UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO 
DE MESQUITA FILHO” 

FACULDADE DE MEDICINA 
 
 
 
 
 
 
 
 
 

Débora de Fátima Almeida 
 
 
 
 
 
 
 
 
 
 
 

Fibrose pulmonar na paracoccidioidomicose: influência do 
fungo e do hospedeiro na fibrogênese 

 
 
 
 
 
 
 
 
 
 

Dissertação apresentada à Faculdade de 
Medicina, Universidade Estadual Paulista “Júlio 
de Mesquita Filho”, Câmpus de Botucatu, para 
obtenção do título de Mestre em Doenças 
Tropicais.  

  
  
  
 

 
 
 

Orientador: Prof. Dr. James Venturini 
 
 
 

 

Botucatu 
2017 

 
 



 

 
 

Débora de Fátima Almeida 
 
 
 
 
 
 
 
 
 

Fibrose pulmonar na paracoccidioidomicose: influência do 
fungo e do hospedeiro na fibrogênese 

 
  
  

 
 
 
 
 
 
 
 
 
 
 

Dissertação apresentada à Faculdade de 
Medicina, Universidade Estadual Paulista 
“Júlio de Mesquita Filho”, Câmpus de 
Botucatu, para obtenção do título de 
Mestre em Doenças Tropicais.  

  
 
 
 
 
 
 
 
 
 
 
 

Orientador: Prof.Dr. James Venturini 

 
 
 
 

Botucatu 
2017



 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 



 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“A verdadeira riqueza de um homem é o bem que ele faz ao 

seu semelhante.” 

(Mahatma Gandhi)



 
 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dedicatória 
 

Dedico este trabalho a todos os pacientes portadores da 
Paracoccidioidomicose. Espero que estes achados possam 

auxiliar futuros estudos. 



 

 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Agradecimentos 



 

 
 

Agradecimentos 
 

À minha mãe Fátima e ao meu pai Luiz por todo o amor e cuidado. 
Por sempre acreditarem na minha capacidade e me apoiarem nas 

dificuldades da vida. 
 

Ao meu grande amor Bruno Donanzam, por estar do meu lado nos 
momentos bons e ruins sempre me dando colo nos momentos de 

fraqueza, puxão de orelha quando foi preciso e acima de tudo por 
sempre querer o meu bem. Obrigada por me fazer rir, por me dar 

esperança e por todo o amor. 
 

Ao meu orientador Dr. James Venturini por tornar possível todas as 
minhas conquistas, me permitir ser sua primeira orientada na 

iniciação cientifica e me acompanhar também no mestrado. Foi uma 
honra fazer parte do grupo tão bonito que é o LIPE. Conviver com 

você me fez amadurecer tanto na vida profissional como na pessoal. 
Hoje eu percebo o quanto eu cresci convivendo com você e te desejo 

cada vez mais sucesso.  
 

À Dra. Tatiani Donato por me ajudar na fase mais difícil do Mestrado 
e acima de tudo pela amizade. Você não só me ajudou a superar as 

dificuldades, você tornou esse trabalho possível. Saiba que me inspiro 
muito no seu jeito leve de viver e sou muito agradecida por toda 

ajuda. 
 

Às minhas amigas e companheiras de longa data Amanda Ribeiro e 
Angela Finato que estão comigo desda iniciação cientifica. Somos 

pessoas tão diferentes e como foi bom conhecer vocês! Foram longos 
anos de amizade, aprendizado, superação de dificuldades e trabalho 

em equipe. Crescemos juntas nesse mundo acadêmico. Vocês marcaram 
a minha vida e quero que saibam que mesmo que sigamos caminhos 

diferentes no futuro, sempre guardarei vocês no meu coração. 
 

Aos meus companheiros do Lipe e todos os “agregados”: Barbara Mello, 
Angela Liberali, Raul, Caio, Rodolfo Ferreira, Victor Resende, Karol, 

Adriely, Bia, Jonatas, Luiz Gustavo, Vitória, Silas, Thainá, Gaby, 
Anne, Laysla Leite e Pedro Hori, vocês presenciaram todas as coisas 

boas e ruins, partilharam conselhos, ensinamentos e risadas. Estar do 
lado de vocês deixou com certeza as coisas mais divertidas e leves por 

mais que houvesse dificuldades. Muito obrigada por todo o carinho e 



 

 
 

amizade.  
Um agradecimento especial para Babi Amorim e Karoline Reis por 

toda a ajuda em todo o meu percurso e por dividir comigo as 
dificuldades da cultura celular. Vocês foram grandes amigas e me 

ajudaram até o último minuto, nunca me esquecerei de tudo que 
fizeram e sempre guardarei vocês no meu coração. Muito obrigada 

pelo amor, companheirismo e amizade acima de tudo. 
 

À Dr. Maria Sulei Parreira de Arruda pela oportunidade de estagiar 
no LIPE, pelo incentivo a sempre fazer o melhor, e por todo carinho. 

 
Às minhas amigas Thais Fraga, Camila Marchetti e Luiza Mimura 

por todos os ensinamentos, conselhos e apoio em toda a minha jornada 
e formação. Aprendi muito com vocês.  

 
À Dra. Priscilla Maria Ponce Vareda e ao Dr. José Roberto Bosqueiro 

pelos ensinamentos de Western Blot e pelo carinho. 
 

Ao Prof. Dr. Fábio Porto-Foresti e ao LaGenPe por abrir as portas 
sempre quando precisei. 

 
Ao Dr. Willian Fernando Zambuzzi e ao Labio pelos ensinamentos e 

ajuda com o Western Blot no início do mestrado. 
 

À Heloisa e Dra. Elenice Deffune por me acolherem no início do 
mestrado e ensinar as boas práticas da cultura celular.  

 
Ao Dr. Rinaldo Poncio Mendes por disponibilizar o soro de pacientes 

para este estudo e toda a ajuda concedida. E também, ao grupo de 
micoses sistêmicas da Faculdade de Medicina de Botucatu. Thaty, 

Drica, Lari, Bia e Camila por toda a ajuda e risadas nas disciplinas do 
mestrado. 

 
Aos amigos do Instituto Lauro de Souza Lima Mariane, Keren, 

Giovanna, Elderson, e Rodrigo pela parceria e em todas as fases do 
mestrado e pelas viagens à Botucatu. Às Dra.Vania, Dra. Ana Carla e 
Dra. Maria Renata pelo acolhimento e ajuda nos momentos de sufoco. 

 
Aos funcionários da seção técnica de pós-graduação e à Bruna Quirino 

Jorgetto pela atenção a nós destinada. 
 

Ao programa de Pós-Graduação em Doenças Tropicais e a todos os 



 

 
 

funcionários da Faculdade de Medicina de Botucatu por toda atenção 
e ajuda quando necessário. 

 
Aos funcionários da Faculdade de Ciências da UNESP de Bauru. 

 
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 

(CAPES) pela concessão da bolsa de estudos. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 
 

 

 

Lista de abreviaturas e siglas 
 

ATP: Trifosfato de adenosina 

BSA: Albumina do soro bovino  

CMX: Cotrimoxazole 

COL I: Colágeno tipo I 

COL III: Colágeno tipo III 

COL IV: Colágeno tipo IV 

CONCEA: National Council for the Control of Animal Experimentation 

CXCL: C-X-C motif chemokine ligand  

DMEM: Dulbecco's Modified Eagle's Medium 

DMSO: Dimetilsulfóxido 

EMT: Transição epitelial-mesenquimal 

EndMT: Transição endotelial-mesenquimal 

FA: Forma aguda 

FC: Forma crônica 

FGFb: Fator de crescimento de fibroblasto básico/ Basic fibroblast growth factor 

FP: Fibroblastos pulmonar/ Pulmonary fibroblast 

FR: Forma residual 

gp43: Glicoproteína de 43KDa 

GPY: Glucose-peptone-yeast extract agar 

IFN-β: Interferon-beta 

IFN-γ: Interferon-gamma 

Ig: Imunoglobulina 

IL: Interleucina 

ITC- Itraconazole 

MMP- Metaloproteinase de matriz 

MRC-5- Linhagem cellular de fibroblastos pulmonar humano 

NFkB: fator nuclear kappa B 

PAMPS- Padrões moleculares associados a patógenos 

PBS: Phosphate buffered saline 

PbSAP: aspartil protease 

PCM: Paracoccidioidomicose 

PGE2: Prostaglandina E2 

SBF: Soro bovino fetal 

SDF-1: Stromal cell-derived factor 1 

TBST: Tris Buffered Saline with Tween 20  

TGF-β1: Fator de transformação do crescimento beta 1 

Th: T helper 

TIMP: Inibidor tecidual de metaloproteinase/ Tissue inhibitor of metalloproteinase 

TLR: Receptor tipo Toll 

VEGF: Fator de crescimento endotelial vascular  

α-SMA: Alpha-actina de músculo liso / Alpha-Smooth Muscle Actin 

 

 



 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Resumo 



 

 
 

Resumo  
 

ALMEIDA, D. F. Fibrose pulmonar na paracoccidioidomicose: influência do fungo e do 

hospedeiro na fibrogênese. 2017. Dissertação (Mestrado) – Faculdade de Medicina de 

Botucatu, Universidade Estadual Paulista, Botucatu, 2017. 

 

Paracoccidioidomicose (PCM) é uma micose sistêmica causada por fungos do gênero 

Paracoccidioides; suas principais formas clínicas são aguda/subaguda e crônica (FC). Restrita 

à América Latina, a PCM apresenta alta incidência no Brasil, Colômbia e Venezuela, 

especialmente entre os trabalhadores rurais. A maioria dos pacientes com a forma crônica da 

doença, mesmo após tratamento eficaz, apresentam sequelas, incluindo fibrose pulmonar 

(FP). Os problemas sociais, econômicos e psicológicos desencadeados pela FP são 

subestimados. Apesar da fibrogênese na PCM ser reconhecida como um processo precoce, 

seus mecanismos não estão totalmente conhecidos. Assim, o presente estudo tem por objetivo 

avaliar a influência de componentes dos isolados fúngicos e do hospedeiro sobre fibroblastos 

pulmonares. Foram utilizados fibroblastos pulmonares humanos da linhagem MRC-5 e 

fibroblastos pulmonares murinos, isolados de camundongos BALB/c. Fibroblastos foram 

cultivados in vitro com exoantígenos de isolados das espécies P. brasiliensis (Pb18 e Pb326) e 

P. lutzii (Pb01, Pb8334 e Pb66); gp43 purificada, e soros de pacientes com FC em diferentes 

momentos do tratamento (antes do tratamento, cura clínica, cura sorológica e cura aparente). 

Após 24 horas de cultivo, foram avaliadas a proliferação celular e produção de citocinas e 

fatores de crescimento por fibroblastos murinos e humanos. Nossos resultados demonstraram 

que componentes do fungo interferem em fibroblastos pulmonares, pela indução da 

proliferação, aumento dos níveis de TGF-β1 e diminuição dos níveis de IL-6 e VEGF e FGFb. 

