the cultivation of these parasites is
not fully standardized inmost research laboratories, especially in the veterinary field. The aim
of this review is to relate the most important microsporidia of veterinary interest and
demonstrate how these pathogens can be grown and propagated in cell culture for diagnostic
purposes or for pathogenesis studies. Cultivation of microsporidia allowed the study of its life
cycle, metabolism, pathogenesis, diagnosis, and may also serves as a repository for these
pathogens for molecular, biochemical, antigenic, and epidemiological studies.
Key words: animals, cell culture, diagnosis, microsporidia, zoonosis
12
Introduction
Microsporidia are obligate intracellular parasites that are found in both invertebrate
and vertebrate hosts [1]. These organisms were described as Protozoa, however it is now
recognized that they are atypical fungi without mitochondria [7]. Of over 1200 species of
organisms classified in the phylum Microsporidia, 17 species of microsporidia are known to
infect animals and humans [6]. The species of microsporidia described simultaneously in
humans and other animals have great genotypic diversity, therefore epidemiological studies
are conducted to understand the dynamics of infection and the zoonotic potential from
animals to humans (Table 1) [1, 5]. Early knowledge of human and animal microsporidiosis is
limited because of difficulties identifying the microscopic spores of microsporidia, which are
small even at the highest magnification (1000x) by light microscopy [6] (3-5 µm) therefore,
the cultivation microsporidia cells is very important for the improvement of diagnostic
techniques or for studies about the pathogenesis of diseases.
Microsporidia infection has been described in a wide variety of domestic and wild
mammals, birds, amphibians, reptiles and fish [5]. The first descriptions in mammals
identified Encephalitozoon cuniculi in commercial breeding rabbits and laboratories rodents.
E. cuniculi is the most microsporidian species identified in non-human vertebrates [17]. More
recently, E. hellem has been identified in wild birds and pigeons [16], with or without clinical
disease [9, 13] and Enterocytozoon bieneusi has been identified in cattle [34].
The cultivation of microsporidia started with the inoculation of Nosema bombycis, an
important parasite to silkworms, causing pebrine disease and is cultivated in silkworm ovarian
tube lining cells [38]. Interest in microsporidia cultivation increased greatly with continuous
cultivation of Encephalitozoon cuniculi in rabbit kidney cell cultures (RK cell) [35], but it
was in the 90s that cultivation of microsporidia really improved due to of increased reports of
human disease [40]. In vitro cultivation continues to provide confirmatory diagnosis of
13
microsporidiosis in both humans and animals, as well as providing spores of microsporidia for
experimental infections, which have provided relevant data regarding immune response
against these pathogens [23]. In vitro culture has also been used to determine the efficacy of
antimicrobial agents against several microsporidia, including Encephalitozoon cuniculi,
Encephalitozoon hellem, and E. intestinalis [44]. Although microsporidia cannot be grown
axenically without the host cell, Encephalitozoon sp., Trachipleistophora hominis, Vittaforma
corneae, and Brachiola algerae have been successfully grown in vitro cell cultivation.
Cultivation of microsporidia has allowed the study of the life cycle, metabolism,
pathogenesis, diagnosis, and also serves as a repository for these pathogens for molecular,
biochemical, antigenic, and epidemiological studies [24]. The study of microsporidiosis in
veterinary medicine is still incipient, therefore, this paper aims to (i) describe the most
important microsporidia to the veterinary area and (ii) describe in vitro cultivation of these
pathogens.
Microsporidia in Animals
Encephalitozoonosis caused by E. cuniculi is a naturally occurring disease in domestic
rabbits and laboratory rodents. Many rabbits have a latent chronic form but only part of them,
develop clinical disease. Three clinical syndromes are recognized and include encephalitis,
renal and ocular lesions (uveitis and cataracts). The animals infected with neurological
involvement are inactive, have torticollis, ataxia, circling, paresis and progress to death. When
the pathogen affects the kidney, it causes chronic interstitial nephritis with kidney failure and
death [17].
Among domestic animals, the dog is the most affected by Encephalitozoon infection.
Moreover, encephalitozoonosis due to E. cuniculi genotype/strain I has been described as the
cause of fatal encephalitis and nephritis in domestic dogs [5]. Moreover, E. cuniculi was
14
detected in a cat with cerebral hypoplasia, causing granulomatous encephalitis, hepatitis, and
mild interstitial nephritis, myocarditis and enteritis [30].
The first descriptions of microsporidia in cattle were mostly in Europe and North
America over the past decade [32]. Enterocytozoon bieneusi was the microsporidia identified
in 23% of 571 cattle examined from 14 farms [34]. Moreover, in South Africa the presence of
E. bieneusi in domestic cattle in rural communities was confirmed by PCR, with a genotype
pathogenic for humans [33]. E. bieneusi was detected in pigs [4] and a prevalence of 35%
determined in Switzerland [3], affecting especially piglets. The pathogen was detected in pigs
of all ages with varying prevalence, but the data suggest that E. bieneusi is not pathogenic to
swine, the same was observed in cattle.
Several reports described microsporidian infections in wild carnivores (red fox, arctic
foxes, wild dogs) maintained as captive or in Zoos [10, 28, 39]. Disseminated natural
infections resulting in high morbidity and marked encephalitis caused by Encephalitozoon
like organism have been reported in stillborn and young squirrel monkeys (Saimiri sciureus)
in the United States [45]. Recently, strain III (“dog strain”) of E. cuniculi was identified in
tamarin colonies (Saguinus imperator, Oedipomidas oedipus, and Leontopithecus rosalia
rosalia) in two Zoos in Europe, causing marked disseminated infection with high mortality in
infants [8]. In monkeys, experimental or even naturally acquired infection by transplacental
transmission resulted in fatal multifocal granulomatous encephalitis [45].
The most common human microsporidian species, E. bieneusi, E. intestinalis, E.
hellem and E. cuniculi have been reported in various species of birds. The first case of
microsporidiosis in birds was reported in masked lovebirds (Agaponis personta) in 1975 [13].
E. bieneusi was detected in chickens (Gallus gallus) in a poultry abattoir in Germany [31], in
captive falcons in the Arab Emirates [27], and in pigeons [9]. In Brazil, we found 24.5% of
positivity among 196 fecal samples analyzed by PCR- and Gram-Chromotrope staining
15
technique; the prevalence was higher in pigeons (31.1%) than the free-ranging birds (18.8%)
[12].
