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CARINA SOLON DA SILVA
A INTERLEUCINA-17 É PRODUZIDA PELO INTESTINO EM RESPOSTA
A ÁCIDOS GRAXOS DA DIETA E REGULA A SECREÇÃO DE INSULINA
INTERLEUKIN-17 IS PRODUCED IN THE GUT IN RESPONSE TO
DIETARY FATS AND REGULATES INSULIN SECRETION
CAMPINAS
2015
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CARINA SOLON DA SILVA
A INTERLEUCINA-17 É PRODUZIDA PELO INTESTINO EM RESPOSTA A ÁCIDOS GRAXOS DA DIETA E REGULA A SECREÇÃO DE INSULINA
INTERLEUKIN-17 IS PRODUCED IN THE GUT IN RESPONSE TO DIETARY FATS AND REGULATES INSULIN SECRETION
Orientador: Licio Augusto Velloso Tutor: Licio Augusto Velloso
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA CARINA SOLON DA SILVA
E ORIENTADA PELO PROF.DR. LICIO AUGUSTO VELLOSO
Assinatura do Orientador
_________________________________
CAMPINAS
2015
Tese apresentada a Faculdade de Ciências Médicas da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Ciências.
Doctoral Thesis presented to the Physiopathology Pos-Graduation Program of the Faculty of Medical Sciences, University of Campinas – UNICAMP to obtain the title of Doctor in Science.
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE CIÊNCIAS MÉDICAS
iv Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Ciências Médicas Maristella Soares dos Santos - CRB 8/8402
Silva, Carina Solon, 1983-
Si38i lA interleucina-17 é produzida pelo intestino em resposta a ácidos graxos da dieta e regula a secreção de insulina / Carina Solon da Silva. -- Campinas, SP : [s.n.], 2015.
Orientador : Lício Augusto Velloso.
Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas. 1. Interleucina-17. 2. Diabetes Mellitus. 3. Incretinas. 4. Pancrêas. I. Velloso, Lício
Augusto,1963-. II. Universidade Estadual de Campinas. Faculdade de Ciências Médicas. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Interlukin-17 is produced in the gut in response to dietary fats and
regulates insulin secretion
Palavras-chave em inglês:
Interleukin-17 Diabetes Mellitus Incretins
Pancreas
Área de concentração: Fisiopatologia Médica Titulação: Doutora em Ciências
Banca examinadora:
Lício Augusto Velloso [Orientador] Adriana de Souza Torsoni
Antônio Carlos Boschiero
Silvana Auxiliadora Bordin da Silva Luciana Chagas Caperuto
Data de defesa: 03-03-2015
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RESUMO
A interleucina-17 (IL17) está envolvida na resposta imune contra agentes patogénicos intestinais, e a sua expressão anómala no intestino pode ocorrer em condições tais como diabetes do tipo 1 (DM1), encefalomielite auto-imune e doença de Crohn. Fatores dietéticos podem alterar a microbiota intestinal desencadeando doenças metabólicas. Nossa hipótese é de que IL17 poderia ser diretamente modulada por nutrientes e pode desempenhar um papel na obesidade e diabetes tipo 2 (DM2). Aqui, nós demonstramos que as gorduras da dieta induzem a expressão IL17, predominantemente no íleo. In vivo, ilhotas pancreáticas isoladas estimuladas com IL17 apresentaram um aumento na secreção de insulina quando comparado a ilhotas não estimuladas, enquanto que a sua inibição sistémica resultou em intolerância à glicose. Animais knockout para o receptor de IL17 (IL17RA) eram intolerantes à glucose devido ao desenvolvimento embrionário anómalo das ilhotas pancreáticos, que eram menores e foram depletados de células produtoras de insulina. Nos seres humanos, os níveis circulantes de IL17 aumentaram após uma refeição. Este aumento foi significativamente maior nos indivíduos obesos normoglicêmicos do que em indivíduos obesos com diabetes. Semelhantes aos roedores, as ilhotas humanos também foram estimulados a secretarem insulina na presença de IL17. Assim nós identificamos a IL17 como um sensor intestinal de gorduras alimentares, que exerceram um efeito semelhante a hormônios incretínicos. Além disso, a presença IL17RA é importante para o desenvolvimento normal das ilhotas pancreáticas.
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ABSTRACT
Interleukin-17 (IL17) is involved in the immune response against intestinal pathogens, and its anomalous expression in the gut can occur in conditions such as type 1 diabetes (T1D) 1,
autoimmune encephalomyelitis 2 and Crohn’sdisease 3. Because dietary factors can change the
gut microbiota, impacting metabolic diseases 4, we hypothesized that IL17 could be directly modulated by nutrients and might play a role in obesity and type 2 diabetes (T2D). Here, we show that dietary fats induced IL17 expression, predominantly in the ileum. Both in vivo and in isolated pancreatic islets, IL17 stimulated insulin secretion, while its systemic inhibition resulted in glucose intolerance. Mice KO for the main IL17 receptor (IL17RA) were glucose intolerant due to anomalous embryonic development of the pancreatic islets, which were smaller and were depleted of insulin-producing cells. In humans, blood IL17 increased following a meal. This increase was significantly higher in obese normoglycemic individuals than in obese subjects with diabetes. Similar to those of rodents, human islets were also stimulated to secrete insulin in the presence of IL17. Thus, we identified IL17 as a gut sensor of dietary fats, which exerted an incretin-like effect. In addition, the presence IL17RA was important for normal development of the pancreatic islets.
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SUMÁRIO
Agradescimentos...xiii Lista de Abreviaturas...xv 1. Introdução.. ... 1 2. Objetivos ... 10 3. Artigo. ... 114. Resumo dos resultados...40
5. Conclusão...41
6. Referências...42
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AGRADECIMENTOS
Aos meus familiares, que me apoiam em todos os momentos de minha vida. Pela segurança que me oferecem ao saber que tenho sempre com quem contar. Principalmente a minha mãe, exemplo de vida para mim, que me ensinou que o que aprendemos é um bem que nunca podem nos tirar. À minha querida irmã Carol, companheira de toda a vida. Somos muito próximas desde a infância e nada nos separa, dividimos até a pós graduação. Obrigada por estar sempre presente na minha vida e em todos os experimentos, os bem sucedidos, os nem tanto, os feitos muito cedo e os até tarde. E lá vamos nós dar mais uma vez trabalho para os revisores entenderem que não foi um erro de digitação, e sim duas autoras com quase o mesmo nome.
Ao Anthony, meu noivo, sempre paciente e compreensivo com os horários estranhos, finais de semana de experimentos e a “dureza” finaceira.
Aos amigos de laboratório, pela convivência que faz ser tão prazeroso o dia-a-dia, cafés a qualquer hora e uma palavra de apoio na hora de uma revelação de blot infeliz.
Aos meus colaboradores, sem os quais não seria possível a realização deste trabalho. Pincipalmente a Nathália e Albina “paus para toda obra”.
À professora Adriana Torsoni que, na graduação, me guiou nos primeiros passos na Ciência. Apresentou-me ao laboratório de sinalização celular, foi meu primeiro exemplo de cientista, me acolheu como uma grande mestra, me ensinou experimentos durante seu pós-dourotado e me tornou quase como um membro de sua família.
Ao Professor Lício, pela orientação deste trabalho. Pelo belo exemplo que nos proporciona, motivando e inspirando-nos a cada dia..
Aos funcionários do Laboratório de Sinalização Celular, pelo suporte técnico. À Universidade Estadual de Campinas, pela infraestrutura.
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À Fundação de Amparo à Pesquisa do Estado de São Paulo e ao Conselho Nacional do Desenvolvimento Científico e Tecnológico, pelo financiamento.