Além disso, nós observamos que exoantigenos induziram altas produções de pró-colágeno I 

por fibroblastos humanos. Gp43 induziu aumento nos níveis de TGF-β1 em fibroblastos 

humanos. Em conclusão, nossos resultados demonstram pela primeira vez que componentes 

de P.brasiliensis e P.lutzzi interferem na fibrogênese por agir diretamente na biologia de 

fibroblastos pulmonares. Além disso, evidenciamos que soros de pacientes com FC interferem 

diferentemente na proliferação e ativação de fibroblastos pulmonares de acordo com cada 

momento do tratamento. 

 

Palavras-chave: paracoccidioidomicose, fibroblasto, fibrose pulmonar. 



 

 
 

 

Abstract  

 
ALMEIDA, D. F. Pulmonary Fibrosis in Paracoccidioidomycosis: Influence of fungus 

and host in Fibrogenesis. 2017. Thesis (Master) – Faculty of Medicine of Botucatu, 

Universidade Estadual Paulista, Botucatu, 2017. 

 
Paracoccidioidomycosis (PCM) is an endemic mycosis caused by fungi of genus 

Paracoccidioides; its main clinical forms are acute/subacute form and the chronic form (CF). 

Endemic in Latin America, PCM presents high incidence in Brazil, Colombia and Venezuela, 

especially among rural workers. Most part of patients with FC exhibits sequelae even after an 

effective fungal treatment, presenting pulmonary fibrosis (PF). The social, economic and 

psychological issues caused by PF are still underestimated. In PCM, fibrogenesis is 

recognized as an early process; however, its mechanisms are unknown. Thus, the present 

study aimed to evaluate the modulatory and functional influence of fungal and host 

components on pulmonary fibroblast. For this, it was utilized human pulmonary fibroblasts of 

cell line MRC-5 and murine pulmonary fibroblasts isolated of BALB/c mice. Next, cells were 

cultivated with different concentrations of isolates of P. brasiliensis (Pb18 and Pb326) and P. 

lutzii (Pb01, Pb8334 and Pb66); purified gp43, and serum of patients with CF in different 

moments of treatment (before treatment, clinical cure, serological cure and apparent cure). 

After 24 hours, cellular proliferation and production of cytokines and grow factors by murine 

and human pulmonary fibroblasts were evaluated. Our results showed that the fungal 

components interfered in fibroblast by induce proliferation and increased levels of TGF-β1 

and decreased IL-6, VEGF and bFGF. In addition we observed that exoantigens induced high 

production of pro-collagen I by human fibroblasts. Gp43 induced increased levels of TGF-β1 

by human cells. In conclusion, our findings showed at first time that components of 

P.brasiliensis and P.lutzii interfere in the fibrogenesis by directly act in the biology of 

pulmonary fibroblast. Furthermore, we verified that serum of FC-patients interfere in 

proliferation and activation of pulmonary fibroblasts according to each moment of treatment. 

 

 

Keywords: paracoccidioidomycosis, fibroblast, pulmonary fibrosis 

 



 

 
 

 

 

 

 

 

Sumário 
 

1. INTRODUÇÃO ....................................................................................................... 1 

1.1. Paracoccidioidomicose .......................................................................................... 1 

1.2. Fungos do gênero Paracoccidioides....................................................................... 2 

1.3. Resposta imune na PCM ........................................................................................ 3  

1.4. Fibrose pulmonar na PCM..................................................................................... 4 

1.5 Mecanismos relacionados à fibrose pulmonar........................................................ 5  

1.6. Papel dos fibroblastos na inflamação...................................................................... 6 

1.7. Racional do estudo.................................................................................................. 7 

2. OBJETIVOS ............................................................................................................ 8 

2.1. Objetivo Geral......................................................................................................... 8 

2.2 Objetivos Específicos............................................................................................... 8 

3. REFERÊNCIAS BIBLIOGRÁFICAS  ................................................................. 9 

4. MANUSCRITO I.................................................................................................... 16 

5. MANUSCRITO II …………………………………………………….................. 31 

6. CONCLUSÃO......................................................................................................... 56 

 

 

 

 

 

 



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introdução 
 



1 
 

 

1.INTRODUÇÂO 

 

1.1. Paracoccidioidomicose 

A Paracoccidioidomicose (PCM) é uma micose sistêmica granulomatosa causada por 

fungos termo-dimórficos do gênero Paracoccidioides [1,2]. A PCM foi descrita pela primeira 

vez, em 1908, por Adolfo Lutz. O termo paracoccidioidomicose foi instituído em 1971 na 

reunião de micologistas das Américas em Medellin (Colombia) e persiste, até hoje, como 

nomenclatura oficial. 

O nicho e habitat deste gênero de fungos ainda não foram totalmente caracterizados, 

porém, sabe-se que colonizam solos úmidos na fase micelial e podem ter como hospedeiro 

natural o tatu Dasypus novemcintus [3,4]. A PCM representa grande impacto na saúde pública 

em países da América Latina, sendo Brazil, Venezuela e Colômbia os mais acometidos com 

prevalência de mais de 15.000 casos relatados entre 1930 e 2012 [5]. E no Brasil, foi 

considerada em 2009 a nona maior causa de mortalidade entre doenças parasitárias [6]. São 

considerados fatores de risco para a manifestação da doença a desnutrição, tabagismo e 

consumo de álcool [7] 

A infecção ocorre através da inalação de conídeos e/ou fragmentos de hifas (forma 

infectante) que atingem os pulmões e se transformam em leveduras (forma patogênica) [8]. 

Uma vez dentro do hospedeiro pode ocorrer disseminação do parênquima pulmonar para os 

linfonodos regionais, podendo também se disseminar via corrente sanguínea e/ ou linfática 

[9]. 

As principais formas clínicas são a forma aguda/subaguda (FA) e forma crônica (FC). 

A FA é caracterizada por acometer principalmente crianças e adultos jovens, apresenta 

história clínica de curta duração (mediana de dois meses) e atinge órgãos do sistema 

fagocítico mononuclear como fígado, baço e linfonodos. A FC acomete em geral adultos com 

30 anos ou mais, apresenta história clínica de longa duração (acima de 6 meses) e acomete 

principalmente pulmões e mucosa das vias aero-digestivas. A maioria dos pacientes 

acometidos é do sexo masculino, trabalhadores rurais, que assim se tornam impossibilitados 

de exercerem suas atividades devido aos problemas respiratórios decorridos da doença, 

levando a diversos problemas socioeconômicos e psicológicos ainda subestimados. Pacientes 

que apresentam sequelas após o tratamento são classificados como FR [10].  

Para o tratamento da PCM são utilizadas drogas como os derivados azólicos 

cetoconazol e itraconazol (ITC) e também a associação sufametoxazol-trimetoprim, também 

chamada de cotrimoxazol (CMX) [11]. A sulfadiazina é pouco utilizada e a anfotericina B é 



2 
 

 

reservada para casos mais graves, devido à sua toxicidade. O ITC é atualmente, a melhor 

escolha para o tratamento da PCM, Cavalcante et al. evidenciaram que sua utilização 

propiciou cura clínica mais precoce e uma melhor tolerância [12]. 

 

1.2 Fungos do gênero Paracoccidioides 

O gênero de fungos Paracoccidioides pertence à ordem Onygenales e a família 

Ajellomycetaceae [13] e é composto por duas espécies: Paracoccidioides brasiliensis e o 

Paracoccidioides lutzii. Sendo ainda a espécie P.brasiliensis divida em 5 espécies crípiticas: 

S1a e S1b que compreende isolados distribuídos por toda a América Latina, principalmente 

Brasil, Argentina e Paraguai [14,15], PS2 composta por isolados venezuelanos e brasileiros; 

PS3 restrita na Colômbia e PS4 restrita na Venezuela. O isolado Pb01 é filogeneticamente 

distinto das outras cepas, definido como Paracoccidioides lutzii e principalmente isolado em 

pacientes da região Centro-Oeste [2,14] e região Norte do Brasil, mais precisamente no 

Estado do Pará [16].  

Este gênero ainda possui a característica do termo-dimorfismo, ou seja, possui a 

capacidade de mudar morfologicamente dependendo da temperatura. O fungo se encontra na 

forma micelial (forma infectante) e a 37°C no interior do hospedeiro transforma-se na forma 

leveduriforme (forma patogênica) [8]. 

Em relação ao metabolismo, são capazes de secretar de 30 à 35 proteases como 

ubiquitinil hidrolase (família C19) e metaloendopeptidase dependente de ATP (família M41) 

que são exclusivas desses fungos. Além disso, secretam enzimas que degradam açúcares 

abundantes em paredes celulares de plantas, indicando que esse grupo pode ser capaz de se 

alimentar de celulose, enquanto crescem no solo [17]. 

Em cultura, estes fungos na fase micelial apresentam o crescimento lento (20 – 30 

dias), a colônia apresenta micélio aéreo curto de cor branca ou creme e, muitas vezes, 

apresenta pigmento castanho na parte reversa, quando aderidas ao ágar, apresentam 

rachaduras e irregularidades no contorno. À temperatura entre 35-37°C, em meio de cultura, a 

colônia se desenvolve mais rapidamente (8-12 dias), apresenta um aspecto cerebriforme e 

consistência macia, cor creme e pode ser facilmente removida do substrato. [18].  

Sua parede celular é composta principalmente por polissacarídeos como quitina, 

polímeros de glicose, proteínas e lipídeos podendo variar de acordo com sua morfologia. A 

quitina apresenta-se em maior quantidade na forma leveduriforme, podendo alcançar 43% do 

peso seco da parede celular e no máximo 13% da parede celular micelial. A camada externa 

da parede na fase leveduriforme é constituida por α-1-3glucana predominante e por β-1-3-



3 
 

 

glucana, na fase de micélio [19]. Especula-se que essa dinâmica de composição de parede 

representa um mecanismo de escape do fungo, uma vez que fagócitos humanos são incapazes 

de digerir a α-1,3 glucana, por produzirem apenas a enzima β- glucanase, sendo capazes de 

digerir a parede somente quando o fungo se encontra na fase micelial [20]. 

Os vários isolados podem apresentar diferenças em sua composição e também podem 

produzir quantidades variáveis de antígenos. Entre as proteínas intracelulares estão: a 

glicoproteína de 43 kDa (gp43) principal antígeno secretado e o mais específico para o 

diagnóstico de infecção por P. brasiliensis [20–22]; aspartil protease (PbSAP) de 66-kDa 

[23]; chaperona mitocondrial de choque térmico Mdj1 [24] e proteína gp75 que tem atividade 

fosfatase [25]. 

 

1.3. Resposta Imune na PCM 

A resposta imune do indivíduo à infecção pelo Paracoccidioides spp determina a 

evolução da infecção. A resposta pode variar de acordo com o estado nutricional, tamanho do 

inoculo, carga genética e gênero do indivíduo. A ação do peróxido de hidrogênio (H2O2) 

produzido pelos macrófagos [26], é a principal causa da morte do Paracoccidioides spp. A 

produção deste metabólito é potencializada pela resposta imune adaptativa do tipo Th1, 

caracterizada pela produção de IFN-γ pelos linfócitos ativados, atraídos para o sítio da lesão. 