Microsporidia are a major cause of disease in fish and may have an economically
important impact on fish stocks [19, 20]. Many fish diseases of considerable economic
importance have been attributed to microsporidia, including diseases that impede aquaculture
of fish and have been a factor in the weakening or collapse of several fisheries [2]. Fish are
hosts of 156 species of microsporidia allocated to 14 genera, as well known: Glugea,
Heterosporis, Ichthyosporidium, Kabatana, Loma, Microfilum, Microgemma,
Neonosemoides, Nosemoides, Nucleospora, Ovipleistophora, Pleistophora, Spraguea,
Tetramicra and the collective group Microsporidium, which is not considered a genus [19].
Microsporidiosis is highly destructive to the infected tissue, resulting in high mortality rates in
fish [14]. Microsporidia are widely distributed in sea water, estuaries and freshwater. Ambient
temperature affects the development of microsporidia, (eg. infections by Glugea stephani and
Loma salmonae), which increases during summer, when water temperatures rise [29].
Prevalence is especially high when the density increases, raising both morbidity and mortality
of young fish [29].
Organism and life cycle
Microsporidia are nucleated, single-celled, obligate intracellular pathogens that were
considered to be early-branching eukaryotic organisms based on the presence of prokaryote-
like ribosome, and the apparent absence of true Golgi, peroxisomes, and mitochondria [5, 41].
The life cycle of microsporidia is characterized by three phases: the infective or
environmental phase (spores); the proliferative phase, identified by merogony; and the
sporogonic or spore-forming phase [12].
16
Spores of microsporidia are generally small, oval or pyriform-shaped with 1 to 12 μm
in length (Fig. 1, 2). Microsporidian spores contain a long and convoluted tubular extrusion
apparatus (polar tubule), which distinguishes them from other organisms (Fig. 1). The polar
tube has a crucial role in invasion of host cell (Fig. 1). Spore approaches the host cell and its
polar tubular is everted to enter the cell and inject its sporoplasm into the cell cytoplasm (Fig.
1). Phagocytosis of spores by host cells or cell culture by Encephalitozoon involves binding
of the spores to the host cell surface glycosaminoglycans (GAGs) [12].
The proliferative phase (schizogony and merogony) includes all cell growth and
division from sporoplasm until spore formation. The sporoplasm injected into the host cell
becomes meronts and proliferates by repeated binary or multiple fission or by plasmotomy
[7]. The sporogony includes sporonts (cells produce two to more sporoblasts), sporoblasts
(cells undergo metamorphosis into spores), and spores. Meronts develop into sporonts, which
is the stage that divides into sporoblasts. The beginning of sporogony to microsporidia which
have diplokaryotic nuclei takes place after meiosis, whereas for other species sporogony
occurs in the presence of plasmalemmal thicking. Sporonts are usually characterized by the
development of a thick, electron-dense surface coat that will later become the exospore layer
of the spore wall (Fig. 1). Sporonts can multiply by binary fission or multiple, acquire
specialized organelles and become spores. Subsequently, spores spread through the tissues of
the host by infecting new cells and continuing the cycle [12].
In vitro culture of microsporidia of mammals
Samples for culture.
For diagnosis, reports indicate the use of urine, sputum, bronchoalveolar lavage, feces,
duodenal aspirates, conjunctival scraping, corneal biopsies, cerebrospinal fluids, muscle
biopsies and brain tissue as possible tissues to harvest. The addition of antibiotics
17
(gentamicin, penicillin and streptomycin) and amphotericin B prevents bacterial and fungal
growth without compromising the development of microsporidia [24]. In the case of samples
with mucus (e.g. respiratory secretions), a mucolytic should be used, then the sample will be
centrifuged and its pellet washed with distilled water before inoculating the cell culture [40].
Tissue from biopsies should be macerated mechanically or by enzymatic action before being
used to inoculate cell monolayers. In the case of urine use for initiating a culture, it must be
centrifuged at 1,500 x g for 30 min and the pellet should be suspended in Dulbecco’s
Modified Eagle’s Medium - DEMEM containing penicillin, streptomycin and amphotericin B
and subsequently incubate for 4 hr. Afterwards, a new centrifugation is carried and the pellet
with spores should be inoculated in monolayers of Madin-Darby Canine Kidney cell line
(MDCK cells) with Modified Eagle’s Medium - MEM and 10% fetal bovine serum [24, 40].
Maintenance and preservation of microsporidian cultures.
Maintaining microsporidia cell cultures is relatively simple. Cultures should be
microscopically examined every week before the change of medium (Fig. 2, 3). An inverted
microscope equipped with differential interference contrast optics or phase is needed, where
cells filled with spores or free spores can be observed [22]. The culture medium should
preferably be exchanged twice a week and this favors the development of microsporidia and
host cells, however in our experience, a medium exchange weekly is sufficient to culture
flasks and spores can survive for several months to a year or even more.
In case of need to rapidly expand the cultures to obtain a large number of spores, new
cultures inoculated with spores can be obtained from the supernatant of infected cultures.
Cultures that have more than 80% of infected cells may be scraped or trypsinized and the cells
transferred to new flasks for expansion cultures [24, 40]. The culture medium collected from
the inoculated flasks should be centrifuged (1,500 x g for 20 min at 4ºC) and the pellet
18
containing spores should be suspended in culture medium and stored at 4°C or used to
inoculate new cell cultures [12, 42].
A number of cell lines have been used (Table 2), including monkey and rabbit kidney
cell lines (Vero and RK-13), a human fetal lung fibroblasts cell line (MRC-5), and the MDCK
cell line. Unfortunately, one of the most common human microsporidial pathogens, E.
bieneusi, has been propagated only in short-term cultures [17].
Culture of cells infected with microsporidia can be scraped or trypsinized and mixed
with an equal volume of culture medium containing 20% DMSO. This mixture should be
aliquoted in a 1.0 mL volume into plastic cryovials. Freezing of the samples at room
temperature should be done in controlled-rate freezing unit for cooling -1°C to -40°C, and
only then they can be transferred to liquid nitrogen. The flasks should be placed at -80°C for 2
hr to overnight period and then they should be transferred to liquid nitrogen [24, 40].
Cell culture of fish microsporidia.
Compared to microsporidia of mammals, in vitro culture of fish microsporidia has a
limited success (Table 2). Although primary cultures and cell lines are available for culturing
various tissues and organs of fish, microsporidia cultures typically are of short duration (about
48 hr or less). Therefore, culture of fish microsporidia has been employed to study their
interaction with cells of the innate immune system, macrophages and neutrophils [15].