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LISTA DE ABREVIATURAS
AUC – Área sob a curva
CEBP – Enhancer-binding proteins
CTLA8 – Cytotoxic T lymphocyte antigen 8 DM2 – Diabetes Mellitus tipo 2
DNA – Ácido Desoxirribonucléico
ERE – Estresse de Retículo Endoplasmático
GCSF - Fator Estimulante de Eolônias Granulocíticas GI – Gastro Intestinal
GIP – Polipeptídeo Inibitório Gástrico GLP-1 – Peptídeo Glucagon-Like 1 GTT – Teste de Tolerância à Glicose HFD – Dieta Hiperlipídica
IFNInterferon gamma
IL1β – Interleucina-1β IL6 – Interleucina-6 IL17 – Interleucina-17
IL17R – Receptor de Interleucina-17 IL21 – Interleucina-21
IL23 – Interleucina-23 IL4 – Interleucina-4
ILR1s – Receptores de Interleucina 1 IKK – IB kinase
xvi KDA - Quilodáltons
LPS – Lipopolissacarídeo
MAPKs - Mitogen Activated Protein Kinases NF-kB – Fator Nuclear kappa B
PCR – Reação em Cadeia da Polimerase
RE –Retículo Endoplasmático
RNAm – Ácido Ribonucléico Mensageiro RORRAR-related orphan receptor gamma TGI – Tratogasto Intestinal
TGFb - Transforming growth factor beta Th1 - Linfócitos T helper 1
Th17 - Linfócitos T helper 17 Th2 - Linfócitos T helper 2 TLR4 – Receptor Toll-Like 4
TNFα – Fator de Necrose Tumoral-α IMC - Índice de Massa Corpórea UA – Ácido usólico
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1.INTRODUÇÃO
A interleucina-17 (IL17) pertence a uma família de citocinas pró-inflamatórias que atuam em diferentes tipos de tecidos e células, induzindo a expressão de fator estimulante de colônias granulocíticas (G-CSF), interleucina-6 (IL6), quimiocinas, citocinas, mucinas, metaloproteínas de matriz e peptídeos antimicrobianos (Gaffen, 2008; Chang & Dong, 2011). Como resultados dos efeitos da IL17 no sistema imune são regulados diversos fenômenos, tais como: a expansão e o recrutamento de neutrófilos durante a resposta imunológica, ligando a resposta inata com a adaptativa (Kolls, J.K et al., 2004; XU et al., 2010); resposta a vários tipos de agentes infecciosos (Ye et al., 2001; Huang et al., 2004; Hamada et al., 2008; LU et al., 2008; Raffatellu et al., 2008; Algood et al., 2009; Conti et al., 2009; Ishigame et al., 2009; Da Matta Gedes et al., 2010; Cho et al., 2010; Miyazaki et al., 2010; Xu et al., 2010; Suryawanshi et al., 2011); e processos inflamatórios outros, tais como os presentes em doenças autoimunes nas desordens metabólicas e no câncer (Ye et al., 2001; Chung et al., 2003; Huang et al., 2004; Ouyang et al., 2008; Ishigame et al., 2009; Ahmaed et al., 2010; Milner, 2011; Kuchroo et al., 2012; Trinchieri 2012; Gallimore et al., 2013).
Em humanos, o gene codificador da IL17 foi mapeado no cromossomo 6 (6p12) (Moseley et al., 2003), seu produto corresponde a uma proteína constituída de 155
aminoácidos, com peso molecular de aproximadamente 15 quilodaltons (kDa), que é secretada
como glicoproteína dimérica de 30-35 kDa, ligadas por pontes dissulfeto. O polipeptídeo precursor da IL17 é composto por uma sequência sinal de 19 aminoácidos, seguido de um fragmento maduro de 136 aminoácidos. Este fragmento contém pelo menos um sítio de glicosilação ligado ao N-terminal, e seis resíduos de cisteína que formam ligações intermoleculares durante a dimerização (Yao et al., 1995).
A identificação da IL17 foi obtida em 1993 pelo pesquisador Rouvier, recebendo a denominação original de CTLA8 (Rouvier, E. et al., 1993). Em decorrência desta importante
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identificação, sucederam-se vários estudos que levaram à quebra de um paradigma, que durou
vinte anos, ao revelarem a presença de um novo subconjunto de células auxiliares T CD4 +, distinto dos linfócitos T helper clássicos Th1 e Th2 (Mosmann, T. R. et al., 1986).
A característica predominante dos linfócitos com os perfis clássicos, Th1 e Th2, é a produção de IFN-ɣ e IL4, respectivamente. As citocinas produzidas pelas células Th1 ativam macrófagos e participam na geração de uma resposta imune mediada por células. Contrariamente, as citocinas produzidas pelas células Th2 ajudam a ativar linfócitos B, resultando na produção de anticorpos. Após a sua ativação, as duas subpopulações, Th1 e Th2, influenciam-se mutuamente e de forma antagônica (Mosmann, T. R. et al., 1989).
Na época, a subpopulação de linfócitos T recém-descoberta, foi denominada Th17, pois sua citocina de assinatura é a IL17. A diferenciação das células T CD4 + naive em células Th17
é induzida e controlada por um conjunto de citocinas, que incluem IL6, IL1, IL21, IL23, e TGF,
levando à ativação de RORt que, por sua vez, atua como fator de transcrição que coordena o
programa a diferenciação de linfócitos com perfil Th17 (Korn T, et al., 2009).
A IL17 pertence a uma família de seis proteínas identificadas até o presente; IL17A a IL17F (Kolls, J.K et al., 2004). Todos os membros da família IL17, com exceção da IL17E,
podem induzir a expressão de citocinas pró-inflamatórias, tais como: fator de necrose
tumoral-alfa (TNF) e IL1. Alternativamente, a IL17E parece estar relacionada à promoção de
respostas imunes de tipo Th2. No entanto, o membro da família da IL17 mais bem caracterizado é a IL17A. Em camundongos, IL17A e IL17F podem atuar com homodímeros ou ainda como heterodímero com cerca de 35 kDa (Liang, S.C. et al., 2007).
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Fig.1 Familia IL17R, relação receptores-ligantes (Gaffen, S.L., 2009).
Existem vários receptores da família de IL17, a saber: IL17RA, IL17RB, IL17RC, IL17RD e IL17RE, os quais foram identificados devido a homologia da estrutura típica entre os membros. O IL17RA é expresso numa ampla gama de tecidos e diferentes tipos de células. Após estímulo com IL17, o IL17RA inicia a ativação das vias de sinalização a jusante, desencadeando ativação das vias NFkB, MAPKs e CEBP, cascatas que levam à regulação positiva de genes pró-inflamatórios (Zhu S. et al., 2012). No entanto, o IL17RA por si só é insuficiente para a sinalização mediada por IL17, sendo necessária a formação um complexo heterotrimérico composto por IL17, IL17RA e IL17RC (Toy D. et al., 2006). Embora a IL17A e IL17F homodímeros e IL17A/IL17F heterodímeros possam estimular múltiplas vias sobrepostas nas células-alvo, a sua potência varia, com um homodímero de IL17A exibindo maior potência do que o heterodímero ou o homodímero de IL17F (Liang et al., 2007; Wright et al., 2007; Wright et al., 2008; Chang et al., 2007).
As regiões de barreiras mucosas, como intestino e brônquios, são sítios de importantes ações desempenhadas pelo sistema IL17 (Dubin, P.J. et al., 2008). O complexo IL17RA/IL17RC é expresso em um grande número de tipos de células encontrado na mucosa,
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incluindo células epiteliais e fibroblastos, desempenhando assim importante papel na defesa do hospedeiro contra certos patógenos em barreiras mucosas.