Qualquer descompasso, deficiência ou alteração nesse processo leva ao estabelecimento da 

doença e a sua progressão.  

O comportamento imunológico é distinto entre as formas clínicas da doença [27–30]. 

Em pacientes com FA, observa-se produção de citocinas do perfil Th2/Th9 (IL-4, IL-5 e IL-9). 

A produção elevada dessas citocinas acentua a deficiência da resposta imune celular, levando 

à ausência de reatividade frente a antígenos de Paracoccidioides spp. Além disso, as citocinas 

de perfil Th2/Th9 induzem produção muito elevada de anticorpos do isotipo IgG4 [31] que 

apresentam reduzida capacidade de fixação de complemento e pequena afinidade pelos 

receptores FcR, fagocitose e, consequentemente, a eliminação do fungo, tornam-se 

comprometidas. 

A FC, que ocorre devido à reativação de focos latentes e, portanto, após o hospedeiro 

ter organizado uma resposta imune adaptativa eficiente frente ao Paracoccidioides spp, 

apresenta instalação lenta e progressiva. À exceção dos que apresentam a forma grave, 

pacientes com a FC apresentam resposta Th1 preservada, isto é, são reatores ao teste 

intradérmico com paracoccidioidina. Apresentam ainda, intensa produção de citocinas pró-

inflamatórias como TNF-α, IL-1β, IL-17 e de peróxido de hidrogênio. Embora esses 



4 
 

 

mediadores sejam importantes para a eliminação do fungo, sua superprodução induz efeitos 

deletérios e não conferem proteção. A produção de anticorpos também pode estar elevada, e 

se caracteriza por imunoglobulinas dos isotipos IgG1 e IgG2 [31], que possuem maior 

capacidade de fixação de complemento e maior afinidade pelos receptores FcR (IgG1 > IgG2 

> IgG4). Embora esse conjunto de elementos seja importante para a eliminação do fungo, a 

capacidade de lise microbiana não acompanha a multiplicação do fungo e o indivíduo adoece. 

Por se tratar de processo inflamatório crônico, esses pacientes, em geral, já apresentam fibrose 

logo no primeiro atendimento clínico, quando se observa produção mais acentuada de TGF-β1 

e do fator de crescimento de fibroblasto básico (FGFb) [32]. 

 

1.4. Fibrose Pulmonar na PCM 

Após tratamento antifúngico eficaz, pacientes com FC podem apresentar fibrose nos 

diferentes órgãos comprometidos, os pulmões, particularmente, também exibem o enfisema. 

Essas sequelas levam ao comprometimento funcional e incapacitação do paciente. A função 

pulmonar poucas vezes é normal, revelando padrão obstrutivo em 85% dos casos, com 

frequências iguais de obstrução leve, moderada e intensa [33–35]. Os achados necroscópicos 

de pacientes com PCM revelam que a fibrose pulmonar é caracterizada por extensas áreas de 

depósito de colágeno próximas à região hilar, envolvendo outras estruturas como linfonodos, 

brônquios e artérias. As fibras colágenas se encontram na periferia dos granulomas e se 

estendem a brônquios e vasos sanguíneos próximos. Além disso, Tuder et al. [36] observaram 

proliferação de fibras reticulares em áreas distintas do processo granulomatoso, indicando que 

o próprio fungo ou seus componentes podem induzir a produção de colágeno.  

Apesar da sua importância, poucos estudos têm focado especificamente a fibrogênese 

pulmonar que ocorre na PCM. Cock et al.[37] demonstraram em modelo experimental murino 

que esse processo é precoce. Araujo [38], em estudos necroscópicos demonstrou a presença 

de fibrose em pacientes que não receberam tratamento antifúngico, mais recentemente, 

Venturini et al. [32] demonstraram que a produção de TGFβ1 e FGF por monócitos de 

pacientes com FC já se encontra elevada no momento do diagnóstico, e em outro estudo, foi 

demonstrado que após a introdução do tratamento antifúngico, ocorre aumento da fibrose 

pulmonar [39]. Em geral, durante a evolução da PCM, a função pulmonar encontra-se alterada 

observando-se hipoxemia com predominância da perfusão sobre a ventilação pulmonar [40]. 

De acordo com os autores, é possível que o envolvimento pulmonar ocorra na fase precoce da 

doença, uma vez que pacientes com padrão obstrutivo e misto apresentam envolvimento 

precoce das vias respiratórias com alterações na difusão e ventilação. Mesmo com a regressão 



5 
 

 

das lesões radiológicas após o tratamento, não há recuperação da função pulmonar sendo 

comum a dispneia a grandes e pequenos esforços [34].  

 

1.5. Mecanismos relacionados à fibrose pulmonar   

Fibrose é uma patologia associada com um amplo espectro de injúrias que podem ser 

causadas por diversas razões, incluindo tratamento antibiótico, infecção e exposição 

ambiental [41–43].  

Denomina-se fibrose a formação ou desenvolvimento de tecido conectivo em órgãos 

ou tecidos quando em processo de cicatrização. Ocorre em determinadas circunstâncias em 

que há aumento do estroma conjuntivo de um órgão decorrente de cicatrização ou de um 

processo reacional em que a produção de matriz extracelular pode não estar relacionada com 

o processo reparativo. Em consequência das modificações na arquitetura do órgão e das 

alterações na função das células parenquimatosas, podem surgir distúrbios funcionais [44]. 

Os mecanismos que induzem a fibrose ainda não foram elucidados, porém, acredita-se 

que seja resultado de uma desregulação durante o reparo tecidual [45]. O reparo tecidual 

ocorre em 4 estágios: 1- Coagulação, 2- Homeostase, 3-Inflamação, 4-Proliferação e 

remodelação [46]. Durante a proliferação e remodelação, os fibroblastos proliferam no local 

da injúria [47] e secretam citocinas incluindo o TGF-β1 que atua de forma autócrina e 

parácrina para induzir proliferação e/ou eventual diferenciação de fibroblastos em 

miofibroblastos [48–50]. Estas células secretam matriz extracelular e proteínas incluindo 

colágeno e fibronectina [45,47,51] que são entrelaçadas para proporcionar um substrato de re-

epitelização do tecido [47]. Miofibroblastos também expressam α-SMA, proteína que integra 

filamentos de actina, proporcionando as células o fenótipo contrátil [52,53]. Embora 

inicialmente benéfico, o processo de regeneração tecidual pode se tornar patogênico se este 

continuar desenfreadamente, resultando em remodelamento da matriz extracelular e formação 

de cicatriz permanente. Em alguns casos, pode até evoluir para falha do órgão e morte. 

No ápice da interação célula-célula durante a cura tecidual há duas classes chave de 

citocinas: mediadores pró-fibróticos e mediadores antifibróticos. O TGF-β1 é o mais bem 

caracterizado mediador pró-fibrótico na fibrose pulmonar este é um fator de crescimento 

secretado por um amplo espectro de células, incluindo fibroblastos, com efeitos que são 

específicos para o tipo celular e tecidual [49,54–58]. 

Em oposição ao TGF-β1, a prostaglandina E2 (PGE2) é um medidor lipídico anti-

fibrótico predominantemente secretado por células epiteliais como indutor de quiêscencia de 

fibroblastos [59,60]. Ele ainda atua como regulador negativo de TGF-β1, inibindo a 



6 
 

 

proliferação de fibroblastos e diferenciação em miofibroblastos e secreção de matriz 

extracelular [48,50,60–62]. 

Acredita-se que o balanço de reguladores positivos e negativos (TGF-β1 e PGE2, por 

exemplo) seja necessário para o alcance da homeostase e assim evitar a ativação excessiva de 

fibroblastos [63]. A rede de colágeno depositada por fibroblastos é regulada continuamente 

pela síntese do colágeno e seu catabolismo. O “desligamento” do colágeno e outras proteínas 

da matriz extracelular são controlados por várias metaloproteinases (MMPS) e seus inibidores 

de metaloproteinases (TIMPs) que são produzidas por granulócitos, macrófagos, células 

epidermais e miofibroblastos. 

Pacientes com fibrose pulmonar apresentam dificuldade ao respirar, obtêm um mau 

prognóstico e consequentemente uma redução da qualidade de vida [64–68]. Tratamentos 

para a fibrose pulmonar são limitados. Transplante de pulmão foi considerado a única 

intervenção até recentemente. Em outubro de 2015 duas drogas Nintedanib (lheim, Ingelheim 

am Rhein, Alemanha) e perferidone (Shionogi & Co., Ltd., Osaka, Japan; Marnac Inc., 

Dallas, TX, USA) foram aprovadas nos EUA para o tratamento para a fibrose pulmonar 

idiopática [69]. Nenhuma dessas terapias é curativa, ambos os tratamentos diminuem, mas, 

não param ou revertem o progresso da fibrose pulmonar idiopática marcada pela redução do 

declínio da capacidade vital dos pacientes [70–72]. Ambas as drogas atuam na dinâmica dos 

fibroblastos inibindo proliferação, diferenciação e produção de TGF-β1. Contudo, nenhuma 

destas drogas demonstrou promover a sobrevivência ou regeneração das células epiteliais em 

pulmões fibróticos. 

 

1.6. Papel dos fibroblastos na inflamação 

Os fibroblastos são células presentes em diversos tecidos e possuem a capacidade de 

se diferenciar em outras células, incluindo cartilagem, osso, adipócito e células de músculo 

liso. Fibroblastos são uma população heterogênea e se origina através de diferentes processos. 

Entre esses processos estão a migração e diferenciação de células da medula óssea, 

denominadas de fibrócitos; a diferenciação de células epiteliais através da transição epitelial-

mesenquimal (EMT), e sendo recentemente sugerido a diferenciação de células endoteliais 

através da transição endotelial-mesenquimal (EndMT) [73]. Apesar dos fibroblastos serem 

uma das células mais abundante do estroma, estas células permanecem relativamente mal 

caracterizadas em termos moleculares [74]. Esta ausência de marcadores específicos dificulta 

a caracterização dos subconjuntos dessas células. Porém, algumas proteínas específicas 



7 
 

 

possibilitam a fenotipagem de fibroblastos como fibronectina, CD90, COL I, III e IV e pró-

colágeno [75]. 

Em resposta a estímulo microbiano, injúria mecânica, sinais derivados de linfócitos e 

macrófagos, fatores autócrinos e padrões moleculares associados a patógenos (PAMPS) que 

interagem com receptores de reconhecimento (TLR), fibroblastos podem secretar citocinas, 

quimiocinas e prostaglandinas [76]. A interação entre fibroblastos e leucócitos através de 

receptores CD40/CD40L, resulta na ativação do fator nuclear NFkB com níveis elevados de 

IL-6, IL-8 e ciclooxigenase 2 [77]. Além disso, essas células são responsáveis por definir 

ativamente o microambiente dos tecidos e desempenharem papel importante na transição da 

inflamação, visto que a produção inapropriada de quimiocinas como SDF-1, IFN-β e 

componentes da matriz extracelular por fibroblastos pode afetar diretamente as células do 

sistema imune levando ao estabelecimento da inflamação crônica [78]. 