Primary cultures of salmonids of leukocytes can give relatively greater longevity to
the development of microsporidia in fish. In vitro cultures of fish microsporidian have been
successfully demonstrated by using in epithelial cell cultures of Aedes albopictus at 28°C
[21]. Culture of Glugea sp. was obtained from specimens of the fish Hyperoplus lanceolatus
that was captured by commercial boats in the coast of Andalusia, Spain. The same
microsporidian was not able to develop more than 48 hr in epithelial cells from salmon
19
embryo at 21°C. It is worth mentioning that the cell lines of fish promotes the growth of
Anncaliia (Brachiola/Nosema) algereae, a microsporidia that was first described in insects,
but is currently found in many vertebrate hosts, including humans [25]. Epithelial-like cells
(PE-1) derived from tissues of eel (Anguilla japonica), are considered immortal lineages and
have allowed the development of Heterosporis (Pleistophora) anguillarum. In this
cultivation, microsporidian spores are not formed, however inoculation of infected culture in
eel causes muscular disease and spore formation [26].
Conclusion
Several species of microsporidia have been identified in domestic and wild animals
and many are zoonotic and lethal pathogens. However, aspects related to the pathogenesis of
infections in animals remain unknown, therefore microsporidia should be further studied in
animals. In conclusion, cell culture is an essential tool for the study of microsporidia
important for veterinary medicine, and can be implanted in research and diagnostic
laboratories using already established cell lines culture. Therefore, the establishment of
cultures is essential for acting an implementation of knowledge of these emerging and
opportunistic pathogens in different areas of scientific knowledge.
Acknowledgements
We thank Magna Aparecida Mautauro Soares (Butantan Institute) for making the
material for light microscopy and Michelle Sanchez Freitas Correia (Paulista University) for
performing the scanning electron microscopy. We thank the Butantan Institute for allowing us
to use their electron microscope obtained by FAPESP project number 18070806074.
20
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25
Table 1. Microsporidia identified in animals and in man.
Species Animals and other hosts Clinical Presentation in
humans
Encephalitozoon
cuniculi
Mammals (rabbits, rodents,
dogs, blue fox, cats, cows,
horses, pigs, primates)
Birds
Eye, respiratory,
gastrointestinal, genitourinary
and disseminated infection
Encephalitozoon
hellem
Birds (psittacine birds, ostrich,
finches, pigeons)
Eye, gastrointestinal and
disseminated infection
Encephalitozoon
intestinalis*
Mammals (dogs, pigs, donkeys,
cows, goats, primates)
Birds
GI infection, ocular,
genitourinary and respiratory
tracts
Enterocytozoon
bieneusi
Mammals (dogs, pigs, donkeys,
cows, goats, primates)
Birds
GI and respiratory infection
Anncalia algerae Mosquitos Eye, muscular and skin
infection
Pleistophora sp. Fish, amphibians and reptiles Muscular infection
Tubulinosema
acridophagus
Insects (grasshoppers) Disseminated and muscular
infection
Endoreticulatus sp. Lepidopteran insects Muscle infection
26
Table 2. Examples of cell line culture used in the cultivation of microsporidia from mammals,
birds and fish.
Microsporidian species Cell culture
Mammals and birds
Encephalitozoon
cuniculi
Encephalitozoon hellem
Monkey kidney (E6); Madin-Darby canine kidney
(MDCK); Rabbit kidney (RK-13); Human lung fibroblast
(HLF); Human lung fibroblast (MRC-5)
Encephalitozoon
intestinalis
Monkey kidney (E6); Madin-Darby canine kidney
(MDCK); Rabbit kidney (RK-13); Human lung fibroblast
(HLF); Human colorectal adenocarcinoma (HT-29);
Human colorectal adenocarcinoma (CACO-2)
Anncaliia algerae Monkey kidney (E6); Human lung fibroblast (HLF)
Trachipleistophora
hominis
Madin-Darby canine kidney (MDCK); Rabbit kidney
(RK-13); Monkey kidney (COS-1)
Vittaforma corneae Monkey kidney (E6); Rabbit cornea (SIRC); Madin-Darby
canine kidney (MDCK); Human lung fibroblast (HLF);
Human lung fibroblast (MRC-5)
Fish
Nucleospora salmonis Primary culture of leukocytes from peripheral blood of
chinook salmon
Nucleospora salmonis Primary culture of epithelial-like cell from kidney of
rainbow trout
Glugea sp. Cell line CHSE-214 from salmon embryo
Pseudoloma neurophilia Cell line CCO- fibroblast from ovary of channel catfish;
Cell line SJD.1- fibroblast from fin of zebrafish; Cell line
EPC- epithelial –like from skin of carp; Cell line EPC-
epithelial –like from connective tissue and muscle of
fathead minnow
Heteroporis anguillarum Cell line EP-1 – epithelial like from infected tissues of
elves of japanese eel
27
Figures
Fig. 1. Encephalitozoon sp. spores from Vero cell. a) Transmission electron microscopy
(TEM) of young E. intestinalis spore with five filament (arrow) coils of polar filament (scale
bar = 0.4µm); b) Extruded (arrow) polar filament of E. cuniculi spore (TEM, scale bar =
0.6µm); c) Scanning electron microscopy of E. cuniculi (scale bar = 3µm).
28
Fig. 2. a) Parasitophorous vacuole (arrow) in a distended RK-13 cell. b) Fully formed
parasitophorous vacuole with sporogonial stages and spores in Vero cell (TEM, Scale bar = 2
μm). c) Scanning electron microscopy of Vero cell culture infected with E. cuniculi (scale bar
= 5µm).
29
Fig. 3. Culture smears of Encephalitozoon cuniculi spores stained with Chromotrope (a) and
Gram-Chromotrope technique (b).