As interleucinas IL17A e IL17F podem atuar diretamente no tecido epitelial induzindo várias respostas antimicrobianas contra agentes patogênicos extracelulares, além de promover o remodelamento do tecido envolvido na resposta. Dentre as respostas induzidas pela sinalização da IL17, destacam-se a indução de quimiocinas, em particular quimio-atratores de neutrófilos como fator de estimulação de colônias de granulóciticas (G-CSF), quimiocinas da
família CXC, citocinas como a IL6, e peptídeos antimicrobianos, tais como -defensinas e
Proteínas S100 (Iwakura Y et al., 2011). Em diferentes contextos, essas substâncias induzidas pela IL17 contribuem para o clearance de bactérias extracelulares com S. aureus que infectam a pele, C. rodentium no cólon intestinal e K. pneumoniae no pulmão (Ishigame H. et al., 2009; Cho JS et al., 2010).
Estudos recentes revelaram também a função crucial da microbiota do intestino na homeostase imune intestinal, incluindo o desenvolvimento e a regulação das células Th17 intestinais. Algumas bactérias filamentosas segmentadas podem induzir a ativação de células Th17, enquanto bactérias intestinais comensais, como Bacteroides fragilis, reprimem a produção de IL17, sugerindo que a microbiota intestinal desempenha papel importante no equilíbrio entre a produção e a resposta à IL-17 (Gaboriau-Routhiau et al., 2009; Ivanov et al., 2009; Round et al., 2011). Além disso, animais tratados com antibióticos ou criados em ambientes livres de microorganismos apresentam redução significativa das células Th17 intestinais (Atarashi et al., 2008; Ivanov et al., 2008). A manutenção da integridade da mucosa intestinal é uma das funções desempenhadas pela IL17 que tem grande impacto na regulação e resposta a mudanças da microbiota. Pelo menos em parte, a IL17 controla a integridade da barreira mucosa por controlar a síntese de proteínas das tight junctions, como aumentando a expressão de claudina (Kinugasa T. et al., 2000).
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Distúrbios da microbiota assim como distúrbios da produção de IL17 no intestino resultam em redução da expressão das proteínas tight junction, o que favorece o aumento da permeabilidade intestinal. Uma das consequências deste aumento de permeabilidade é o aumento da absorção de lipopolissacarídeo (LPS) produzidos por bactérias Gram negativas. Dependendo da magnitude deste fenômeno pode ocorrer um quadro de endotoxemia metabólica associada a um quadro de inflamação subclínica que favorece o desenvolvimento de resistência à insulina. Estudos recentes têm atribuído às alterações da microbiota um dos papéis mais importantes na gênese da resistência à insulina associada a distúrbios nutricionais, obesidade e DM2 (Cani, P. D. et al., 2207).
O trato gastrointestinal (TGI) dispõe de um sistema sensorial, que engloba o sistema imunológico, humoral e nervoso, especializado em detectar e responder a mudanças que ocorrem constantemente no intestino (Dubin, P.J. et al., 2008). Tal sistema sensorial tem sido bem estudado no que diz respeito a detecção da presença de nutrientes na dieta. Em resposta a determinados nutrientes, células especializadas do intestino produzem peptídeos ou outras substâncias que atuam regulando respostas sistêmicas a determinados nutrientes. Dentre os diversos efeitos exercidos por estes agentes sensores, destata-se o efeito incretínico caracterizado pela liberação de substancias que, em resposta a glicose na dieta, atuam na ilhota pancreática promovendo um aumento na secreção de insulina (Creutzfeldt, W. et al., 1985). Hormônios incretínicos promovem, direta ou indiretamente, um aumento na secreção de insulina. Apenas dois desses hormônios podem agir de forma direta, tornando-os mais relevantes para as investigações, sendo eles: o peptídeo insulinotrópico dependente de glicose (GIP) e o peptídeo similar ao glucagon 1 (GLP1) (McIntyre N. et al., 1965; Shuster L.T. et al., 1988).
As células L, enterócitos endócrinos, são as responsáveis pela síntese e liberação do GLP1. Essas são encontradas dispersas por todo o intestino grosso e delgado, com uma densidade mais elevada no íleo distal e no cólon proximal. As células K, que por sua vez
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produzem GIP, são amplamente encontradas no duodeno e no jejuno proximal. Isso pode explicar os diferentes perfis de GLP1 e GIP no sangue após a ingestão de alimentos (Parker H.E. et al., 2010).
Fig.2 Localização da expressão de GIP e GLP1 no trato gastrointestinal.
O GIP é rapidamente secretado no duodeno, em resposta a nutrientes, devido à elevada densidade local de células K. O GLP1 é estimulado apenas quando a taxa de entrada de glicose no duodeno excede a sua capacidade de absorção. A secreção desses peptídeos é estimulada não só por carboidratos, mas também por lipídeos e proteínas (Karamanlis A. et al., 2007; Hirasawa A. et al., 2005; Carr R. D. et al., 2008).
O aumento do consumo de alimentos ricos em gordura de alta densidade energética e altamente palatável representa uma das principais causas do aumento da obesidade mundial (Bray G. et al., , 2004; Golay A. et al., , 1997). A exposição crônica a uma dieta rica em gordura leva a alterações morfológicas, fisiológicas e metabólicas que resultam em interrupções nas vias reguladoras da fome e do gasto energético, promovendo a absorção e utilização mais eficiente de gordura, falha nos sinais de saciedade e excesso de adiposidade (Bray G. et al., , 2004; Golay A. et al., , 1997).
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O aumento da adiposidade corporal é o principal fator de risco para o desenvolvimento de doenças metabólicas, particularmente DM2. Estudos epidemiológicos mostram que existem hoje aproximadamente 300 milhões de pessoas com DM2 no planeta e, em decorrência do aumento esperado dos índices de obesidade, o número de pessoas com DM2 deve aumentar proporcionalmente nas próximas décadas (Chen, L. et al., 2011).
DM2 decorre da associação entre resistência à ação da insulina e uma falência relativa da ilhota pancreática em produzir quantidades de insulina que atendam à demanda periférica (Weyer C. et al., , 1999). Nos últimos 20 anos, estudos revelaram que a principal causa da resistência à insulina é a ativação de proteínas inflamatórias intracelulares que tem atividade serina/treonina quinase. Uma vez ativadas, estas proteínas podem levar à fosforilação em resíduos serina e subsequente inibição de alguns dos intermediários da via de sinalização da insulina (Cani P. D. et al., , 2005). Ácidos graxos saturados presentes na dieta são os principais responsáveis pela indução da ativação das serina/treonina quinases inflamatórias (Hotamisligil, G. S. et al., , 1993). Foram identificados dois mecanismos principais responsáveis por mediar os efeitos inflamatórios dos ácidos graxos: ativação de estresse de retículo endoplasmático (ERE) (Ozcan, U., et al., 2004) e ativação de TLR4 (Shi, H. et al., 2006).
O retículo endoplasmático (RE) é a organela responsável pela síntese e enovelamento de proteínas de membrana e proteínas secretórias. Em condições fisiológicas, o RE opera num ritmo necessário para manutenção da homeostase protéica da célula (Ellgaard, L. et al., 2003). Entretanto, em algumas condições patológicas, como infecções e distúrbios nutricionais ou metabólicos, há necessidade de se alterar o ritmo de síntese protéica para que se adeque às novas necessidades da célula, como por exemplo, para aumento da síntese de proteínas que participam da resposta contra os agentes ou fenômenos patogênicos. Existem proteínas sensoras do ritmo de atividade e sobrecarga do RE (Hotamisligil, G. S., et al., , 2010). Uma vez ativadas, tais proteínas acionam uma maquinaria protéica que tem por objetivo biológico
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organizar a atividade de síntese frente ao estímulo patogênico. A sobrecarga da atividade no
RE, induzida por um estímulo potencialmente patogênico, é denominada ERE, enquanto a
resposta induzida pelos sensores do ERE denomina-se resposta a proteínas mal-formadas, ou em inglês, unfolded-protein response (UPR) (Kozutsumi, Y. et al., , 1988).
