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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

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Page 1: UNIVERSIDADE PAULISTA Artigo1: CULTURE AND … · Costa, Lidiana Flora Vidôto da. Culture and propagation of microsporidia of veterinary interest - Culture of microsporidia; B-1

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

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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, 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

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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.

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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

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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!

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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.

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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.

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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

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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

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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

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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.

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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]

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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

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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

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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

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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

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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].

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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

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(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

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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

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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.

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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

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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

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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).

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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).

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Fig. 3. Culture smears of Encephalitozoon cuniculi spores stained with Chromotrope (a) and

Gram-Chromotrope technique (b).

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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]

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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.

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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).

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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

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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

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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.

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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.

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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.

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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

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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).

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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).

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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.

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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

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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

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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.

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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.

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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.

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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 (

%)

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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.

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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.

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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.

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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).

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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.

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