30
2.2 Artigo 2
INFECTION AND IMMUNITY
B-1 CELLS AS A COMPONENT OF THE IMMUNE RESPONSE AGAINST MURINE
ENCEPHALITOZOONOSIS
Running-title: B-1 cells and encephalitozoonosis
Lidiana Flora Vidoto da Costa1, Anuska Marcelino Alvares-Saraiva
1, Paulo Ricardo Dell’Armelina
Rocha1, Diva Denelle Spadacci-Morena
2, Elizabeth Christina Perez Hurtado
1, Mario Mariano
1,3, and
Maria Anete Lallo1
1Environmental and Experimental Pathology, Paulista University (UNIP)
2Fisiopathology Department, Butantan Institute, São Paulo, Brazil
3Microbiology, Immunology and Parasitology Department, Federal University, São Paulo,
Brazil
*Corresponding author: Mailing address: Paulista University, R. Dr. Bacelar 1212, 4th
floor, CEP: 04026002. São Paulo, SP, Brazil. Phone-Fax: 55. 11.5586.4093, Mobile phone:
55. 11.99986.9607. E-mail: [email protected] or [email protected]
31
Abstract
Encephalitozoon cuniculi is an opportunistic pathogen for people with AIDS or other immune
deficiencies. The adaptive immune response is essential to eliminate E. cuniculi, but evidence
indicates that the innate immune response is responsible for initiating and setting up the
pathogen. B-1 cells act as antigen-presenting cells, differentiates into a phagocytes, produce
IL-10 and had other immune functions. The possible role of these cells in the dynamics of the
infectious process of various etiologies is unknown. To understand the role of B-1 cells in E.
cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E.
cuniculi spores by intraperitoneal route (i.p.). After 15 and 21 days post-infection (DPI),
spleen and peritoneal cells were evaluated by flow cytometry. Th1 and Th2 cytokines’profile
were quantified from serum samples by cytometric beads array (CBA). BALB/c XID mice
showed more susceptible to infection evidenced by symptoms and histopathological lesions
presence when compared to BALB/c mice. Populations of B-1 cells and macrophages into the
peritoneum increased significantly in BALB/c mice infected with E. cuniculi compared to
uninfected controls, but in BALB/c XID mice infected or not it wasn´t identified. Despite the
increase in the number of CD4+ and CD8
+ lymphocytes in BALB/c XID mice, these animals
were still more susceptible to infection. After the infection, there was an increase of IFN-γ,
IL-6 and TNF-α cytokine levels in the sera of BALB/c mice. In animals BALB/c XID besides
IFN-γ, IL-6 and TNF-α, IL-4 was detected after infection. Thus, we can concluded that the
absence of B cells (B-1 and B-2) increases the susceptibility of BALB/c XID mice to
infection by E. cuniculi, evidenced their participation in the immune response against this
microsporidian, probably as a component that interacts with macrophages and becomes in
phagocytes.
Keywords: B-cell, B-1 cell, Encephalitozoon cuniculi infection, macrophage, XID mice.
32
Introduction
Microsporidia are small obligate intracellular parasites that, until a short time, were
thought to be protozoans; however, evidence now confirmed that they are related to Fungi (1).
Before AIDS advent, the microsporidia was recognized as important pathogens in agriculture
because it may cause disease in fish, fur bearing animals, silkworm and laboratory animals
(rabbits, rodents and primates) (2). In the mid-80s, microsporidia was evidenced by the
occurrence in HIV-AIDS-patients. The main symptoms are diarrhea, hepatitis, pneumonia,
peritonitis, keratitis and other clinical manifestations (3). Immunosuppressed patients by
drugs or any other immunosuppressive condition are also target of this opportunistic infection
(4).
Cellular immunity is essential for the resolution of microsporidia infection (5,6).
Studies with BALB/c mice demonstrated that resistance to lethal disease produced by E.
cuniculi was T cell-dependent (7). Phenotypic analysis of spleen cells from animals infected
with E. cuniculi showed a significant increase in the population of CD8+ T cells compared to
other subtypes (7). Knockout mice lacking CD8+ T cell died after inoculation with E. cuniculi
and developed a severe and lethal form of encephalitozoonose as opposed to CD4-/-
mice,
which exhibit few symptoms and no mortality when infected by this parasite (7). In spite of
the infection with E. cuniculi causes chronic infection associated with high and persistent
antibodies occurrence and continuous inflammatory process in rabbits (8) and foxes (9), the
adoptive transfer of B lymphocytes into BALB/c nude or SCID mice does not protect from
death following E. cuniculi infection (10). Transfer of maternal antibodies can protect
newborn rabbits against infection with microsporidia in the first 2 weeks of life (10). It has
been demonstrated that monoclonal antibodies against spore coat or monoclonal and
polyclonal antibodies against polar tubule protein reduce microsporidia infectivity (10).
33
The X-linked immunodeficiency (XID) phenotype in mice is due to partial block of B-
lymphocyte development caused by a missense mutation (R28C) in the N-terminal PH
domain of Btk, leading to a total lack of B-1 lymphocytes and substantial decrease in the
number of conventional B-2 lymphocytes (11). The phenotype of Btk deficiency is mainly
related to B-lymphocytes and its various functions. However Btk is also expressed and have
function in myeloid lineage cells (12). B-1 cells are essential not only for T-independent
innate host response but also for adaptive immunity (12, 13). Cells known as B-1 cells are
preferentially located in the peritoneal and pleural cavities (14), and can be expanded through
self-renewal (15), produce natural IgM antibodies (16) and are antigen-presenting cells - APC
(17). They are able to migrate to a non-specific inflammatory focus and differentiate into
macrophage-like cells (18), producing IL-10 which acts as a potent down-regulator of cell-
mediated immunity (19). B-1 cells are involved in specific functions in autoimmunity (20),
antigen tolerance (21), and increasing metastatic behavior and the malignancy of murine
melanoma cells (22, 23).
Here, we showed the participation of B-1 and B-2 cells in the development of the
immune response against Encephalitozoon cuniculi and its relations with the effectors cells,
especially macrophages and CD8+ T cells responsible for immunity and the elimination of this
pathogen. In addition, we demonstrated that BALB/c XID mice were more susceptible to
experimental infection with E. cuniculi and could be a new candidate as an animal model for
experimental encephalitozoonosis.
Material and methods
Mice. Female, 6-8 weeks old BALB/c and BALB/c XID were obtained from the animal
facilities of the Development Center of Experimental Models (CEDEME) of Federal
University of São Paulo, Brazil. During the experimental period the animals were housed in
34
sterilized isolators under the standard pathogen free condition. All experiments were
conducted with the approval of the institutional animal care and ethics committee (Ethics
Committee on Animal Research of Paulista University - CEUA protocol number
CEP138/2012).
Parasites and infection. E. cuniculi (genotype I) (from Waterborne Inc., New
Orleans, LA, USA) were grown in a rabbit kidney cell lineage (RK-13, ATCC CCL-37) in
DMEM supplemented with 10% of fetal calf serum (FCS), pyruvate, nonessential amino
acids, and gentamicin at 37°C in 5% CO2 and harvested from tissue culture supernatants.
Spores were washed 3 times in Phosphate buffered Saline (PBS) and counted with a
hemacytometer. Mice were infected i.p. with 1x107 E. cuniculi spores. Uninfected animals
were maintained as control for all the groups.