Em 2004, Ozcan e colaboratores demonstraram que ácidos graxos presentes na dieta eram capazes de induzir ERE em tecidos insulino sensíveis. Como consequência da indução de ERE, ocorria a ativação de proteínas com atividade serina/treonina quinase, como JNK e IKK as quais, uma vez ativadas, levavam à fostorilação em serina do principal substrato do receptor de insulina, IRS1, resultando em resistência à insulina. O uso de chaperonas químicas, que revertem o ERE, promovem melhora considerável da intolerância à glicose (Ozcan, U., et al., 2006).
Ácidos graxos saturados podem ativar vias inflamatórias por outro mecanismo que tem efeitos que se sobrepõem àqueles ativados por ERE. Neste caso, os ácidos graxos ativam um receptor de membrana pertencente ao sistema imune inato, denominado TLR4 (Shi, H. et al., 2006). De acordo com estudos realizados nos últimos 10 anos, o aumento de ácidos graxos
séricos, induzidos pelo consumo elevado de alimentos ricos em gorduras, ativa a sinalização de
TLR4 em vários tecidos do organismo, mas predominantemente em macrófagos e micróglia (Milanski, M. et al., 2009; Razolli, D. S. et al., , 2015). Neste contexto, macrófagos produzem citocinas inflamatórias como TNFα, IL1β e IL6, as quais atuam em células insulino-sensíveis induzindo a ativação das serina/treonina quinases, JNK e IKK, que são ativadas na vigência de ERE (Ozcan, U., et al., 2006). De modo similar ao que ocorre durante o ERE, a ativação do TLR4 acaba por reduzir a atividade da via de sinalização da insulina por mecanismos inflamatórios.
Estudos recentes colocaram mais um componente importante na complexa rede de mecanismos que leva à resistência à insulina. Estudos demonstraram que o consumo de dietas ricas em gorduras é capaz de modificar a microbiota intestinal e promover danos na integridade
9
da mucosa do trato digestório (Cani, P. D. et al., 2007). Neste contexto, há aumento da absorção de nutrientes, entre ele ácidos graxos, o que aumenta a absorção calórica e contribui para aumento de ganho de massa corpórea (Bray G. A. et al., 2004). Alem disso, há aumento da transposição de LPS do lumen intestinal para a circulação. O LPS atuando sistemicamente, sinergiza com ácidos graxos saturados para ativar TLR4, fechando assim um círculo de fenômenos que se combinam para levar à inflamação subclínica característica das doenças metabólicas associadas à obesidade (Cani, P. D. et al., 2007).
Partindo das premissas que a dieta altera a microbiota e que a IL17 participa do controle da resposta contra potenciais patógenos intestinais, sendo assim um fator imunológico importante envolvido no controle da homeostase do trato digestório (Cani, P. D. et al., 2007; Dubin, P.J. et al., 2008), aventamos a hipótese que componentes da dieta poderiam diretamente modular a expressão de IL17 no trato digestório e, caso tal premissa fosse correta, talvez esta citocina pudesse colaborar com o quadro de inflamação subclínica presente em condições metabólicas.
Na primeira etapa do estudo, confirmamos que de fato a dieta é capaz de modular a expressão da IL17 no intestino, porém, diferente do que inicialmente supúnhamos, a IL17 não tem efeito indutor de resistência à insulina, mas sim, age de forma similar a uma substância com atividade incretínica, aumentando a secreção de insulina induzida por glicose, além de atuar durante o período embrionário estimulando o desenvolvimento adequado da ilhota pancreática.
10
1. OBJETIVOS
Geral
Avaliar a expressão da IL17 no trato gastrointestinal em resposta a nutrientes e sua repercussão no controle metabólico.
Específicos (muitos deles propostos em decorrência de resultados obtidos no
decorrer do desenvolvimento do estudo)
Avaliar a expressão gênica da IL17 em diferentes regiões do TGI em resposta a ração
convencional e dieta rica em gordura;
Caracterizar metabolicamente camundongos knockout para o receptor da IL17 (KO
IL17RA);
Avaliar morfológica e funcional do pâncreas endócrino adulto de animais IL17RKO;
Avaliar morfológica do pâncreas endócrino durante o desenvolvimento embrionário de
animais IL17RKO;
Avaliar os efeitos da secreção de insulina e homeostase sistêmica da glicose dos
animais IL17RKO;
Avaliar a resposta de ilhotas pancreáticas humanas a IL17;
Avaliar os níveis sanguíneos de IL17 em humanos magros, obesos com tolerância a
11
3. ARTIGO
Letter
Interleukin-17 is produced in the gut in response to dietary fats and regulates insulin secretion
Carina Solon, Nathalia R. Dragano, Ana C. Vasques, Albina F. Ramalho, Carolina S. Sollon, Junia C. Santos-Silva, Fabio A. Grieco, Joseane Morari, Daniela S. Razolli, Everardo M. Carneiro, Decio L. Eizirik, Bruno Geloneze, Licio A. Velloso
Affiliations
Laboratory of Cell Signaling and Obesity and Comorbidities Research Center, University of Campinas, 13084-970 – Campinas-SP, Brazil
CS, NRD, CSS, JM, DSR & LAV
Laboratory of Investigation in Metabolism and Diabetes, University of Campinas, 13084-970 –
Campinas-SP, Brazil ACV & BG
Department of Structural and Functional Biology and Obesity and Comorbidities Research Center, University of Campinas, 13083-865 – Campinas-SP, Brazil
JCS-S & EMC
Center of Diabetes Research, UniversiteLibre de Bruxelles (ULB), 1070 Brussels, Belgium FAG & DLE
Competing Financial Interests
The authors declare no competing financial interests
Corresponding Author Licio A Velloso
Laboratory of Cell Signaling, University of Campinas 13084-970 Campinas-SP, Brazil
12 Abstract
Interleukin-17 (IL17) is involved in the immune response against intestinal pathogens, and its anomalous expression in the gut can occur in conditions such as type 1 diabetes (T1D) 1, autoimmune encephalomyelitis 2 and Crohn’sdisease 3. Because dietary factors can change the gut microbiota, impacting metabolic diseases 4, we hypothesized that IL17 could be directly modulated by nutrients and might play a role in obesity and type 2 diabetes (T2D). Here, we show that dietary fats induced IL17 expression, predominantly in the ileum. Both in vivo and in isolated pancreatic islets, IL17 stimulated insulin secretion, while its systemic inhibition resulted in glucose intolerance. Mice KO for the main IL17 receptor (IL17RA) were glucose intolerant due to anomalous embryonic development of the pancreatic islets, which were smaller and were depleted of insulin-producing cells. In humans, blood IL17 increased following a meal. This increase was significantly higher in obese normoglycemic individuals than in obese subjects with diabetes. Similar to those of rodents, human islets were also stimulated to secrete insulin in the presence of IL17. Thus, we identified IL17 as a gut sensor of dietary fats, which exerted an incretin-like effect. In addition, the presence IL17RA was important for normal development of the pancreatic islets.
Recent studies have identified gut dysbiosis as an important mechanism connecting the consumption of dietary fats to obesity and insulin resistance 5,6. Upon changes in the gut microbiota, there is increased energy harvesting from the diet, accompanied by increased uptake of fats and increased leakage of lipopolysaccharides (LPS) from the gut to the
bloodstream 5,6. These factors contribute to a progressive increase in body adiposity and to the
development of metabolic inflammation and insulin resistance. A large number of studies have evaluated different aspects of gut dysbiosis in obesity and T2D,4,7 but little is known about the
13
cross-talk between nutrients and the regulation of gut immune factors that provide surveillance against potential gut pathogens.
IL17 has emerged as a major player in gut immunity,8 and the anomalous regulation of its production can impacthealth, predisposing to diseases such as bacterial or fungal infections,
inflammatory bowel disease, autoimmunity and cancer 1-3,9,10. The expression of IL17 is
implicated in the response against intestinal microbes, and changes in the gut microbiota can
affect or be affected by IL17-driven responses 2,9-11. Nutritional factors are important
determinants of the gut microbiota landscape 12; however, it is not clear whether these factors act directly or indirectly to promote these changes. Here, we explored the hypothesis that nutrients modulate the gut production of IL17.