 

1.7. Racional científico do estudo 

A PCM é uma doença endêmica da América latina, e apesar de representar problemas 

na saúde pública, é negligenciada. Mesmo após o tratamento antifúngico finalizado com 

sucesso, a maior parte dos pacientes portadores da forma crônica apresenta sequelas, dentre 

elas a fibrose pulmonar. Como consequência esses pacientes apresentam problemas 

respiratórios que acabam incapacitando-os muitas vezes de exercerem suas profissões e 

atividades. Em alguns casos, essa condição pode desencadear problemas psicológicos, e 

intensificar o quadro de alcoolismo já frequente nesses pacientes. 

Até o momento, ainda não foram totalmente elucidados os mecanismos que levam a 

fibrose durante a PCM, e poucos são os estudos que focam no papel das células pulmonares 

durante esse processo. Na literatura estudos focam no mecanismo de adesão e infecção de 

células por parte do fungo [79,80]. Uma lacuna ainda não explorada é o papel do fungo e seus 

componentes no aspecto funcional e modulador nas células responsáveis pela instalação da 

fibrose, como fibroblastos e miofibroblastos. Esse racional tem por fundamento estudos 

patológicos que verificaram que áreas de deposicição de colágenos em septo alveolar ocorrem 

à distância da lesão granulomantosa, sugerindo que componentes do fungo poderiam atuar 

nesse processo. 

 



1 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Objetivos 



 
 

 

2. OBJETIVOS 

 

2.1. Objetivo Geral 

Avaliar a influência do fungo Paracoccidioides spp. e componentes do hospedeiro sobre 

aspectos funcionais e moduladores de fibroblastos pulmonares. 

 

2.2 Objetivos Específicos 

 

 Isolar fibroblastos pulmonares murinos de camundongos BALB/c e obter cultura 

primária; 

 Produzir exoantígenos dos isolados pertencentes às espécies P.brasiliensis Pb326, 

Pb18 e P. lutzii Pb66, Pb8334 e Pb01; 

 Avaliar a proliferação de fibroblastos pulmonares murinos e humanos da linhagem 

MRC-5 frente a diferentes concentrações de exoantígenos, gp43 e soro de pacientes 

com FC da PCM nos momentos pré-tratamento, cura clínica, cura sorológica e cura 

aparente; 

 Avaliar a produção de citocinas IL-6, IL-1β, VEGF e TGF-β1 por fibroblastos 

pulmonares murinos frente aos exoantígenos e gp43 e avaliar a produção de IL-1β, 

TGF-β1, FGFb e pró-colágeno I por fibroblastos pulmonares humanos frente aos 

exoantígenos, gp43 e soro de pacientes com FC da PCM nos momentos pré-

tratamento, cura clínica, cura sorológica e cura aparente. 

 



9 
 

 

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64.  De Vries J, Kessels BL, Drent M. Quality of life of idiopathic pulmonary fibrosis patients. Eur 
Respir J. 2001;17: 954–961.  

65.  Nishiyama O, Taniguchi H, Kondoh Y, Kimura T, Ogawa T, Watanabe F, et al. Health-related 
quality of life in patients with idiopathic pulmonary fibrosis. What is the main contributing 
factor? Respiratory Medicine. 2005;99: 408–414. doi:10.1016/j.rmed.2004.09.005 

66.  Swigris JJ, Kuschner WG, Jacobs SS, Wilson SR, Gould MK. Health-related quality of life in 
patients with idiopathic pulmonary fibrosis: a systematic review. Thorax. 2005;60: 588–594. 
doi:10.1136/thx.2004.035220 

67.  Tzanakis N, Samiou M, Lambiri I, Antoniou K, Siafakas N, Bouros D. Evaluation of health-related 
quality-of-life and dyspnea scales in patients with idiopathic pulmonary fibrosis. Correlation 
with pulmonary function tests. Eur J Intern Med. 2005;16: 105–112. 
doi:10.1016/j.ejim.2004.09.013 

68.  Tomioka H, Imanaka K, Hashimoto K, Iwasaki H. Health-related quality of life in patients with 
idiopathic pulmonary fibrosis--cross-sectional and longitudinal study. Intern Med. 2007;46: 
1533–1542.  

69.  George G, Vaid U, Summer R. Therapeutic advances in idiopathic pulmonary fibrosis. Clin 
Pharmacol Ther. 2016;99: 30–32. doi:10.1002/cpt.283 

70.  King TE, Bradford WZ, Castro-Bernardini S, Fagan EA, Glaspole I, Glassberg MK, et al. A phase 3 
trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med. 2014;370: 
2083–2092. doi:10.1056/NEJMoa1402582 



14 
 

 

71.  Kreuter M. Pirfenidone: an update on clinical trial data and insights from everyday practice. Eur 
Respir Rev. 2014;23: 111–117. doi:10.1183/09059180.00008513 

72.  Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, et al. Efficacy and safety of 
nintedanib in idiopathic pulmonary fibrosis. N Engl J Med. 2014;370: 2071–2082. 
doi:10.1056/NEJMoa1402584 

73.  Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin 
Invest. 2003;112: 1776–1784. doi:10.1172/JCI20530 

74.  Hughes CCW. Endothelial-stromal interactions in angiogenesis. Curr Opin Hematol. 2008;15: 
204–209. doi:10.1097/MOH.0b013e3282f97dbc 

75.  Pilling D, Fan T, Huang D, Kaul B, Gomer RH. Identification of markers that distinguish 
monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS ONE. 
2009;4: e7475. doi:10.1371/journal.pone.0007475 

76.  Ekuni D, Yamanaka R, Yamamoto T, Miyauchi M, Takata T, Watanabe T. Effects of mechanical 
stimulation by a powered toothbrush on the healing of periodontal tissue in a rat model of 
periodontal disease. J Periodont Res. 2010;45: 45–51. doi:10.1111/j.1600-0765.2009.01195.x 

77.  Smith RS, Smith TJ, Blieden TM, Phipps RP. Fibroblasts as sentinel cells. Synthesis of chemokines 
and regulation of inflammation. Am J Pathol. 1997;151: 317–322.  

78.  Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon M. Fibroblasts regulate the 
switch from acute resolving to chronic persistent inflammation. Trends Immunol. 2001;22: 
199–204.  

79.  Ywazaki CY, Maza PK, Suzuki E, Takahashi HK, Straus AH. Role of host glycosphingolipids on 
Paracoccidioides brasiliensis adhesion. Mycopathologia. 2011;171: 325–332. 
doi:10.1007/s11046-010-9376-4 

80.  Mendes-Giannini MJS, Monteiro da Silva JL, de Fátima da Silva J, Donofrio FC, Miranda ET, 
Andreotti PF, et al. Interactions of Paracoccidioides brasiliensis with host cells: recent advances. 
Mycopathologia. 2008;165: 237–248. doi:10.1007/s11046-007-9074-z 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



15 
 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Manuscrito I 
 



15 
 

 

4. MANUSCRITO I 

 

An easy method for isolation and differentiation of murine pulmonary fibroblasts 

 

Débora de Fátima Almeida 
a,b

, Tatiani Ayako Goto Donato 
a
, Priscilla Maria Ponce Vareda 

a
, 

Karoline Haghata dos Reis 
a,b

, Angela Carolina Finato 
a,b

, Amanda Ribeiro dos Santos 
a,b

, José 

Roberto Bosqueiro
a
, James Venturini

a,b,c
* 

 

a
 Faculdade de Ciências, UNESP – Univ Estadual Paulista, Av. Eng. Luiz Edmundo C. Coube 

14-01, Bauru, SP, Brazil. 

b
 Faculdade de Medicina de Botucatu, UNESP – Univ Estadual Paulista, Distrito de Rubião 

Junior s/n, Botucatu, SP, Brazil. 

c
 Faculdade de Medicina, UFMS – Univ Federal do Mato Grosso do Sul, Cidade universitária, 

s/n, Campo Grande, MS, Brazil. 

 

*Reprints or correspondence: Dr. James Venturini, Faculdade de Ciências, UNESP – Univ 

Estadual Paulista, Av. Eng. Luiz Edmundo C. Coube 14-01, 17033-360, Bauru, SP, Brazil. 

+55(14) 31036078 (james@fc.unesp.br) 

 

Key Words: pulmonary fibrosis; tissue dissociation; pulmonary fibroblasts; cell culture 

 

Journal: Journal of Immunological Methods 

 

Abbreviations: EMT, epithelial-mesenchymal transition; EndMT, endothelial-mesenchymal 

transition; TGF-β, transforming growth factor beta; α-SMA, alpha-smooth muscle actin; PBS, 

mailto:james@fc.unesp.br


16 
 

 

phosphate-buffered saline; DMEM, Dulbecco’s modified eagle medium; TBST, tris buffered 

saline with tween 20; BSA, bovine serum albumin. 



17 
 

 

ABSTRACT 

Fibroblasts play a pivotal role in tissue repair by differentiation in myofibroblasts and 

production of extracellular matrix. Fibroblasts are abundant in many tissues and, in lungs, 

play a critical role in pulmonary fibrosis (PF). The studies of PF are usually restricted to in 

vivo models and, thus, the isolation and study of pulmonary fibroblasts could help in 

researches of cellular mechanisms as well as trials for new drugs. In the present study, we 

report an easy method to efficiently isolate and maturate fibroblasts in culture for subsequent 

functional analysis. 



18 
 

 

1. Introduction 

Fibroblasts are the main cellular constituents of connective tissues that could be originated 

by different process, such as epithelial-mesenchymal transition (EMT) (Kalluri and Neilson, 

2003), endothelial-mesenchymal transition (EndMT) (Frid et al., 2002), and by the 

differentiation from bone marrow cells, denominated fibrocytes (Schmidt et al., 2003).  

Fibroblasts play a central role in tissue repair, proliferating in the injury area (Midwood et 

al., 2004) and producing cytokines, including the TGF-β1 that acts in paracrine and autocrine 

form to induce differentiation of fibroblasts into myofibroblasts (Thannickal et al., 2003). 

These mature cells secrete extracellular matrix and proteins including, collagen and 

fibronectin (Midwood et al., 2004; Witte and Barbul, 1997) that are interlaced to 

proportionate a substrate for re-epithelization (Midwood et al., 2004). Myofibroblasts are 

recognized by expresses α-SMA, a protein that integrates filaments of actin that provide a 

contractile phenotype (Hinz et al., 2001). Due to the central role in the remodeling and tissue 

repair, studies have focused on the biology of these cells in the context of fibrosis. Although 

the fibrosis may present a variety of etiologies, it is generally characterized by the abnormal 

deposition of extracellular matrix and alteration of architecture of the affected organ (Luzina 

et al., 2015). The studies of the process and establishment of fibrosis in deep tissues, such as 

the lungs, have some limitations due to invasive methodologies for collecting patient samples. 

Thus, the development and improvement of in vitro techniques that seek to mimic this 

environment or provide the development of functional, molecular and biochemical studies are 

important. There are, at least, two basic methods for establishing primary fibroblast cultures 

from tissues: the explant method and the tissue dissociation method that may require a 

prolonged time to promote efficient isolation and stability of culture. 