B-1 cells adoptive transfer. Peritoneal cells were obtained from successive washes
with RPMI/1640 medium (Sigma) from non-infected BALB/c mice and cultured for 40
minutes at 37°C with 5% CO2. After discarding the supernatant, RPMI supplemented with
10% of FCS was added on adherent cells which were kept under the same conditions. On the
5th
day, the float B-1 cells were collected and re-suspended in PBS. A total of 1x106 B-1 cells
in 100 µL PBS were i.p. route in BALB/c XID mice 7 days before infection, as previously
described (24).
Light microscopy analysis. Animals were euthanized at 14 and 21 days post-infection
(DPI), necropsied and tissue samples were collected. Fragments of lung, brain, liver, kidneys,
spleen and intestines were removed and fixed in 10% buffered formalin solution (pH 7.2 to
35
7.4). Tissue fragments were then routinely prepared for histological analysis embedding in
paraffin and stained with Hematoxylin-Eosin and Gram-Chromothrope stain (25). In order to
compare the different groups, morphometric analysis was perform from histological sections
of liver fragments taken from each animal and stained with H-E. For this procedure, we used
the MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices,
California, USA) and we were evaluated the average area of lesions to compare the
microsporidian infection in animals.
36
Analysis of peritoneal and spleen cells by flow cytometry. Peritoneal cells were
obtained by successive washes from peritoneal cavity (PerC) with 10 mL of PBS solution
(Sigma, St. Louis, MO, USA). Spleen was processed and red blood cells removed with a lysis
buffer. Cell suspension was centrifuged, subsequently cells were washed with PBS 1x and re-
suspended in 100 L PBS supplemented with 1% bovine serum albumin (BSA) (PBS-BSA
1%). Each sample was incubated at 4ºC for 20 minutes with anti-CD16/CD32 to block the Fc
II and III receptors. After incubation, cells were washed, divided in two aliquots and re-
suspended in 1% PBS-BSA; then each sample was incubated with monoclonal antibodies for
surface marker analysis. The following monoclonal antibodies were used: Fluorescein-
isothiocianate- (FITC) -conjugated rat anti-mouse CD23, Peridinin Chlorophyll - (PerCP) -
conjugated rat anti-mouse CD19, Pacific Blue-conjugated rat anti-mouse CD11b,
Allophycocyanin- (APC) -conjugated rat anti-mouse F4/80 (BD-Pharmingen, San Diego, CA,
USA) for the first aliquot; and Peridinin Chlorophyll- (PerCP) -conjugated rat anti-mouse
CD19, Pacific Bblue-conjugated rat anti-mouse CD8a and Phycoerythrin- (PE) -conjugated
rat anti-mouse CD4 for the second aliquot. The cell pellet was incubated with the
fluorochrome-conjugated antibodies for 20 minutes at 4ºC, washed with PBS-BSA 1%, re-
suspended in 500 L of PBS and analysed with a FACS Canto II flow cytometer (BD
Biosciences, Mountain View, CA, USA). Cells were characterized according their surface
markers as indicated in Table 1.
37
Table 1. Surface marker combination used in this study for cytometric immunophenotyping of
peritoneal and spleen cells.
Phenotype Surface marker combination
B-1 cell CD23- CD19
+
B-2 cell CD23+ CD19
+
CD 4+ cell CD19
- CD4
+
CD 8+ cell CD19
- CD8
+
Macrophage CD19- CD11b
+ F4/80
+
Cytokines quantification. Approximately 1 mL of blood was collected from each
mice and the serum of the animals was stored at −80°C. Then, they were thawed, and IL-2,
IL-4, IL-6, IFN-γ, TNF-α, IL-17 and IL-10 were detected using the BD CBA Mouse
Th1/Th2/Th17 Cytokine Kit (BD Biosciences, CA, USA) according the manufactures’
instruction. Briefly, 25 µL of each sample was added to capture beads specific for the
cytokines and PE labeled secondary antibodies. Samples were incubated for 2 hours at room
temperature in the dark. Two-color flow cytometric analysis was performed using FACS
Canto II (BD Biosciences, Mountain View, CA) and analyzed using CBA analysis software.
Statistical analysis. Histological data were submitted to descriptive statistics and to
Kruskall Wallis and Mann-Whitney tests. For cells evaluation, the statistical program SPSS
was used (SPPS Inc., Chicago, IL, USA). The significance of differences between groups of
animals was tested with analysis of variance calculated with the minimal square method. P
values under 0.05 were considered statistically significant.
38
Results
B cells favor susceptibility for E. cuniculi infection. To understand the effect of E. cuniculi
infection in the presence or absence of B-1 cells, respectively BALB/c and BALB/c XID (B-1
cells-deficient) mice were infected by i.p. route and disease progression was followed for 14
and 21 DPI. BALB/c XID mice were more susceptible to E. cuniculi infection than BALB/c
animals, which did not show symptoms of infection and had mild lesions. Infected BALB/c
XID mice showed lethargy and increased of abdominal volume. We observed E. cuniculi
spores free or within peritoneal cells in BALB/c XID animals, but in BALB/c mice the spores
were not found. All animals showed hepato- and splenomegaly at 14 DPI, but only BALB/c
XID mice presented mild ascites with small collection of serous-bloody fluid at 14 DPI, all
these changes were more remarkable at 21 DPI. Macroscopically were also found necrotic
area in the liver, which was the most affected organ by the pathogen. Microscopy examination
of these tissues revealed the widespread presence of extracellular E. cuniculi spores with an
associated inflammatory response or the presence of parasites contained within intact
parasitophorous vacuoles without change in the surrounding tissue (Figure 1). It was also
observed discrete multifocal interstitial nephritis and pneumonia with parasitophorous
vacuoles and free spores were present in the walls of the alveoli. The quantification by
morphometric analysis of granulomatous lesions of the liver showed a significant increase of
lesion area in the BALB/c XID mice compared to BALB/c (P = 0.05) (Table 2). This data
show the higher susceptibility of animals deficient in B-1 cells to infection.
B cells and macrophages in E. cuniculi infected mice. E. cuniculi inoculation in
BALB/c XID mice did not change B-1 cell population by comparing the peritoneum in
infected and non-infected animals at 14 DPI (Figure 2B and 2C), although it was observed an
increase in the absolute number B-1 cell in BALB/c mice after infection (21 DPI) (Figure
39
2D). In the spleen, where the percentage of B-1 cells decreased significantly in BALB/c and
BALB/c XID mice infected, considering that its expression in the spleen is low (date not
shown). However, the B-2 cell populations were significantly decreased in the spleen of
infected animals by comparing BALB/c and BALB/c XID (Figure 2E). We identified
significant increase in macrophages population in the peritoneal cavity of infected BALB/c
mice, but there was no difference in BALB/c XID infected or not by E. cuniculi (Figure 2F
and G).