In fasting mice, IL17 mRNA is detected in all parts of the gut, with the highest expression in the jejunum and ileum (Fig. 1A). In the jejunum, the ingestion of chow following a period of overnight fasting leads to a discrete (not significant) increase in IL17 expression (Fig. 1B). However, in the ileum, chow intake leads to a significant and sustained increase in IL17 mRNA expression (Fig. 1C). As an experimental control, we evaluated the gut expression of GLP1. As depicted in Figures 1D-1F, the highest expression of GLP1 occurred in the ileum and colon (Fig. 1D), and the most relevant increase following the ingestion of chow occurred in the ileum (Fig. 1E), whereas in the colon, there was only a non-significant oscillation, in accordance with previous
studies 13. To evaluate the impact of nutrient type and route of administration on IL17
expression, mice were submitted to an ivGTT, an oGTT or a chow refeeding after an overnight fast, and blood was sequentially collected for the determination of the serum levels of IL17, insulin and GLP1. IL17 was not affected by either intravenous or oral glucose (Fig. 1G); there was, however, a significant increase in serum IL17 60 min after chow intake (Fig. 1G). As expected, both insulin (Fig. 1H) and GLP1 (Fig. 1I) were most significantly increased following an oral ingestion of glucose.
14
Next, we evaluated the impact of a fat-enriched diet (high-fat diet, HFD) on the gut production of IL17. When the fasting mice, previously fed on chow, were refed on the HFD, the expression of IL17 mRNA in the ileum (Fig. 2B), but not in the jejunum (Fig. 2A), wasrapidly and significantly increased. Refeeding with HFD was significantly more effective to stimulate the expression of IL17 in the ileum than refeeding with chow (Fig. 2C).
Previous studies have identified lymphocytes from the gut lymphoid tissue as the main source of intestinal IL17 14. Here, we used nude mice to test the hypothesis that lymphocytes were the main source of diet-induced gut IL17. Nude mice are athymic due to a spontaneous deletion of
the foxn1 gene, resulting in a severe reduction of T lymphocytes 15. We refed fasting nude mice,
which were previously fed on chow, with either chow (Fig. 2D) or a HFD (Fig. 2E) and determined IL17 mRNA expression in the duodenum, jejunum and ileum. As depicted in Figures 2D and 2E, the wild-type mice presented detectable expression of IL17 mRNA in both regions of the gut, and IL17 expression was particularly high in the ileum following the refeeding with a HFD (Fig. 2E). Conversely, no IL17 mRNA was detected in the duodenum and jejunum of nude mice following refeeding with either chow (Fig. 2D) or the HFD (Fig. 2E). Very low levels of IL17 mRNA were detected in the ileum of nude mice, but refeeding with the HFD had no impact on IL17 expression (Fig. 2E).
Because gut IL17 was particularly responsive to dietary fat, we hypothesized that this cytokine is involved in the control of metabolic functions. To test this hypothesis, we employed a mouse knocked out for the main IL17 receptor (IL17RA),16 to determine the impact of a HFD on a number of metabolic parameters. Upon feeding for five weeks on either chow or HFD, IL17RA-KO mice presented no significant differences in body mass gain (Fig. 3A and 3B) or caloric intake (Fig. 3C and 3D) compared withwild-type controls. However, upon feeding with the HFD, the IL17RA-KO mice had smaller epididymal fat pad depots (Fig. 3E). Interestingly, IL17RA-KO mice, fed either chow or a HFD, presented higher fasting blood glucose levels, which tended to
15
be accentuated in mice fed a HFD (Fig. 3F and 3G). Moreover, during an ipGTT, IL17RA-KO mice displayed a glucose intolerance phenotype with increased area under the glucose curve when fed either chow or the HFD (Fig. 3H and 3I). All of these experiments were performed both with male and with female mice, and the results were similar, independent of gender. Because the fasting blood glucose levels of IL17RA-KO mice were significantly higher at the beginning of follow-up, when the mice were 6weeks old (Fig. 3F), we evaluated blood glucose in newborn mice. As depicted in Figure 3J, IL17RA-KO mice were hyperglycemic both at birth and from day 25 on. Fasting serum insulin was significantly lower in IL17RA-KO mice at days 5 and 15 of life (Fig. 3K).
To understand further the observed hypoinsulinemic phenotype of IL17RA-KO mice, we performed hematoxylin-eosin staining of adult mouse pancreatic sections, which revealed a consistent reduction in pancreatic islet size (Fig. 4A). Next, pancreatic sections were employed in immunofluorescence staining with anti-insulin and anti-glucagon antibodies. As depicted in Figure 4B, the reduction of pancreatic islets was due to a reduction in -cell mass. Importantly, pancreatic islets of wild-type mice expressed IL17RA, and most of its immunoreactivity coincided with the cells (Fig. 4C). Morphometric evaluation of pancreatic islets revealed that islets from IL17RA-KO mice had a 60% reduction in size compared withislets from wild-type mice
(8,345±1,331 vs. 3,355±0,882 m2 for the control and IL17RA-KO mice, respectively; p<0.05).
Defective regulation of IL17 is known to play a role in inflammatory and autoimmune diseases, including T1D 17,18. However, in immunofluorescence studies, we found no signs of pancreatic islet infiltration by CD4, CD8 or CD11b cells in IL17RA-KO mice (Fig. 4D), thus virtually excluding the possibility of a T1D-like autoimmune process as the explanation for the observed reductions in pancreatic islet size and function.
Next, we evaluated whether the defective IL17 signaling resulted in pre-natal abnormalities in pancreatic islet ontogenesis. Immunofluorescence studies reveled that, at embryonic day 15.5
16
(E15.5) in wild-type mice, the expression of the pancreatic islet markers Maf-A and Pdx-1 were mostly detectable in scattered cells distributed in the exocrine tissue; however, some clusters of Maf-A/insulin and Pdx-1/insulin were also detectable, indicating the early formation of pancreatic islets (Fig. 5A). Conversely, in IL17RA-KO mice, we found no clustering of cells expressing Maf-A/insulin or Pdx-1/insulin (Fig. 5B). At E18, the differences in islet development were even more evident between wild-type and IL17RA-KO mice (Fig. 6A-6B). In wild-type mice, Nkx2.2 and Ngn3 co-localized with insulin, forming almost-round clusters of cells, which, in some cases, already resembled mature islets (Fig. 6A), while in IL17RA-KO mice, Nkx2.2/insulin and Ngn3/insulin cells were more scattered and tended to organize into elongated clusters with no resemblance to pancreatic islets (Fig. 6B). The morphological differences between E18 developing islets in wild-type and IL17RA-KO mice were better observed on higher magnification microphotographs of Pdx-1/insulin-stained tissues (Fig. 7A-7B).
The potential physiological rolesofIL17 in the regulation of islet function and glucose metabolism were further explored by inhibiting steady-state IL17 by two distinct approaches. First, we acutely immunoneutralized IL17 with a specific antibody, which resulted in long-lasting but transient fasting hyperglycemia in wild-type mice (Fig. 8A). Moreover, acute treatment with an anti-IL17 immunoneutralizing antibody induced glucose intolerance, as determined by anipGTT (Fig. 8B-8C). Next, we treated wild-type mice with ursolic acid (UA), which is known to reduce
the expression of IL17 by inhibiting the transcription factor ROR t19. Chronic treatment with UA
resulted in a progressive increase in fasting glucose levels in wild-type mice, which became significantly higher than the levels in the control mice after three weeks (Fig. 8D). In addition, treatment with UA was also capable of producing glucose intolerance, as evaluated by anoGTT (Fig. 8E-8F). We further explored the glucose-modulating actions of IL17 by designing and performing an IL17-tolerance test. The test was designed to be similar to a traditional ITT, with the exception that IL17, and not insulin, was acutely injected. Under the IL17-tolerance test,
17
blood glucose area under the curve was significantly decreased (Fig. 8G-8H). In addition, we evaluated the direct effect of IL17 on insulin secretion by isolated pancreatic islets. For this experiment, pancreatic islets were exposed either to low (2.8 mM) or high (11.1 mM) glucose levels and then were acutely treated with IL17. As depicted in Figure 8I, IL17 at a concentration of 100 ng/ml was capable of significantly increasing glucose-induced insulin secretion. Finally, we observed that pancreatic islets from IL17RA-KO mice presented impaired glucose-stimulated insulin secretion at different glucose concentrations (11 – 22 mM) (Fig. 8J).