Here, we describe a modified protocol of tissue dissociation based on protocols 

previoulsy reported in the literature and utilizing the association of type II collagenase and 



19 
 

 

trypsin, in order to increase the efficiency isolation of mice pulmonary fibroblast. In addition, 

we also validated the use of these cells in an assay for maturation. 

 

2. MATERIALS AND METHODS 

 

2.1. Mice. Male BALB/c mice were o purchased from Instituto Lauro de Souza Lima (Bauru, 

Brazil). All mice aged 21 to 45 days old and received a sterile balanced diet, water ad libitum 

and were kept in a ventilated shelf ALERKS-56 housing system (Alesco®, Monte Mor, 

Brazil). The experimental protocol was performed in accordance with the ethical principles 

for animal research adopted by the National Council for the Control of Animal 

Experimentation (CONCEA). This study was approved by the Ethical Committee of School 

of Sciences (Proc. #760/2016, UNESP, Bauru, Brazil). 

 

2.2. Mouse lung fibroblast isolation and culture. Naïve mice were randomly selected and 

euthanized by intraperitoneal administration of ketamine and xylazine by overdose. After 

thoracotomy, in aseptic conditions, the lungs were perfused through inoculation of sterile 

PBS. The lungs were then removed and cut into small pieces and washed three times in PBS 

plus 1% penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA) during 5 minutes each. 

After, the pieces were washed in PBS plus 2% antibiotics and 9% DMEM during 5 minutes 

each. After, the fragments were underwent to two steps of enzymatic digestion. Each 

digestion consisted in the incubation of lung fragments with solution of 1mg/ml of type II 

collagenase (Gibco® Life Technologies, Paisley, UK) and Trypsin 0,25% (Gibco
®
 Life 

Technologies,), under stirring for 45 minutes at 37°C. The result of digestion was centrifuged 

for 10 minutes at 1500 rpm, re-suspended in DMEM 20% heat-inactivated fetal calf serum 

(Sigma-Aldrich), penicillin (100 IU/ml), and streptomycin (100 mg/ml) (Gibco
®

 Life 



20 
 

 

Technologies,). Afterwards, the material was centrifuged and re-suspended in 5 ml of DMEM 

and filtrated in 70 µm cell strainer. The viability was determined by 0.1% trypan blue 

staining. Cells were aliquoted into cell culture flasks with 25 cm
2
 surface area (Corning 

Costar
® 

New York, NY, USA) following a proportion of 1 lung/flask (1 ml/ flask) with 4.0 ml 

complete medium and incubated at 37°C, 5% CO2 in a humidified chamber. After 24 hours, 

medium was changed to remove the non-adherent cells. When the cell culture reached 70% of 

confluence, cells were dispersed by adding trypsin-EDTA (Sigma-Aldrich) for 5 minutes and 

then transferred to new culture flasks. 

 

2.3. Maturation of fibroblasts. After the 70% of confluence, cells were dispersed by trypsin-

EDTA (Sigma-Aldrich) for 5 minutes and then adjusted to 2x10
4
 cells cultivated above round 

coverslips (13 mm
2
) in 24-well plate. After 24 hours, the cells were washed with sterile PBS 

and stimulated with 10 ng/ml of recombinant TGF-β1 (R&D Systems, Inc. Minneapolis, MN, 

USA). The maturation process was evaluated 24 hours after the stimuli for α-SMA 

expression. 

 

2.4. Immunofluorescence. After 24 days of treatment, cells were fixed with methanol at 

20°C for 6 minutes and washed with PBS. After, cells were permeabilized with 0.3% of triton 

X-100, followed by blocking with 5% of skimmed milk for 30 minutes at room temperature. 

Cells were incubated with the primary antibodies anti CD90 (1:100) (Thy1.2) (Acris 

Antibodies, Inc, San Diego, CA, USA) and anti α-SMA (1:50) (Santa Cruz, Dallas, Texas, 

USA) overnight at 4°C in humidified chamber. After, cells were incubated with the secondary 

antibody anti-mouse biotinylated (1:50) (Vector Laboratories Inc, Burlingame, CA, USA) for 

1 hour, and incubation with conjugated streptavidin with fluorescein (Vector Laboratories 

Inc) at room temperature for 30 minutes. Coverslips were mounted on glass slides using 



21 
 

 

Fluroshield™ with DAPI (4’,6’-diamino-2-phenylindole, dihydrocloride) (Sigma-Aldrich). 

The observations and photomicrographs were performed under a fluorescence microscope 

(Olympus), with camera (Q color 5, Olympus).  

 

2.5. Western Blotting. After treatment cells were lysed with RIPA buffer (Sigma-Aldrich) 

containing protease inhibitor (cOmplete ULTRA Tablets, Mini, EASYpack – Roche Applied 

Science, Mannheim, GER), sonicated and maintained on ice for 10 min. Then lysates were 

centrifuged at 14000 rpm at 4°C for 20 minute, the supernatant was collected and protein 

estimation was performed by Bradford kit (Bio-Rad Kit, Hercules, CA, USA). Next, samples 

containing 20µg protein were boiled at 100°C for 4 minutes in 30% of the volume in Laemmli 

buffer, separated by 12% SDS PAGE, transferred to a nitrocellulose membrane (Bio-Rad), 

blocked with 5% BSA in 1× TBST for 10 min at room temperature (RT) in SNAP i.d. 2.0 

(Millipore, Billerica, MA, USA) and incubated with primary antibodies includes anti- α-

SMA, (clone B4 - Santa Cruz Biotecnology, Dallas, Tx, EUA) at dilution 1:500 and anti- 

GAPDH (clone I-19 - Santa Cruz Biotecnology), at 1:200 dilution for 10 min at RT in SNAP 

i.d. 2.0 (Millipore).  After, the samples were incubated with an appropriate secondary 

antibodies, anti-mouse (sc-2060 - Santa Cruz Biotecnology), conjugated with peroxidase-HRP 

and anti-goat (sc-2768 – Santa Cruz Biotrcnology) in 3% BSA in 1x TBST for 10 min in RT 

in SNAP i.d. 2.0 (Millipore). Then protein expressions were visualized with 

chemiluminescence substrate (SuperSignal
™

 West Pico Chemiluminescent Substrate, Thermo 

Fisher Scientific, Waltham, MA, USA) and read in a fluorescence system (G:BOX, Syngene, 

Beacon House, Cambridge, UK ). At last, the protein band intensity was quantified by using 

Image J software.  

 

 



22 
 

 

3. Results 

 

3.1. Mains steps of fibroblast isolation 

The main steps to pulmonary fibroblasts isolation are summarized in Figure 1. First 

step was the perfusion to eliminate the excess of blood cells from the lungs. We used at least 

the injection of 40 ml of sterile PBS in the heart to effectively remove erythrocytes. In the 

second step, lungs were fragmented in small pieces and put them in a solution of sterile PBS 

with antibiotics to remove all contaminants. In the third step, fragments were soaked in a 

solution of DMEM, antibiotics and PBS in order to allow tissue to slowly absorb culture 

medium.. The fourth step was the digestion promoted by trypsin and type II collagenase 

association. After this process, cells are dispersed at supernatant. In the fifth step, cellular and 

tissue debri were eliminated by filtration in cell strainer. The viability reached more than 

95%. 

After 24 hours of the isolation, the culture medium was replaced to eliminate the non-

adherent cells. The DMEM medium will only support the growth of fibroblasts and other cell 

types will die or stop proliferating. 

After three days of the isolation it was possible to observe cells with morphology 

similar to fibroblasts. (Figure 2A). The confluence of 70% was observed after 8-10 days. 

(Figure 2B). 

 

3.2. Identification of fibroblasts and myofibroblats 

To confirm the isolation of fibroblasts we determined the expression of CD90 (Thy 

1.2) by immunofluorescence, as show in Figure 3A.  

Next, we evaluated the ability of these cells to differentiate in myofibroblasts in vitro.  

 



23 
 

 

Fibroblasts were cultivated and stimulated with recombinant TGF-β1 for 24 hours. 

Differentiation was confirmed by α-SMA expression by immunofluorescence (Figure 3B) and 

western blotting (Figure 4). Immunofluorescence allowed to observe actin filaments in cell 

cytoskeleton. 

 

4. Discussion 

As above mentioned, there are two basic methods for establishing primary fibroblast 

cultures from tissues: the explant method and the tissue dissociation method. In the literature, 

we found two protocols used to isolate pulmonary fibroblasts. Seluanov et al. (2010) 

described a protocol using two steps: the first using Liberase™ (Sigma-Aldrich),  highly 

purified type I and II collagenases trigger tissue digestion, and the second step in which the 

lung-digested fragments are cultivated without any dissociation process. According to the 

authors, the fibroblasts migrate out of the tissue with 3-5 days as expected to the explant 

methodology. In the present study, we isolated pulmonary fibroblast by dissociation method 

using the enzymatic digestion with type II collagenase and trypsin. Although digestion step is 

composed of two steps, in only 24 hours the fibroblast are adhered, and after 8-10 days it is 

possible to observe 70% of cell confluence. Trentin et al. (2015) utilized a classic dissociation 

method with type I collagenase; however, there is no detailed description of the used method. 

In the present protocol, we utilized the association of type II collagenase, that degrade the 

triple-helical native collagen fibrils, and trypsin, that is composed by a mixture of proteases 

and has digestive strength, promoting the advantage of increased tissue dissociation. 

Therefore, we consider that the combination of collagenase and trypsin favors the dissociation 

of fibroblast from tissue without interfere in cell viability. 

One of the purposes of using pulmonary fibroblast culture is the possibility to differentiate 

these cells in myofibroblasts. Myofibroblasts are cells usually involved in establishment of 



24 
 

 

fibrosis by the ability to produce of extracellular matrix, fibronectin and collagens, also 

presenting contractile phenotype (Hinz et al., 2001; Peyton et al., 2008). In the present study 

we confirmed that using this protocol, fresh isolated fibroblasts were able to differentiate in 

myofibroblasts. Thus, further in vitro studies may be performed using our protocol to 

evaluate, for instance, new therapeutic drugs for pulmonary fibrosis. One important advantage 

of primary culture is the heterogeneity of genetic background that favors, for instance, 

mechanistic investigations. Besides the alternative method to in vivo studies, primary culture 

provides better cost-benefit in comparison to cell lines. 

Taken together, the use of an easy method to isolate a primary pulmonary fibroblast 

culture and the possibility to promote the differentiation in myofibroblasts phenotype in vitro 

may be important to better understand the biology of pulmonary environment affected by 

fibrosis. Moreover it may promote new strategies to test novel anti-fibrotic drugs and 

pathogen influence on these cells.  

 

5. Acknowledgments 

We thank the Laboratório de Genética de Peixes (LAGENPE), School of Sciences, São Paulo 

State University (UNESP), Bauru, SP, Brazil for the images acquired using the digital 

microscope camera. 

 

 

6. Funding 

This work was supported by the Conselho Nacional de Desenvolvimento Científico e 

Tecnológico (CNPq) (Grant #470221/2014-3). 