Increase of T lymphocytes in infected mice with E. cuniculi. Considering the
already known about T cell response against E.cuniculi infections; we investigated if there
was a relationship between theses populations and the susceptibility observed in XID mice.
Analysis for T cells’populations in PerC revealed increase in CD4+ and CD8
+ T cells both in
BALB/c and BALB/c XID mice after the infection with E. cuniculi (Figure 3), the same was
observed at 21 DPI (data not shown). At 21 DPI, we also observed an increase in CD4+ and
CD8+ T cells in the spleen analysis of the BALB/c mice infected with E. cuniculi (Figure 3).
Increase of cytokine levels in infected mice. We investigate the cytokines levels in
the blood serum of the animals. After inoculation of E. cuniculi at 14 DPI, in BALB/c mice
we found increased levels of IFN-γ and IL-6. At 21 DPI, IL-6 levels decreased and IFN-γ and
TNF were increased. In BALB/c XID mice besides IFN-γ and IL-6, IL-4 and TNF-α also
increased after E. cuniculi infection at 14 DPI, but at 21 DPI, IL-4 decreases and other
cytokines remain above normal in infected animals. There were no significant changes
observed for IL-2 in groups (Figure 4). The cytokines IL-10 and IL-17 were tested but
detectable levels of them were not found (data not shown).
40
B-1 cells adoptive transfer to mice. To further access if the lack of B-1 cells was
responsible for the susceptibility of XID mice to E. cuniculi infection; we performed adoptive
transfer of B-1 cells by i.p. route to BALB/c XID mice (XID+B-1) 7 days before being
inoculated with E. cuniculi. Although adoptive transfer of B-1 cells have become the BALB/c
XID mice (XID+B-1) slightly more resistant to infection, still have fewer symptoms and
lesions than BALB/c mice. There was statistically significant difference between the XID +
B-1 mice with control group (BALB/c) in area of lesions produced by infection by
morphometric analysis (Table 2), confirming the lower susceptibility of mice after adoptive
transfer of B cells -1. Analyzing the XID+B-1 mice, we observed that E. cuniculi infection
significantly decreased the population of B-1 and B-2 cells both in the peritoneal cavity and
spleen compared to non-infected control groups (Figure 5A-D). Similarly that occurred in
BALB/c mice, XID+B-1 mice also infected with E. cuniculi showed an increase in the
number of peritoneal macrophages (Figure 5E). The same was not observed in BALB/c XID
mice. This data shows the regulation of macrophages population of the PerC by B-1 cells.
Further, the number of T cells was increased in the peritoneal cavity of these mice after the
infection, especially CD8+ T cells. The same not occurs in the spleen (Figure 6). When we
compared the B and T cells populations after B-1 cell transfer we found higher number of
these cells in the peritoneal cavity before or after E. cuniculi infection. These results suggest
that B-1 cells can regulate the peritoneal cells population.
Surprisingly, in XID+B-1 mice all cytokines detected (IFN-γ, IL-6, TNF-α, IL-2 and
IL-4) were increased significantly in infected mice (Figure 7). The increase observed in the
cytokines levels shows a changed inflammatory milieu after E. cuniculi infection when mice
were adoptively transferred with B-1 cells. This result reinforces the role played by B-1 cells
to promote the inflammatory response by regulating the peritoneal cells populations. IL-10
and IL-17 cytokines were not detected in the process (data not shown).
41
Discussion
Successful defense against pathogens require the coordinated function of multiple host
cells. Among infections with intracellular pathogens, T cells, macrophages and dendritic cells
all play well described role in the resolution of infection. B cells primary function is generally
attributed to their ability to secrete antibodies for neutralization and/or opsonization of the
antigens. However, B cells have multiple functions, such as antigen presentation, co-
stimulation of T cells, and secretion of both pro- and anti-inflammatory cytokines (26). Herein
we demonstrated that BALB/c XID mice are more susceptible to infection with E. cuniculi
than BALB/c mice, thereby may constitute an animal model for the study of
encephalitozoonosis and their interactions with the immune system. The results suggest that
B1 and B-2 cells promote resistance to E. cuniculi infection. Adoptive transfer of B-1 cells to
BALB/c XID (XID+B-1) mice slightly more resistant to infection these mice had similar
pattern of infection in to the BALB/c XID. Bruton’s tyrosine kinase (Btk) is a cytoplasmic
kinase that is essential form mediating signals from B cell receptor and is critical for
development of B-1 cells. Animals lacking Btk have few B-1 cells, minimal antibody
responses, and can preferentially generate Th1 type immune responses following infection.
The same susceptibility was observed in X-linked immunodeficient mice (CBA/XID mice),
which were unable to control Cryptococcus neoformans dissemination to the brain during
chronic infection. Adoptive transfer of B-1 cells to XID restored peritoneal B-1 cells but did
not restore Ig M levels, as the animals continue to brain infection (27). Similar results were
observed in BALB/c XID mice infected with BCG, which were more susceptible to infection
when compared to BALB/c controls (24). We results suggest that B-1 and B-2 cells may be
related to increased susceptibility evidenced in BALB/c XID mice and XID+B-1 to
experimental infection with E. cuniculi, and may also be demonstrated by clinical and
histopathological findings.
42
After infection with E. cuniculi, the amount of macrophages increased significantly in
BALB/c mice, however in BALB/c XID mice the population of peritoneal macrophages
decreases, not significantly. These results strongly suggest that macrophage response have a
connection with the presence of B cells. To enhance these results, the B-1 cells population in
XID+B-1 mice decreases while of macrophages population increase. The B-1 lymphocytes
are involved in essential immune mechanisms. It is important to note that B-1 cell response to
pathogens is poorly understood and there are no studies related to the role of these cells in
microsporidia infections, especially by E. cuniculi like performed in this study. Almeida et al.
(18) demonstrated that B-1b cells differentiate into a mononuclear phagocyte acquiring a
myeloid phenotype following attachment to a substrate in vitro. It has been shown that the
presence of Propionibacterium acnes induces B-1 cells of myeloid lineage differentiate into
phagocytes (28). B-1 cell differentiation into phagocytes also occur in vivo using
monocyte/macrophage depletion by clodronate advice (29). It was observed LPS-elicited
macrophages in BALB/c XID mice only after the transfer of B-1 cells. Together, these results
strongly suggest that B-1 cell-derived phagocytes are a component of the LPS-elicited
peritoneal macrophage population (29).