In the final part of the study, we evaluated the metabolic actions of IL17 in humans. First, using isolated human pancreatic islets, we confirmed the capacity of IL17 to increase insulin secretion to the medium (Fig. 9A). Next, we observed that obese subjects had higher fasting blood levels of IL17than lean controls (Fig. 9B). Upon a meal-tolerance test, the lean subjects presented the greatest increase in IL17 levels, obese non-diabetic presented an intermediary increase, and obese individuals with diabetes presented the smallest increase in IL17 levels (Fig. 9C-9F). This study revealed novel and physiologically relevant functions of IL17. First, we showed that nutrients, particularly dietary fats, stimulated IL17 expression in the gut. This finding has potential impact on two different aspects of metabolic regulation. The first regards a putative indirect role of fat in the gut microbiota via regulation of IL17 and the consequent immune response against gut pathogens or commensals. The second aspect regards the previously unknown effects of IL17 on the regulation of islet function and glucose metabolism. Because it responded to dietary fats, gut-produced IL17 could be added to the list of gut peptides involved in the regulation of glucose metabolism, such as GLP1 and GIP, with the difference that, instead
of responding primarily to carbohydrates 20, IL17 responds to fat.
Another important finding of this study was the potential impact of IL17RA on pancreatic islet development, as evidenced by the smaller and dysfunctional islets present in IL17RA-KO mice. To our knowledge, there is no other ligand for IL17RA than IL17, suggesting that IL17 acts
18
throughout embryonic life as an important factor in the organization and maturation of the endocrine pancreas. This effect continues during post-natal life,with IL17 providing a rather powerful stimulus to enhance glucose-stimulated insulin secretion. Finally, by inhibiting physiological levels of IL17 by two distinct approaches, we provided evidence for a role of IL17 in glucose homeostasis.
The results obtained in humans suggested that anomalous regulation of IL17 could play a role in the disturbed glucose homeostasis of obese diabetic subjects compared withobese non-diabetic subjects. T2D results from the combination of insulin resistance and a relative deficiency in insulin 21. Metabolic inflammation is a key phenomenon leading to the impairment of insulin
signal transduction, and it has emerged as a unifying mechanism to explain insulin resistance 22.
Defective pancreatic islet function, however, could result from a number of distinct mechanisms, such as lipotoxicity23, glucotoxicity24, altered neural control of insulin secretion 25 and inflammation 26,27, implying that the deficiency in insulin accounts for most of the mechanistic
variability behind T2D 21. Our results suggested that anomalous regulation of gut IL17 in patients
with T2D might be another mechanism contributing to the defective pancreatic islet function in this disease.
In conclusion, we described a novel and pivotal role for the cytokine IL17 in connecting the gut sensing of nutrients to the regulation of pancreatic islet physiology.
Acknowledgements. We thank Erika Roman, GersonFerraz, Antonio Calixto and Marcio Cruz,
from the University of Campinas, for providing technical support for the study. Grants were
provided by Sao Paulo Research Foundation and ConselhoNacional de
DesenvolvimentoCientifico e Tecnologico to LV and the European Union (project BetaBat, in the Framework Program 7 of the European Community) and Fonds National de la RechercheScientifique (FNRS), Belgium, to DLE.
19 Figures
Fig.1
Figure 1. Expression of IL17 in response to nutrients. IL17 and GLP1 (A and D, respectively)
transcripts were determined by real-time PCR in tissue fragments obtained from distinct gut regions of wild-type mice. IL17 and GLP1 transcripts were determined by real-time PCR in the jejunum (B, for IL17), ileum (C and E for IL17 and GLP1, respectively) and colon (F, for GLP1) of wild-type mice after an overnight fast, immediately after chow offering (refed) and following 30, 60 and 120 min of fasting. IL17 (G), insulin (H) and GLP1 (I) serum levels were determined by ELISA in wild-type Wistarrats submitted to an intravenous glucose tolerance test (ivGTT), an oral glucose tolerance test (oGTT) or a refeeding following an overnight fast (refed). In all of the experiments, n=6. In C and E, *p<0.05 vs. overnight fast. In G-I, *p<0.05 vs. respective group at
20
0 min. In the real-time PCR experiments, GAPDH was the housekeeping gene, and the results are presented as mRNA fold variationsrelativeto the controls; in A and D, the stomach was the baseline, and in B, C, E, and F, overnight fasting was the baseline.
Fig.2
Figure 2. Expression of IL17 in response to dietary fat and expression of IL17 in the gut of nude mice. IL17 transcripts were determined by real-time PCR in the jejunum (A) and ileum (B)
of wild-type mice after an overnight fast, immediately after the offering of a high-fat diet (refed) and following 30, 60 and 120 min of fasting after the offering of a high-fat diet. The difference between fasting and the highest diet-stimulated transcript levels of IL17 in the ilea of chow- or high-fat diet (HFD)-fed mice is depicted in (C). IL17 transcripts were determined by real-time PCR in the duodenum, jejunum and ileum of wild-type or nude mice following overnight fast (D) or a high-fat diet (E). In all experiments, n=6. In C, *p<0.05 vs. chow.
21 Fig.3
Figure 3. Metabolic characterization of knockout mice. Wild-type (Ctr) and
IL17RA-knockout (IL17R-KO) mice were fed either chow or a high-fat diet (HFD) and were evaluated for body mass (A), body mass change over a 5-week period (B), daily food intake (C), cumulative food intake over a period of 5weeks (D), epididymal fat pads (E), fasting blood glucose during the period of evaluation (F), fasting blood glucose level at the end of the experimental period (G), blood glucose levels during an intraperitoneal glucose tolerance test (H) and area under the glucose curve during the intraperitonealglucose tolerance test (I). The fasting blood glucose
22
levels were determined in wild-type (Ctr) and IL17RA-KO newborn mice, from day 0 to day 80 (J). The fasting blood insulin levels were determined in wild-type (Ctr) and IL17RA-KO newborn mice, at days 5 and 15 of life (K). In all of the experiments, n=6. In B, D, E, G and I, *p<0.05 vs. respective strain fed on chow; in E, G and I, §p<0.05 for comparisons between conditions as depicted. In J and K *p<0.05 vs. Ctr. Multiple comparisons were performed,so ANOVA was required.
Fig.4
Figure 4. Characterization of the pancreatic islets of IL17RA-knockout mice. In A,
hematoxylin-eosin staining of 5.0-m sections of pancreata from wild-type (upper panel) and IL17RA-knockout mice (lower panel); the red arrows depict the limits of pancreatic islets. In B,
23
immunofluorescence staining using antibodies to detect insulin (rhodamine, red) and glucagon
(fluorescein, green) in 5.0-m sections of pancreata from wild-type (upper panels) and
IL17RA-knockout mice (lower panels). In C, immunofluorescence staining using antibodies to detect IL17RA (fluorescein, green) and insulin (rhodamine, red) in 5.0-m sections of pancreata from wild-type mice. In D, immunofluorescence staining using antibodies to detect insulin (rhodamine, red) and CD4 (fluorescein, green), CD8 (fluorescein, green) or CD11b (fluorescein, green), as
depicted in the respective microphotographs, in-5.0 m sections of pancreata from wild-type
(upper panels) and IL17RA-knockout mice (lower panels); only the merges are shown. All of the microphotographs are representative images obtained from preparations of 3-5 distinct mice, from which at least three sections were stained and analyzed.