 

 



25 
 

 

 

 

 

 

 

Figure 1. Schematic presentation of six steps for pulmonary fibroblasts isolation. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



26 
 

 

 

 

 

 

 

 

 

 

Figure 2. Fresh isolated pulmonary fibroblasts. (A) Third day after isolation, showing a large 

amount of cells (light microscopy, objective of 10X). (B) Eighth day after isolation, 

presenting 80% confluence (light microscopy, augment 40X). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



27 
 

 

 

Figure 3. (A) Isolated pulmonary fibroblasts. Pulmonary fibroblasts were harvested after 8 

days of culture and seeded in round coverslips for 24 hours and submitted to 

immunofluorescence determined CD90 expresssion (objective of 40X; green: CD90; blue: 

nuclei stained with DAPI). (B) Myofibroblast expressing α-SMA by immunofluorescence. 

Pulmonary fibroblasts were stimulated with 10ηg/ml of recombinant TGF-β1 for 24 hours 

(objective of 40X; red: α-SMA; blue: nuclei stained with DAPI). 

 

 

 



28 
 

 

 

Figure 4. Expression of α-SMA by immunoblotting. Pulmonary fibroblasts were cultivated in 

the presence or absence of 10ηg/ml of recombinant TGF-β1 for 24 hours. Results are 

expressed as fold change represented by ratio between the tested culture and control culture 

(CC).  

 

 

 

 

 

 

 

 

 

 



29 
 

 

7. References 

 

Epstein Shochet, G., Brook, E., Israeli-Shani, L., Edelstein, E., Shitrit, D., 2017. Fibroblast 

paracrine TNF-α signaling elevates integrin A5 expression in idiopathic pulmonary 

fibrosis (IPF). Respir. Res. 18, 122. doi:10.1186/s12931-017-0606-x 

Frid, M.G., Kale, V.A., Stenmark, K.R., 2002. Mature vascular endothelium can give rise to 

smooth muscle cells via endothelial-mesenchymal transdifferentiation: in vitro analysis. 

Circ. Res. 90, 1189–1196. 

Hetzel, M., Bachem, M., Anders, D., Trischler, G., Faehling, M., 2005. Different effects of 

growth factors on proliferation and matrix production of normal and fibrotic human lung 

fibroblasts. Lung 183, 225–237. doi:10.1007/s00408-004-2534-z 

Hinz, B., Celetta, G., Tomasek, J.J., Gabbiani, G., Chaponnier, C., 2001. Alpha-smooth 

muscle actin expression upregulates fibroblast contractile activity. Mol. Biol. Cell 12, 

2730–2741. 

Kalluri, R., Neilson, E.G., 2003. Epithelial-mesenchymal transition and its implications for 

fibrosis. J. Clin. Invest. 112, 1776–1784. doi:10.1172/JCI20530 

Liu, M.-H., Lin, A.-H., Ko, H.-K., Perng, D.-W., Lee, T.-S., Kou, Y.R., 2017. Prevention of 

Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice by Paeonol. Front 

Physiol 8, 193. doi:10.3389/fphys.2017.00193 

Liu, Y., Lu, F., Kang, L., Wang, Z., Wang, Y., 2017. Pirfenidone attenuates bleomycin-

induced pulmonary fibrosis in mice by regulating Nrf2/Bach1 equilibrium. BMC Pulm 

Med 17, 63. doi:10.1186/s12890-017-0405-7 

Luzina, I.G., Todd, N.W., Sundararajan, S., Atamas, S.P., 2015. The cytokines of pulmonary 

fibrosis: Much learned, much more to learn. Cytokine 74, 88–100. 

doi:10.1016/j.cyto.2014.11.008 

Midwood, K.S., Williams, L.V., Schwarzbauer, J.E., 2004. Tissue repair and the dynamics of 

the extracellular matrix. Int. J. Biochem. Cell Biol. 36, 1031–1037. 

doi:10.1016/j.biocel.2003.12.003 

Peyton, S.R., Kim, P.D., Ghajar, C.M., Seliktar, D., Putnam, A.J., 2008. The effects of matrix 

stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D 

biosynthetic hydrogel system. Biomaterials 29, 2597–2607. 

doi:10.1016/j.biomaterials.2008.02.005 

Schmidt, M., Sun, G., Stacey, M.A., Mori, L., Mattoli, S., 2003. Identification of circulating 

fibrocytes as precursors of bronchial myofibroblasts in asthma. J. Immunol. 171, 380–

389. 

Seluanov, A., Vaidya, A., Gorbunova, V., 2010. Establishing primary adult fibroblast cultures 

from rodents. J Vis Exp. doi:10.3791/2033 



30 
 

 

Thannickal, V.J., Lee, D.Y., White, E.S., Cui, Z., Larios, J.M., Chacon, R., Horowitz, J.C., 

Day, R.M., Thomas, P.E., 2003. Myofibroblast differentiation by transforming growth 

factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion 

kinase. J. Biol. Chem. 278, 12384–12389. doi:10.1074/jbc.M208544200 

Trentin, P.G., Ferreira, T.P.T., Arantes, A.C.S., Ciambarella, B.T., Cordeiro, R.S.B., Flower, 

R.J., Perretti, M., Martins, M.A., Silva, P.M.R., 2015. Annexin A1 mimetic peptide 

controls the inflammatory and fibrotic effects of silica particles in mice. Br. J. 

Pharmacol. 172, 3058–3071. doi:10.1111/bph.13109 

Witte, M.B., Barbul, A., 1997. General principles of wound healing. Surg. Clin. North Am. 

77, 509–528. 

 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 



31 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Manuscrito II 



30 
 

 

5. MANUSCRITO II 

 

Exoantigens of Paracoccidioides spp promote proliferation and modulation of human 

and mouse pulmonary fibroblasts 

 

Débora de Fátima Almeida 
1,2

, Tatiani Ayako Goto Donato
1
, Karoline Haghata dos Reis 

1,2
, 

Angela Carolina Finato 
1,2

, Amanda Ribeiro dos Santos 
1,2

, Ricardo Souza Cavalcante
2
, 

Rinaldo Poncio Mendes
2
, Anamaria Mello Miranda Paniago

3
, James Venturini

1,2,3
* 

 

 

1
 Faculdade de Ciências, UNESP – Univ Estadual Paulista, Av. Eng. Luiz Edmundo C. Coube 

14-01, Bauru, SP, Brazil. 

2
 Faculdade de Medicina de Botucatu, UNESP – Univ Estadual Paulista, Distrito de Rubião 

Junior s/n, Botucatu, SP, Brazil. 

3
 Faculdade de Medicina, UFMS – Univ Federal do Mato Grosso do Sul, Cidade universitária, 

s/n, Campo Grande, MS, Brazil. 

 

*Reprints or correspondence: Dr. James Venturini, Faculdade de Ciências, UNESP – Univ 

Estadual Paulista, Av. Eng. Luiz Edmundo C. Coube 14-01, 17033-360, Bauru, SP, Brazil. 

+55(14) 31036078 (james@fc.unesp.br) 

 

Short title: Paracoccidioides ssp. induce pulmonary fibroblasts proliferation  

Key Words: Pulmonary Fibroblasts, Pulmonary Fibrosis, Paracoccidioidomycosis 

Journal: PLoS Neglected Tropical Diseases 

mailto:james@fc.unesp.br


31 
 

 

ABSTRACT 

Paracoccidioidomycosis (PCM) is an endemic mycosis caused by fungi of genera 

Paracoccidioides; its main clinical forms are acute/subacute (AF) and the chronic form (CF). 

Endemic in Latin America, the PCM presents high incidence in Brazil, Colombia and 

Venezuela, especially among rural workes. Almost part of patients with CF demonstrated 

sequelae even after an effective antifungal treatment, such as pulmonary fibrosis. In PCM, 

fibrogenesis is recognized as an early process; however, its mechanisms are unknown. Thus, 

the present study aimed to evaluate the modulatory and functional influence of fungal and 

host components on pulmonary fibroblast. Human pulmonary fibroblasts (MRC-5 lineage) 

and murine pulmonary fibroblasts isolated from Balb/c mice were cultivated with different 

concentrations of P. brasiliensis (Pb18 and Pb326) and P. lutzii (Pb01, Pb8334 and Pb66) 

exoantigens; purified gp43, and serum of patients with CF in different moments of treatment 

(before treatment, clinical cure, serological cure and apparent cure). After 24 hours, cellular 

proliferation and production of cytokines and grow factors by murine and human pulmonary 

fibroblasts were evaluated. Our results showed that the fungal components interfered in 

fibroblast by induce proliferation (p < 0.05) and increased levels of TGF-β1 (p < 0.05) and 

decreased IL-6 (p < 0.05), VEGF (p < 0.05) and bFGF (p < 0.05). We also observed that 

exoantigens induced high production of pro-collagen I (p < 0.05) by human fibroblasts. Gp43 

induced increased levels of TGF-β1 (p = 0.02) by human cells. In addition, serum of patients 

with serological cure induced proliferation (p =0.01) and production of pro-collagen I (p = 

0.007) by human cells In conclusion, our findings showed at first time that components of 

P.brasiliensis and P.lutzii interfere in the fibrogenesis by directly act in the biology of 

pulmonary fibroblast. Furthermore, we verified that serum of CF-patients interfere in 

proliferation and activation of pulmonary fibroblasts according to each moment of treatment. 

 

 

.



32 
 

 

INTRODUCTION 

 

Paracoccidioidomycosis (PCM) is a systemic and granulomatous mycosis caused by 

thermally dimorphic fungi of the genus Paracoccidioides [1]. The infection occurs by 

inhalation of conidia or hypha fragments that reach the lungs and change morphologically to 

yeast form [2]. 

The mainly clinical forms are acute/subacute (AF), and chronic (CF) form. The AF is 

characterized by involvement of organs rich in the mononuclear phagocytic system such as 

bone marrow, liver, spleen and lymph nodes. CF usually affects male adults, with more than 

30 years old and exhibit predominant pulmonary and mucocutaneous involvement. After 

treatment, patients have to live with pulmonary fibrosis (PF) [3] and emphysema due to 

smoke. A total of 85% of patients with CF show pulmonary impairment with obstructive 

pattern [4].  

In general PF is associated with a wide spectrum of injuries such as infection, use of 

antibiotics, and ambient exposure. [5–7]. The establishment of this pathology is still not 

elucidated, but it is related to a deregulation of wound healing [8]. During the wound healing, 

fibroblasts proliferate and produce factors related to tissue repair such as, TGF-β1 that acts in 

paracrine and autocrine form to induce the differentiation of fibroblasts in myofibroblasts 

[9,10]. Myofibroblasts are cells involved in production of extracellular matrix, fibronectin and 

collagen, also are characterized by expression of alpha smooth actin (α-SMA), protein that 

integrates actin filaments and proportionate the contractile phenotype of these cells [11,12]. 

Although benefic in the beginning, tissue repair can become pathogenic if occur rampantly, 

resulting in extracellular matrix remodeling and a permanent scar. 