We believe that the B-1 from peritoneum or spleen transformed into macrophages in
infected animals, capable to promote phagocytosis of E. cuniculi spores. This hypothesis is
reinforced by simultaneous observation of the increase of B-1 cells and macrophages in
infected BALB/c mice. Macrophages are a critical link between innate and adaptive
immunity. This recognition results in a milieu of host defense mediators including
chemokines, cytokines, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and radical
oxygen species. They respond to IFN-γ secreted by activated T cells to kill phagocytized
intracellular pathogens (30), by initiating a respiratory burst. The macrophages recognize the
pathogen and respond by secreting chemoattractants that recruit new cells, including
43
monocytes, to solve the infection (31). We observed that BALB/c mice infected with E.
cuniculi show increasing of the number of macrophages, T cells and plasma levels of IFN-γ,
which is responsible for the activation of macrophages and kill pathogen. These animals were
more resistant to infection than others experimentally infected group (BALB/c XID).
Moreover, adoptive transfer of B-1 cells was unable to protect BALB/c XID mice of E.
cuniculi infection, suggesting the involvement also of the B-2 cells in the defense process.
Future studies should be conducted with the adoptive transfer of B-2 cells so that their
participation should be further clarified. In addition to causing B cell defects, the absence of
Btk signaling can affect myeloid cell function in XID mice. Btk deficiency has been linked to
impaired production of reactive oxygen species (ROS) and nitric oxide (NO) in macrophages
and neutrophils (12, 13), which may have compromised the functional efficiency of the
macrophages to kill spores of E. cuniculi and clear the infection.
It is generally accepted that a protective immune response against this pathogen is
mediated by cytotoxic CD8+ T-lymphocytes (32) and their activation does not appear to be
dependent upon CD4+ T-cells (5). It was found that IFN-γ is the primary mechanism that
mediates partial protection of SCID mice in the absence of CD4+ and CD8
+ T cells (6). This
cytokine can enhance the cytotoxic activity of natural killer cells and activate macrophages to
effectively kill phagocyted microsporidial spores (33). Moreover, activated macrophages also
produce IFN-γ, which amplifies macrophage activation. Furthermore, T-cell-dependent B-cell
activation for antibody production is also important in protection against microsporidia (34,
35). We observed that CD8+ and CD4
+ T cells population increased after inoculation of E.
cuniculi in all infected groups, including in BALB/c XID mice. BALB/c mice had no
symptoms of infection and few histopathological changes were observed by E. cuniculi,
which reinforces the results described by these authors and already observed in our previous
studies (36). Data have demonstrated that resistance to lethal disease produced by E. cuniculi
44
was T cell-dependent (5, 36). However, the increase of CD8+ T cells was not sufficient to
prevent progression of the infection up to 21 days in BALB/c XID mice.
Humoral antibody response to microsporidia do contribute to resistance and facilitate
opsonization, neutralization, and complement fixation that enable phagocyte killing of
microsporidia and inhibit phagosome-lysossome fusion (37). SCID mice treated with CD4+ T
cells and monoclonal antibodies to E. cuniculi spore wall proteins survived significantly
longer than mice reconstituted only within the CD4+ T cells, supporting a contribution of
antibodies to resistance in vivo as well (34). We suggest that the absence of B cells in BALB/c
XID and possible modification of antibody production may have affected the response of T
cells and macrophages, rendering them more susceptible mice with E. cuniculi.
Proinflammatory responses via Th1 cytokines, such as IFN-γ, TNF-α, and IL-12, as
well as reactive oxygen and nitrogen intermediates are important for early stages of resistance
to Encephalitozoon infections, as shown in experiments using murine models and ex vivo
human studies (5, 38, 39, 40). We observed that the BALB/c infected show increased of tree
plasm cytokines, IFN-γ, IL-6 and TNF-α. Already in BALB/c XID mice, plus IFN-γ, IL-6 and
TNF-α was also an increase of IL-4. In addition to these cytokines, XID+B-1 mice infected
had also IL-2 increased. IFN-γ appears to be especially important in both innate and T cell
mediated protection against E. cuniculi (5, 35). This is reinforced by the dates observed study
since all the infected animals had an increase of INF-γ. The mice showed increased
susceptibility to infection, BALB/c XID and XID+B-1 were also increased by other
cytokines. Perhaps this condition indicated a compensatory response since the effectors cells
were unable to remove the agent. The relevance of classical Th2 cytokines during
microsporidia infections is less well understood. Gene expression and circulating levels of IL-
4 during E. cuniculi infection were undetectable in infected mice animals (5), however we
observed an increase of this cytokine.
45
In summary, our results indicate that B cells (B-1 and B-2) play an important role in
the defense response against microsporidiosis since BALB/c XID mice were more susceptible
to infection by E. cuniculi, thus XID mice are a model for the study of encephalitozoonosis.
Acknowledgments. We would like to thank Magna Aparecida Mautauro Soares (Butantan
Institute) for making the material is light microscope.
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Figures
Figure 1. Hepatic granuloma in BALB/c (A) and BALB/c XID (B) mice infected with E.
cuniculi - 21 DPI; (400x, HE), note the difference in lesion size between the groups.
Table 2. Comparison of liver lesions in BALB/c, BALB/c XID and XID+B-1 cell mice
infected with E. cuniculi at 14 and 21 days Post-Infection.
Day Post-Infection BALB/c mice
mean of lesion
areaa (±SD)
BALB/c XID mice
mean of lesion
area (±SD)
XID+B-1 cell
mean of lesion
mean area (±SD)
14 days 42.93 ( 8.8) 100.3 ( 1.6)* 64.5 ( 17.4)
21 days 65.1 ( 14.8) 112.1 ( 16.1)* 85.3 ( 12.1)
Total 54.0 ( 15.7) 106.2 ( 8.3)* 74.8 ( 14.7)
a Measured in pixels/1000
* Means significant difference (p < 0.05) between infected groups.