24
Figure 5. Ontogeny of pancreatic islets in IL17RA-knockout mice at embryonic day 15.5.
Five-micrometer sections prepared from wild-type (A) and IL17RA-KO pancreata were evaluated at embryonic day 15.5 (E15.5) by immunofluorescence staining for the expression of Maf-A (fluorescein, green) and insulin (rhodamine, red) or of Pdx-1 (fluorescein, green) and insulin (rhodamine, red). Yellow arrows indicate clusters of double-positive cells. All of the microphotographs are representative images obtained from preparations of 3-5 distinct mice, from which at least three sections were stained and analyzed.
25 Fig.6
Figure 6. Ontogeny of pancreatic islets in IL17RA-knockout mice at embryonic day 18.
26
at embryonic day 18 (E18) by immunofluorescence staining for the expression of Nkx2.2 (fluorescein, green) and insulin (rhodamine, red) or of Ngn3 (fluorescein, green) and insulin (rhodamine, red). All of the microphotographs are representative images obtained from preparations of 3-5 distinct mice, from which at least three sections were stained and analyzed.
Fig.7
Figure 7. Detail of ontogeny of pancreatic islets in IL17RA knockout mice at embryonic day 18. Five-micrometer sections prepared from wild-type (A) and IL17RA KO-pancreatawere
evaluated at embryonic day 18 (E18) by immunofluorescence staining for the expression of Pdx-1 (fluorescein, green) and insulin (rhodamine, red). Only merged images are depicted. All of the
27
microphotographs are representative images obtained from preparations of 3-5 distinct mice, from which at least three sections were stained and analyzed.
Fig.8
Figure 8. IL17 and glucose homeostasis. Wild-type mice were treated either with a
pre-immune serum or with an immunoneutralizing antibody against IL17 (IL17 antibody, 2.5 g
intraperitoneally, twice per week for four weeks), and fasting glucose levels were measured (A). At the end of the experimental period, the mice were submitted to a glucose tolerance test, and blood glucose levels were measured (B); in addition, the area under the glucose curve was
28
determined (C). Wild-type mice were treated either with diluent or ursolic acid (UA, 2.0 mg), and fasting glucose levels were measured (D); at the end of the experimental period, the mice were submitted to an intraperitoneal glucose tolerance test, and blood glucose levels were measured (E); in addition, the area under the glucose curve was determined (F). Wild-type mice were acutely treated with a single intraperitoneal dose of IL17 (0.5 g) or with a similar volume of saline, and blood glucose levels were determined (G) and employed to calculate the area under the glucose curve (H). Pancreatic islets were isolated from wild-type mice and employed in static insulin secretion experiments (I); for these experiments, groups of five islets were exposed to 2.8 or 11.1 mM glucose for 30 min and then were exposed to saline (Sal) or to increasing concentrations of IL17, as depicted in the panel. After 60 min, medium supernatants were harvested and used to determine the concentration of insulin. Pancreatic islets were isolated from wild-type (Ctr) or IL17RA-knockout mice and were employed in static insulin secretion experiments (J); for these experiments, groups of five islets were exposed to increasing concentrations of glucose as depicted in the panel; after 60 min, medium supernatants were harvested and used to determine the concentration of insulin. In A-H, n=6; in I and J, for each condition, there were six groups of five islets obtained from distinct animals. In A, C, D, F, H and J, *p<0.05 vs. respective controls. In I, *p<0.05 vs. saline under 11.1 mM glucose.
29 Fig.9
Figure 9. Human studies. Groups of 100 pancreatic islets isolated from six human donors were
maintained in culture in medium containing glucose 5.5 mM and were exposed to saline (Sal) or IL17 (20 ng/ml) for 48 h; the supernatants were harvested and used to determine the concentration of insulin (A). The serum concentration of IL17 was determined in the sera of fasting lean subjects, obese subjects with no diabetes (obese) or obese subjects with type 2 diabetes (DM) (B); thereafter, the subjects received a standard meal, and IL17 was determined in sera collected after 15, 30 and 120 min in the lean (C), obese (D) and DM (E) subjects. The difference between fasting and maximal IL17 levels is depicted in F. In A, for each condition, there were 6 groups of 100 islets obtained from 6 distinct subjects; in B-C, there were 12 subjects in each group. In A, *p<0.05 vs. saline; In B and F, *p<0.05 vs. lean subjects.
30 Refecences
1 Alam, C. et al., . Inflammatory tendencies and overproduction of IL-17 in the colon of
young NOD mice are counteracted with diet change. Diabetes59, 2237-2246, doi:10.2337/db10-0147 (2010).
2 Lee, Y. K., Menezes, J. S., Umesaki, Y. & Mazmanian, S. K. Proinflammatory T-cell
responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proceedings of the National Academy of Sciences of the United States of America108
Suppl 1, 4615-4622, doi:10.1073/pnas.1000082107 (2011).
3 Fujino, S. et al., . Increased expression of interleukin 17 in inflammatory bowel disease.
Gut52, 65-70 (2003).
4 Tremaroli, V. & Backhed, F. Functional interactions between the gut microbiota and host
metabolism. Nature489, 242-249, doi:10.1038/nature11552 (2012).
5 Turnbaugh, P. J. et al., . An obesity-associated gut microbiome with increased capacity
for energy harvest. Nature444, 1027-1031, doi:10.1038/nature05414 (2006).
6 Cani, P. D. et al., . Metabolic endotoxemia initiates obesity and insulin resistance.
Diabetes56, 1761-1772, doi:10.2337/db06-1491 (2007).
7 Khan, M. T., Nieuwdorp, M. & Backhed, F. Microbial Modulation of Insulin Sensitivity.
Cell metabolism20, 753-760, doi:10.1016/j.cmet.2014.07.006 (2014).
8 Gu, C., Wu, L. & Li, X. IL-17 family: cytokines, receptors and signaling. Cytokine64,
477-485, doi:10.1016/j.cyto.2013.07.022 (2013).
9 Gaboriau-Routhiau, V. et al., . The key role of segmented filamentous bacteria in the
coordinated maturation of gut helper T cell responses. Immunity31, 677-689, doi:10.1016/j.immuni.2009.08.020 (2009).
31
10 Wu, S. et al., . A human colonic commensal promotes colon tumorigenesis via activation
of T helper type 17 T cell responses. Nature medicine15, 1016-1022, doi:10.1038/nm.2015 (2009).
11 Lemaire, M. M. et al., . Dual TCR expression biases lung inflammation in DO11.10
transgenic mice and promotes neutrophilia via microbiota-induced Th17 differentiation. Journal of immunology187, 3530-3537, doi:10.4049/jimmunol.1101720 (2011).
12 Sonnenburg, E. D. et al., . A hybrid two-component system protein of a prominent human
gut symbiont couples glycan sensing in vivo to carbohydrate metabolism. Proceedings of the National Academy of Sciences of the United States of America103, 8834-8839, doi:10.1073/pnas.0603249103 (2006).
13 Holst, J. J. The physiology of glucagon-like peptide 1. Physiological reviews87,
1409-1439, doi:10.1152/physrev.00034.2006 (2007).
14 Leppkes, M. et al., . RORgamma-expressing Th17 cells induce murine chronic intestinal
inflammation via redundant effects of IL-17A and IL-17F. Gastroenterology136, 257-267, doi:10.1053/j.gastro.2008.10.018 (2009).
15 Balciunaite, G. et al., . Wnt glycoproteins regulate the expression of FoxN1, the gene
defective in nude mice. Nature immunology3, 1102-1108, doi:10.1038/ni850 (2002).
16 Ye, P. et al., . Requirement of interleukin 17 receptor signaling for lung CXC chemokine
and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. The Journal of experimental medicine194, 519-527 (2001).