The sequela in PCM patients is observed even before treatment since necroscopic 

findings of patients revel presence of PF, characterized by extensive areas of collagen 



33 
 

 

deposition, near the hilar region, involving other structures such as lymph nodes, bronchi and 

arteries, collagen fibers are found on the periphery of granulomas and extend to nearby 

bronchi and blood vessels [13]. The fibrotic sequelae alters respiratory function and 

incapacitates the patient [3]. Usually it is observed disproportion between ventilation and 

perfusion and also alveolar-capillary blockade causing dyspnea.[14]. The most common 

abnormalities are architectural distortion and interlobular septal thickening and reticulate [15] 

and residual lesions occurs in up to 53% of treated patients [3].  

Cock et al [16] verified that in experimental model of PCM, the PF is an early process 

with establishment around 8 weeks after infection. Araujo et al [17] verified PF in patients 

that do not received treatment. Furthermore, Tuder et al [13] verified proliferation of reticular 

fibers in remote areas of granulomatous reaction, leading to the hypothesis that components of 

fungus could promote the collagen production. Despite this evidence, the interaction between 

Paracoccidioides ssp and host cells has not yet fully elucidated. 

Considering, the possible influences of the fungus Paracoccidioides ssp on the 

development of pulmonary fibrosis and the influence of host immune response on activity of 

pulmonary fibroblasts, in the present study, we investigated at first time the influence of 

Paracoccidioides ssp components and serum of PCM patients in the proliferation and 

responsiveness of pulmonary fibroblasts. 

 

MATERIALS AND METHODS 

 

Patients. CF-PCM patients were categorized into four groups according the moment of 

antifungal therapy: M1) non-treated patients newly diagnosed (n=12). M2) patients under 

treatment that showed disappearance of the initial symptomatology (clinical cure) but with 

detectable titles of specific antibodies to P. brasiliensis antigen determined by double 



34 
 

 

immunodifusion test (n=5). M3) patients with clinical cure and undetectable titles of specific 

antibodies to P. brasiliensis antigen (serological cure) determined by double immunodifusion 

test and under antifungal therapy (n=8). M4) patients with clinical cure, serological cure and 

without antifungal therapy (n=4). The control group was composed of ten healthy individuals 

of age- and gender-matched, selected among blood donors from the same geographical area. 

This study was approved by the Research Ethics Committees of Botucatu Medical School – 

UNESP (CAEE # 62177516.3.0000.5411). After being informed of the study, written 

informed consent was obtained from all participating adults. 

 

Paracoccidioides ssp. isolates. The isolates of P.brasiliensis (Pb18 and isolate Pb326, 

recently isolated of a patient from Botucatu, SP, Brazil) and P. lutzii (Pb01, Pb8334 and 

Pb66) were maintained by frequent subculture in semi-solid medium containing 2% glucose, 

1% peptone, 0.5% yeast extract and 2% agar (GPY) at 36º C. 

 

Exoantigen production. Total exoantigen was produced according to Camargo et al. [18] 

with modifications. Briefly, yeats forms of Paracoccidioides spp, were subcultured in 

Sabouraud containing 2% of dextrose (Sigma-Aldrich, St Louis, MO, USA) supplemented 

with 0.01% tiamine (Sigma-Aldrich) and 0.14% L-asparagine (Sigma-Aldrich) and 

maintained at 37°C, for 3 days. The fungi were cultivated in supplemented Sabouraud broth 

for three days shaking at 50 rpm at 37°C. Next, more supplemented Sabouraud broth was 

added and the inoculum was cultivated for more seven days shaking 50 rpm at 37°C. The 

fungi were killed by the addition of sodium merthiolate (0.2 g/L) for 24 hours at 4°C and 

filtered in Whatman™ filter paper #1. The filtrate was dialysate against distilled water with 

several exchanges during 24 hours at 4ºC. The dialysate was then filtered and concentrated by 

centrifugation with Amicon® Ultra 15 Filter (Millipore, Billerica, MA, USA) at 4000 rpm, 



35 
 

 

for 30 minutes, at 4ºC. The protein concentration was determined by Pierce™ BCA Protein 

Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). 

 

Human Lung Fibroblasts. Human lung fibroblast cell line MRC5 cells (ATCC CCL 171) 

were purchased from Banco de Células do Rio de Janeiro, RJ, Brazil. Fibroblasts were 

incubated at 37ºC in a humidified 5% CO2 95% air atmosphere in DMEM (Sigma-Aldrich), 

containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/mL penicillin and 100 

µg/mL streptomycin (Sigma-Aldrich). When the cell culture reached 80% confluence, cells 

were dispersed by adding trypsin-EDTA (Sigma-Aldrich) for 5 minutes and then transferred 

to new culture flasks (Greiner BioOne, Frickenhausen, BW, GER). 

 

Mice. Male BALB/c, 8-12 weeks old, mice were obtained from Instituto Lauro de Souza 

Lima, Bauru, SP, Brazil. All mice received a sterile balanced diet, water ad libitum and were 

kept in a ventilated shelf ALERKS-56 housing system (Alesco®, Monte Mor, SP, Brazil). 

The experimental protocol was performed in accordance with the ethical principles for animal 

research adopted by the National Council for the Control of Animal Experimentation 

(CONCEA). This study was approved by the Ethical Committee of School of Sciences (Proc. 

#760/2016, UNESP, Bauru, São Paulo, Brazil).  

 

Murine pulmonary fibroblast culture.  Naïve young mice were euthanized by 

intraperitoneal administration of ketamine and xylazine. After thoracotomy, in aseptic 

conditions, the lungs were perfused through inoculation of sterile PBS. The lungs were then 

removed and cut into small pieces and underwent to two steps of enzymatic digestion using 

collagenase type II (Gibco
®
 Life Technologies, Paisley, UK) and Trypsin 0,25% (Gibco

®
). 

The viability was determined by 0.1% trypan blue staining. The cells were aliquoted into 25 



36 
 

 

cm
2
 cell culture flasks (Corning Costar, New York, NY, USA) following a proportional of 1 

lung/flask (1 mL/ flask) with 4.0 mL complete medium and incubated at 37°C, 5% CO2 in a 

humidified chamber. After 24 hours, the medium was changed to remove the non-adherent 

cells. When the cell culture reached 70% of confluence, cells were dispersed by adding 

trypsin-EDTA (Sigma-Aldrich) for 5 minutes and then transferred to new culture flasks. 

 

Experimental design. The study was performed using three protocols: Protocol I: Human 

and murine fibroblasts were incubated with exoantigens of P.brasiliensis and P. lutzii at 

concentrations of 2.5, 5, 10, 100 and 250µg/mL and gp43, the immunodominant antigen 

purified from exoantigen of P. brasiliensis [19] at concentrations of 5 and 10 µg/mL for 24 

hours, after the cells were submitted to proliferation assay ; Protocol II: Human and murine 

fibroblasts were incubated with exoantigens of P.brasiliensis and P. lutzii at concentrations of 

of 2.5, 5, 10µg/mL and gp43 at concentrations of 5 and 10 µg/mL for 24 hours, after cell-free 

supernatants were submitted to cytokine dosages; Protocol III: Human fibroblasts were 

incubated 10% of serum of health individuals or 10% of serum of CF patients in different 

moments of treatment. After 24 hours of stimuli, cells were submitted to proliferation assay 

and the cell-free supernatants were submitted to cytokine dosages. 

 

Proliferation Assay. Proliferation was measured by the MTT assay according to Mosmann 

(1983) [20]. Briefly, fibroblasts were seeded (2x10
4 

murine pulmonary fibroblast/well and 

1x10
4 

human pulmonary fibroblasts/well) into 96-well culture plates in octuplicate. After 24 

hours, the cells were stimulated with 1) Paracoccidioides ssp exoantigens in the 

concentrations of 2.5, 5, 10, 100 and 250µg/mL, 2) gp43 at concentration of 5 and 10 µg/mL, 

3) 10% of serum of health or 10% of serum of CF patients in different moments of treatment. 

After 24 hours of stimuli, the supernatant was collected and the cells incubated in complete 



37 
 

 

DMEM medium containing MTT (5 mg/mL). The plate was incubated for 2 h at 37 ◦C in 5% 

CO2 and then centrifuged for 5 min at 1500 rpm. The supernatant was removed and the cells 

resuspended in 100 mL of dimethyl sulfoxide (DMSO) per well. After 5 min, the plate was 

read in a spectrophotometer reader at 540 nm. The percentage of proliferation was calculated 

by the ratio between the treated culture and control culture, times hundred. 

 

Functional analyses. Human levels of bFGF, TGF-β1, IL-1β, TNF-α, and pro-collagen I or 

mouse TGF-β1, IL-1β, IL-6, and VEGF were measured in cell-free supernatants and human 

TNF-α and bFGF in serum of health individuals or CF-PCM patients using a the Duo-Set Kit 

(R&D Systems, Minneapolis, MI, USA), according to the manufacturer’s instructions. The 

results were expressed in pg/mL, and determined from standard curves established for each 

assay. 

 

Statistical analyses. The two matched groups were compared using the paired t-test. All 

statistical analyses were carried out using GraphPad Prism 5.0 software (GraphPad Software. 

Inc., San Diego, California, USA) at a significance level of 5 % (p < 0.05) [21]. 

 

RESULTS 

 

Paracoccidioides ssp. exoantigens induced proliferation in murine pulmonary fibroblasts 

and discrete proliferation in human pulmonary fibroblast 

In general, exoantigens of Paracoccidioides ssp. induced proliferation of murine 

pulmonary fibroblast in the lowest concentration (2.5 µg/mL). (Fig 1). The purified gp43 did 

not promote fibroblast proliferation (Fig 1F). We also challenged human pulmonary fibroblast 



38 
 

 

with exoantigens of Paracoccidioides ssp. Our results showed that fibroblasts slightly 

proliferate in the presence of 10 µg/mL of Pb18 (Fig 2A); 2.5, 5 and 10 µg/mL of Pb01 (Fig 

2B); 5 µg/mL of Pb8334 (Fig 2C). Pb66 exoantigens induced decrease viability in all 

concentrations (Fig2D). Pb326 exoantigens promoted proliferation at 2.5 µg/mL (Fig 2E). 

Decrease of viability was also observed in the concentrations of 100 and 250 µg/mL of Pb18, 

Pb01, Pb8334 and MIC (Fig 2A-C, E); 2.5 and 10 µg/mL of Pb8334 (Fig 2C); 10 µg/mL of 

Pb326 (Fig2E); and 5 and 10 µg/mL of gp43. 

 

Paracoccidioides ssp. exoantigens decreased the production of IL-6 and VEGF by 

murine pulmonary fibroblasts and induced intense production of pro-collagen I by 

human pulmonary fibroblast 

To evaluate the effects of exoantigens in the activity of pulmonary fibroblast, we 

measured the levels of cytokines and grow factors involved in inflammation and wound 

healing. We verified that all exoantigens triggered decreased production of IL-6 in lowest 

concentrations in murine pulmonary fibroblasts (2.5, 5 and 10 µg/mL) (Fig 3A-E). We also 

observed decreased levels of VEGF by murine fibroblasts stimulated with 10 µg/mL of Pb18 

(Fig 3A), 2.5 and 10 µg/mL of Pb01 (Fig 3B), 2.5, 5 and 10 µg/mL of Pb66 (Fig 3C), 10 

µg/mL of Pb8334 (Fig 3D). We verified increased production of TGF-β1 by murine 

fibroblasts stimulated with 2.5 µg/mL of Pb66 (Fig 3C), and 10 µg/mL of Pb326 (Fig 3E). 