51
Figure 2. Evaluation of the presence of B cells and macrophages populations in the PerC and spleen of
BALB/c and BALB/c Xid mice. A) Representative dot plot of lymphocytes gate from PerC and spleen
according Sider Scatter (SSC) and Forward Scatter (FSC) parameters. B) The presence of B-1 and B-2
cells populations in the PerC of BALB/c and BALB/c XID mice infected with E. cuniculi - 21DPI or
their uninfected controls mice. C) Representative dot plot of the presence of B-1 and B-2 cells in the
PerC of the BALB/c and BALB/c XID mice infected - 14 DPI or not infected with E. cuniculi. D) The
presence of B-2 cell population in the spleen of BALB/c and BALB/c XID mice E. cuniculi infected -
14 DPI or their uninfected controls mice. E) Representative dot plot of the presence of B-2 cell
population in the spleen of BALB/c and BALB/c XID mice infected or not with E. cuniculi. F)
Macrophage populations in the PerC and spleen of BALB/c and BALB/c XID mice infected with E.
cuniculi or their uninfected controls mice. G) Representative dot plot of the presence of macrophage
populations in the PerC and spleen of BALB/c mice infected with E. cuniculi. Two-way ANOVA test
with multiple comparisons Bonferroni posttests shows *** p<0,001. These results are representative of
2 independent experiments.
52
Figure 3. Evaluation of the presence of T cell populations in the PerC and spleen from BALB/c and
BALB/c XID mice infected with E. cuniculi or their uninfected control group. A) Lymphocytes gate
identified according the Sider Scatter (SSC) and Forward Scatter (FSC) parameters and excluded
CD19+ B cells population gate. B) The presence of CD4
+ and CD8
+ T cells in the PerC of BALB/c and
BALB/c XID mice infected with E. cuniculi - 14 DPI or their uninfected control mice. C)
Representative dot plot of the presence of CD4+ and CD8
+ T cells in the PerC of BALB/c and BALB/c
XID mice infected - 14 DPI or not with E. cuniculi. D) The presence of CD4+ and CD8
+ T cells in the
spleen of BALB/c and BALB/c XID mice infected with E. cuniculi - 21 DPI or their uninfected
control mice. E) Representative dot plot of the presence of CD4+ and CD8
+ T cells in the spleen of
BALB/c and BALB/c XID mice infected - 14 DPI or not with E. cuniculi. Two-way ANOVA test with
multiple comparisons Bonferroni posttests shows * p<0,05, ** p<0,01 and ***p<0,001. These results
are representative of 2 independent experiments.
BALB/c XID0
10
20
30
40
*
CD
4+
T c
ells (
%)
BALB/c XID0
5
10
15
**
CD
8+
T c
ells (
%)
53
Figure 4. Cytokines serum levels (IL-2, IL-4, IL-6, IFN-γ and TNF-α) obtained from BALB/c
and BALB/c XID mice infected with E. cuniculi or their uninfected control groups with 14
DPI (A) and 21 DPI (B) Data are representative of two independent experiments (n=5). Two-
way ANOVA test with multiple comparisons Bonferroni posttests shows *p<0,05, **p<0,01
and ***p<0,001.
54
Figure 5. Evaluation of the presence of B cell and macrophage populations in the PerC and
spleen of BALB/c XID mice (XID+B-1 mice) adoptively transferred with cultured 106 B-1
cells from BALB/c mice. A) B-1 cell populations in the spleen of XID+B-1 mice infected
with E. cuniculi - 14 DPI or their uninfected control mice. B) B-1 cell populations in the
spleen of XID+B-1 mice infected with E. cuniculi - 21 DPI or their uninfected control mice
Representative dot plot of the presence of B-1 and B-2 cells in the PerC of XID+B-1 mice
infected or not with E. cuniculi. C) B-1 cell populations in the spleen of XID+B-1 mice
infected with E. cuniculi - 14 DPI or their uninfected control mice. D) B-2 cell populations in
the spleen of XID+B-1 mice infected with E. cuniculi- 14 DPI or their uninfected control
mice. E) Macrophages populations in the PerC and spleen of E. cuniculi XID+B-1 infected
mice - 14 DPI or their uninfected control mice. Unpaired T test showed *p<0,05, **p<0,01
and ***p<0,001. These data are representative of 2 independent experiments.
55
Figure 6. Evaluation of the presence of T cells populations of BALB/c XID (XID+B-1) mice
adoptively transferred with cultured 106 B-1 cells from BALB/c mice infected with E.
cuniculi – 14 DPI or their uninfected control mice. A) The lymphocytes gate determined
according Sider Scatter (SSC) and Forward Scatter (FSC) parameters and excluded CD19+ B
cells gate. B) The presence of CD4+ and CD8
+ T cells in the PerC of XID+B-1 mice infected
with E. cuniculi or their uninfected control mice. C) Representative dot plot of the presence of
CD4+ and CD8
+ T cells in the PerC from XID+B-1 mice infected or not with E. cuniculi. D)
The presence of CD4+ and CD8
+ T cells in the spleen from XID+B-1 mice infected or not
with E. cuniculi. E) Representative dot plot of the presence of CD4+ and CD8
+ T cells in the
spleen from XID+B-1 mice infected or not with E. cuniculi. Unpaired T test showed *p<0,05
and ***p<0,001.Data are representative of 2 independent experiments.
56
Figure 7. Citokynes serum levels. IFN-γ, TNF-a, IL-2, IL-4 and IL-6 from E. cuniculli
infected XID+B-1 mice and their uninfected control group A) Citokynes serum levels. IFN-γ,
TNF-a, IL-2, IL-4 and IL-6 from E. cuniculli infected XID+B-1 mice – 14 DPI and their
uninfected control group. B) Citokynes serum levels. IFN-γ, TNF-a, IL-2, IL-4 and IL-6 from
E. cuniculli infected XID+B-1 mice – 21 DPI and their uninfected control group Unpaired T
test showed *p<0,05 and **p<0,01. Data are representative of 2 independent experiments
(n=5).
57
3 CONSIDERAÇÕES FINAIS
Independente de quem seja o hospedeiro, a resolução da infecção depende fortemente
da eficiência da imunidade induzida pelo microsporídio, a qual não está ligada somente às
características internas do hospedeiro como também à capacidade de manipulação e evasão do
parasita da resposta imunológica. Enquanto a imunidade adaptativa é claramente essencial
para a eliminação desses parasitas, há evidências de que a resposta iniciada pelo braço da
imunidade inata pode vir a definir a sobrevivência ou não do parasita.
A imunidade celular tem sido considerada o componente central da resposta imune nas
infecções por microsporídios em modelos experimentais e em humanos, e neste sentido o
estudo demonstrou uma tendência participativa das células B-1 e B-2 como células
facilitadoras na progressão da infecção pelo E. cuniculi, contribuindo e abrindo campo para
novas investigações sobre o assunto.
58
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