17 Arif, S. et al., . Peripheral and islet interleukin-17 pathway activation characterizes human
autoimmune diabetes and promotes cytokine-mediated beta-cell death. Diabetes60, 2112-2119, doi:10.2337/db10-1643 (2011).
18 Bending, D. et al., . Highly purified Th17 cells from BDC2.5NOD mice convert into
Th1-like cells in NOD/SCID recipient mice. The Journal of clinical investigation119, 565-572, doi:10.1172/JCI37865 (2009).
32
19 Xu, T. et al., . Ursolic acid suppresses interleukin-17 (IL-17) production by selectively
antagonizing the function of RORgamma t protein. The Journal of biological chemistry286, 22707-22710, doi:10.1074/jbc.C111.250407 (2011).
20 Fehmann, H. C., Goke, R. & Goke, B. Cell and molecular biology of the incretin
hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide. Endocrine reviews16, 390-410, doi:10.1210/edrv-16-3-390 (1995).
21 Kahn, S. E., Hull, R. L. & Utzschneider, K. M. Mechanisms linking obesity to insulin
resistance and type 2 diabetes. Nature444, 840-846, doi:10.1038/nature05482 (2006).
22 Hotamisligil, G. S. Endoplasmic reticulum stress and the inflammatory basis of metabolic
disease. Cell140, 900-917, doi:10.1016/j.cell.2010.02.034 (2010).
23 Cnop, M., Hannaert, J. C., Hoorens, A., Eizirik, D. L. & Pipeleers, D. G. Inverse
relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes50, 1771-1777 (2001).
24 Maedler, K. et al., . Glucose-induced beta cell production of IL-1beta contributes to
glucotoxicity in human pancreatic islets. The Journal of clinical investigation110, 851-860, doi:10.1172/JCI15318 (2002).
25 Calegari, V. C. et al., . Inflammation of the hypothalamus leads to defective pancreatic
islet function. The Journal of biological chemistry286, 12870-12880, doi:10.1074/jbc.M110.173021 (2011).
26 Cnop, M. et al., . Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes:
many differences, few similarities. Diabetes54 Suppl 2, S97-107 (2005).
27 Velloso, L. A., Eizirik, D. L. & Cnop, M. Type 2 diabetes mellitus--an autoimmune
disease? Nature reviews. Endocrinology9, 750-755, doi:10.1038/nrendo.2013.131 (2013).
33 Methods
Experimental animals and protocols. The experimental procedures involving rats and mice
were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the Ethics Committee of the University ofCampinas (#CEUA2329-1). In experiments with Wistar rats, Swiss and Nude mice, we employed always males. In experiments with IL17RA KOand C57BL6 mice (whole body knockout for the IL17RA receptor and its respective control)1 both male and females were employed.Rats were fed on standard rodent chow. Mice were fed either standard rodent chow or a high-fat diet (HFD) (detailed composition of diets is shown in Supplementary Table 1). During the experimental period, the animals were housed at 22°C with a 12-h light/dark cycle andhad access to their respective diets and water ad libitum.
Immunoneutralization of IL17.I n experiments designed to immunoneutralizeIL17, we used a
rabbit anti-IL17 antibody (polyclonal antibody, Santa Cruz Biotechnology, SC-6077). Six-week old male C57BL6mice were randomly selected for the treatment with either vehicle (pre-immune rabbit IgG) or IL17 antibody (2.5 g)viaintraperitoneal (ip) injection twice a week for four weeks.Mice were evaluated weekly fasting blood glucose.
Pharmacological inhibition of IL17. In experiments designed to pharmacologically inhibit IL17,
we used ursolic acid (UA) (Sigma-Aldrich, U6753). Six-week old male C57BL6 mice were selected randomly for treatment with either vehicle (soy oil) or ursolic acid (2.0 mg) via gavage twice a week to four weeks.Mice were evaluated weekly fasting blood glucose.
Exogenous recombinant IL17 treatment. In experiments aimed at evaluating the effect of IL17
on glucose tolerance, we used a recombinant IL17 peptide (Sigma-Aldrich, I4026).For that, C57BL6 mice were randomly selected for treatment with either vehicle (saline) or IL17 peptide (0.5 g) acutely injected ip.
34
Gut preparation. Six-week old mice were used in the experiments. Following a lethal dose of
anesthetic, the entire gut was exposed and fragments of stomach, duodenum, jejunum, ileum and colon were removed. The anatomical limits for the dissection were performed with help of a mouse anatomical atlas. Tissue fragments were homogenized in Trizol and prepared for evaluation of transcript expression by real-time PCR.
Pancreas preparation. Six-week old mice were used in the experiments. In addition, for
evaluation of embryonic development of pancreatic islets, mice on embryonic days 15.5 (E15.5) and 18 (E18) were also employed. Following a lethal dose of anesthetic, the pancreas was removed and fixed in paraformaldehyde (4% final concentration in PBS) for histological evaluation. For the preparation of the fetal pancreata we used magnifying lenses.
Real-time PCR. IL17 and GLP1 transcripts were measured in the distinct gut regions by
real-time PCR (ABI Prism 7500 detection system; Applied Biosystems, Grand Island, NY). The intron-skipping primers were obtained from IDT Biosystems (IL17, mm PT586531092 and GLP1, mm PT5821875990). Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was used as a house-keepingcontrol. The endogenous control for normalization of GAPD was hypoxanthine-guanine phosphoribosyltransferase. No significant change in GAPD expression was detected in the different experimental conditions. The optimal concentrations of the cDNA and primers, as well as the maximum efficiency of amplification, were obtained through a seven-point, 2-fold dilution curve analysis for each gene. Each PCR contained 40 ng of reverse-transcribed RNA and was performed according to the manufacturer's recommendation using the TaqMan PCR master mix (Applied Biosystems). The real-time data were analyzed using the Sequence Detector System 1.7 (Applied Biosystems). All results are presented as mRNA fold-change as compared to the experimental control.
Rodent islet isolation and static insulin secretion.Islets were isolated by collagenase digestion of the pancreas as previously described2. For static insulin secretion, groups of five islets were
35
incubated in the presence of different concentrations of glucose and IL17 was added to the medium. The supernatants were collected after 60 minutes for insulin determination by radioimmunoassay as previously described3.
Determination of insulin, GLP1 and IL17 in response to nutrients.These experiments were
performed with Wistar rats because of the large amount of blood required in each time-point of the test. Six-week old Wistar rats were randomly separated into three groups submitted either to an oral glucose tolerance test (75 mg/Kg), intravenous glucose tolerance test (2.0 mg/g) or refeding with chow. Blood samples were collected at time 0 min (fasting sample) and at 15, 30, 60, 90, 120 and 150 min after administration of glucose or after refeding. Insulin, GLP1 and IL17 were measured by ELISA in the serum samples, according the recommendations of the manufacturers. Insulin (Millipore #EZRMI-13K), GLP1 (Millipore #EGLP-35K) and IL17 (Biolegend #437907).
Metabolic studies. These experiments were performed with six-week old male and female
IL17RA knockout mice and their respective controls, C57BL, which were randomly selected for feeding on either chow or a high-fat diet (composition on Supplementary Table 1). Body mass, food intake and fasting blood glucose (four hours fasting) were measured weekly from six to eleven weeks of live. At the end of the experimental period, mice were submitted to an intraperitoneal glucose tolerance test (ipGTT); finally, mice received a lethal dose of anesthetic and the perigonadal fat mass was measured.
Intraperitoneal Glucose Tolerance Test (ipGTT). Mice were acutely treated with a single ip
injection of glucose 25% (2.0 mg/Kg) and blood glucose was measured at baseline (before glucose administration; 0min) and at 15, 30, 60, 90 and 120 min after glucose administration. Results are presented as glucose levels at each time-point and also as the area under the glucose curve.