Decreased production of TGF-β1 was observed in murine cultures stimulated with 2.5 µg/mL 

of Pb18 (Fig 3A) and 2.5 µg/mL of Pb326 (Fig 3E). No differences were observed in the 

levels of IL-6, VEGF and TGF-β1 in murine fibroblasts stimulated with gp43 (p > 0.05) (Fig 

3F). Levels of IL-1β were not detected in supernatants of murine fibroblast cultures. 

The functional analyses revealed intense production of pro-collagen I in human 

fibroblasts stimulated with 2.5, 5 and 10 µg/mL of Pb18 (Fig 4A), 2.5 µg/mL of Pb01 (Fig 



39 
 

 

4B), 5 µg/mL of Pb66 (Fig 4C). No differences were observed in human fibroblasts 

stimulated with P8334 (Fig 4D) and increased production of pro-collagen I was also observed 

in 2.5 µg/mL of Pb326. We verified that exoantigen of Pb18 decreased the production of 

TGF-β1 in all concentrations (Fig 4A) and Pb01 do not interfere in the normal production 

compared to control culture (Fig 4B). However, related to Pb66, we verified a decrease in 

TGF-β1 levels at 5 µg/mL and an increase at 10 µg/mL (Fig 4C). We also observed an 

increase of TGF-β1 levels in human fibroblasts stimulated with 5 and 10 µg/mL of Pb8334; 5 

µg/mL of Pb326 (Fig 4E). 

Furthermore, we evaluated the production of bFGF by human pulmonary fibroblasts; 

we verified decreased production in cultures stimulated with 2.5, 5 and 10 µg/mL of Pb18 

(Fig 4A) and 5 and 10 µg/mL of Pb326 (Fig 4E). No differences were observed in human 

fibroblasts stimulated with Pb66 (Fig 4C) and Pb8334 (Fig 4D). Gp43 did not interfere in the 

proliferation and production of pro-collagen I of human pulmonary fibroblast but increased 

the production of TGF-β1 at 5 µg/mL (Fig 4F). Levels of IL-1 β were not detected in 

supernatants of human pulmonary fibroblasts 

 

Remarkable alterations in pulmonary fibroblast cell culture are observed after 

incubation with serum of PCM patients that achieved serological cure. 

To evaluate the interference of PCM patient serum in proliferation and activity of 

pulmonary fibroblasts, we cultivated MCR-5 cells in the presence of serum from health 

individual and CF-PCM patients categorized into 4 groups according to moments of 

treatment. The division of the follow-up is useful in both clinical and immunological 

evaluations during the treatment since each moment is based on biological aspects of each 

patient, i.e., it reflects the time each patient required to meet the established criteria. Serum of 

patients staged into M1 induced no alterations in pulmonary fibroblast proliferation as well as 



40 
 

 

in functional activity (Fig. 5). Proliferation of MCR-5 cells (Fig. 5A) and higher production of 

bFGF (Fig. 5C) and pro-collagen I (Fig. 5B) was observed in cultures stimulated with serum 

of patients that reached serological cure (M3), a stage in which the patients show an 

improvement of specific immunity to the fungi. In M2, when the patients present absence of 

the inital symptoms, the serum stimulated the production of bFGF (Fig. 5C). We also 

observed that serum of patients after complete successfully treatment (M4) slightly induced 

decrease in viability of human fibroblasts (p = 0.006) (Fig. 5A). 

 

DISCUSSION 

 

The interaction of P.brasiliensis and host cells has been investigated in the 

mechanisms involved in adherence and mechanism of scape [22–24]. Vicentini et al. [22] 

verified that extracellular matrix protein laminin binds specifically to yeast forms of P. 

brasiliensis and enhances adhesion of the fungus to the surface of epithelial cells. Ywazaki et 

al [23] verified adhesion of P. brasiliensis in GM1 and GM3 gangliosides of human 

pulmonary fibroblasts, and this could be the pathway of bound/infection by the fungus. In 

other study, Mendes-Giannini et al [24] verified interactions between P. brasiliensis and 

epitelial cells of Vero/A549 cell line and these authors suggest that the adhesion and invasion 

of these cells could represent an escape mechanism and corroborate to spread of infection. 

In the present study, we showed at the first time the interaction of P.brasiliensis and 

P.lutzii with human and murine pulmonary fibroblasts in the context of fibrogenisis. We 

verified in the murine cells that low concentrations of exoantigens downregulate production 

of IL-6 and VEGF and increased cell proliferation. We also observed that the isolates Pb01 

and Pb66 of P.lutzii and Pb326 of P. brasiliensis caused a dose-dependent effect, while the 

isolates Pb18 of P.brasilensis and Pb8334 of P.lutzii promoted proliferation independently 



41 
 

 

exoantigen concentration. It is possible that the observed differences among exoantigens are 

associated with the variation in fungal composition between species and isolates, once the 

composition and production of secreted molecules varies among isolates. Machado et al [25] 

verified differences in composition of isolates from P. lutzii (Pb 01 and Pb 8334) and 

P.brasiliensis (Pb18, Epm83 and Pb265) species. Authors suggested that P.lutzii present less 

amount of gp43 compared to P.brasiliensis. In our study, we observed that depending on 

specie, the exoantigen also, promote increase in the production of TGF-β1. This could mean a 

capacity to promote the differentiation of fibroblasts in myofibroblasts and so corroborating to 

fibrosis establishment. Also, we observed that the gp43 did not show an important role in the 

induction of proliferation, but increased the production of TGF-β by human fibroblast. 

Paracoccidioides ssp. exoantigens also promoted proliferation of human pulmonary 

fibroblasts; however, more discreetly. Furthermore, we verified a higher production of pro-

collagen I in these cells, corroborating with the idea that exoantigens could participate on the 

establishment of pulmonary fibrosis. Similar to observed in the murine cells, we verified that 

gp43 did not participate in proliferation of pulmonary fibroblasts, however, we verified that 

gp43 promoted increase in production of TGF-β1 levels in human pulmonary fibroblasts. 

Also, we observed increased production of TGF-β1 in human pulmonary fibroblasts 

stimulated with Pb66, Pb8334, while in other isolates we verified decreased TGF-β1 levels 

compared to control culture. Interestingly, in human fibroblasts we verified proliferation but 

the levels of bFGF were lower than the control. Venturini et al. [26] verified P. brasiliensis 

antigens increased the production of IL1-β, TNF-α, TGF-β1 and bFGF by peripheral blood 

monocytes of CF-PCM patients, suggesting that fungal metabolites play an important role in 

the activation of these cells. Therefore, Paracoccidioides ssp antigens could participate in the 

establishment of fibrosis in PCM, activating monocytes that produce high levels of bFGF and 

TGF-β1, and also, activating fibroblasts that produce TGF-β1 as well, and pro-collagen I. 



42 
 

 

Other authors suggest the main role of fibroblasts during infections. Recently, Kühbacher 

et al [27] using a 3D cellular model culture composed by fibroblasts, keratinocytes TCD4
+
 

lymphocytes and mononuclear cells verified that dermal fibroblast transform to a 

antimicrobial phenotype with high expression of CXCL9, CXCL10 and CXCL11 in the 

presence of TCD4
+
 lymphocytes during Candida albicans infection, and this process is 

dependent of TLR2 pathway and by pro-IL-1β cleavage. Our findings showed that both cell 

types did not produce IL-1 β indicating that exoantigens do not have the capacity alone 

activating inflamassome and producing IL-1β. 

Herein, we also demonstrated that PCM patients serum influence the proliferation and/or 

functional activities of human pulmonary fibroblasts according to treatment moment. Our 

findings demonstrated that no alterations were observed in MCR-5 cells cultured in the 

presence of serum from no treated patients. Considering that before treatment, CF PCM 

patients present high levels of proinflammatory cytokines in the serum [28–30] and 

monocytes are able to produce in vitro growth factors, such as bFGF [26], we had expected 

intense proliferation and alterations in the activity of pulmonary fibroblast. Frankel et al [31] 

demonstrate that MRC-5 cells pre-incubated with TNF-α showed 41% of underwent Fas-

induced apoptosis, suggesting that this pro-inflammatory cytokine is able to sensitize 

fibroblast and induce cell death. We hypothesized that the unresponsiveness of these 

fibroblasts could be a result of the balance of TNF-α-induced apoptosis and proliferation 

triggered by growth factors. Thus, we additionally determined the levels of TNF-α and bFGF 

in the serum of heath individual and PCM patients and, indeed, high levels of TNF-α and 

bFGF were observed in serum of PCM patients in M1 (Suppl. Figure 1).  

Serum levels of patients in M2 showed higher TNF-α levels than normal individuals but it 

was lower than M1. In this scenario, M2 sera induced enhanced production of bFGF by MCR-

5. Therefore, our findings confirm that the TNF-α plays a dual role in fibrogenesis: this 



43 
 

 

cytokine induces tissue damage and, thus, favor fibroblast and myofibroblast persistence and 

accumulation [32]. On the other hand, TNF-α also interferes in lung repair by sensitizing 

fibroblasts and myofibroblasts to cell death [31], and diminishes the pulmonary fibrosis 

induced by bleomycin-induced mice [33]. 

Interestingly, remarkable profibrogenic activity was observed in MCR-5 cells cultivated in 

presence of serum from patients that achieved serological cure (M3). It is well known that 

PCM patients show decreased serological titers of specific antibodies along the treatment 

what indicates improvement of immunity against the fungi [34]. In the serological cure, the 

serum showed the lowest levels of TNF-α and bFGF. Even we failed to determine the levels 

of TGF-β1 in the serum, we shall not rule out the role of others growth factors acting in the 

response of pulmonary fibroblast. Therefore our results suggest that during and/or after the 

recovery of immune response, the process of tissue repair seems to reach its maximal activity. 

It is important to highlight that our suggestion does not allow to correlate our in vitro findings 

with a possible increment of pulmonary fibrosis in patients after serological cure.  

Serum from patients after treatment decreased the proliferation of MRC-5 cell, but 

induced high production of TGF-β1 by these cells. PCM patients exhibit, after treatment, 

completely restored cell-mediated immunity to P. brasiliensis [35] characterized by increased 

levels of IFN-γ and IL-2 by peripheral blood mononuclear cells. Frankel et al. [31] also 

observed that IFN-γ exhibited synergistic activity with TNF-α and promoted increased 

apoptosis of MRC-5 cells. As we detected high levels of TNF-α in these serums (Suppl Figure 

1), it is possible that our findings are associated with the synergism of these cytokines. Intense 

production of TNF-α in PCM patients after treatment has been previously observed [26,28] 

and the authors suggest that hypoxia induced by pulmonary sequelae is responsible for 

continued systemic inflammation. Indeed, several reports have shown that hypoxia continues 

to interfere in inflammation and fibrob