IN ST IT U TO D E CI ÊN CI A S B IO M ÉD IC A S A BE L S A LA ZA R FA CU LD A D E D E CI ÊN CI A S
Kaori
Levy da F
onseca
. Impact of Host Glycosylation in Tuberculosis
Impact of Host Glycosylation in Tuberculosis
Kaori Levy da Fonseca
D.
ICBAS
2019
E AD M IN IS TR AT IV A DOUTORAMENTOBIOLOGIA MOLECULAR E CELULAR
Impact of Host Glycosylation in
Tuberculosis
Kaori Levy da Fonseca
D
Impact of Host Glycosylation in Tuberculosis
Tese de Candidatura ao grau de Doutor em Biologia
Molecular e Celular;
Programa Doutoral da Universidade do Porto (Instituto de
Ciências Biomédicas de Abel Salazar e Faculdade de
Ciências)
Orientador
– Doutora Margarida Sofia da Silva Santos
Saraiva
Categoria – Investigadora Principal
Afiliação – Instituto de Investigação e Inovação em Saúde
(i3S); Instituto de Biologia Molecular e Celular (IBMC)
Coorientador
– Doutora Ana Maria Rodrigues Leite de
Magalhães
Categoria – Investigadora Júnior
Afiliação – Instituto de Investigação e Inovação em Saúde
(i3S); Instituto de Patologia e Imunologia Molecular da
Universidade do Porto (IPATIMUP)
Coorientador
– Professor Doutor Pedro Nuno Simões
Rodrigues
Categoria – Professor Associado com Agregação
Afiliação –Instituto de Ciências Biomédicas Abel Salazar da
Universidade do Porto
Coorientador –Professor Doutor José Carlos Machado
Categoria – Professor Associado
Afiliação
– Faculdade de Medicina da Universidade do
Porto
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This work was funded in part by the grants 01-0145-FEDER-028955 and POCI-01-0145-FEDER-028488 supported through European Regional Development Fund (FEDER) and Fundação para a Ciência e Tecnologia (FCT), by the grant NORTE-01-0145-FEDER-000012 supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the FEDER. The author was supported by a PhD scholarship from FCT, reference SFRH/BD/114405/2016.
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O autor esclarece que na elaboração desta tese foram incluídos os artigos publicados ou manuscritos submetidos para publicação abaixo indicados, e declara ter participado ativamente na conceção e execução das experiências que estiveram na origem da mesma, assim como na sua interpretação, discussão e redação.
The author declares that in the elaboration of this thesis published articles or manuscripts submitted for publication were included as listed below, and declares that she participated actively in the conception and execution of the experiments that produced such data, as well as in their interpretation, discussion and in the manuscript writing.
PUBLICATIONS
Fonseca KL, Rodrigues PNS, Olsson IAS, Saraiva M (2017) Experimental study of tuberculosis: From animal models to complex cell systems and organoids. PLoS Pathogens 13(8): e1006421. https://doi.org/10.1371/journal.ppat.1006421.
Fonseca KL, Maceiras AR, Matos R, Simoes-Costa L, Sousa J, Cá B, Barros L, Fernandes AI,Mereiter S, Reis R, Gomes J, Tapia G, Rodríguez-Martínez P, Martín-Céspedes M, Vashakidze S, Gogishvili S, Nikolaishvili K, Appelberg A, Gärtner F, Rodrigues PNS, Vilaplana C, Reis CA, Magalhães A and Saraiva M. (2020) Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils. This work has been accepted for publication in the
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CKNOWLEDGMENTSO resultado desta tese é o culminar de muitas aprendizagens e de encontros com pessoas que permitiram que este projecto fosse realizado da melhor forma possível.
Em primeiro lugar, gostaria de agradecer à minha orientadora, Dra. Margarida Saraiva, por me receber no laboratório e pela oportunidade que me deu de trabalhar numa área, até então, nova para mim. Agradeço também pelo constante apoio e acompanhamento, pelos ensinamentos, pela motivação, dedicação, total disponibilidade, pelas palavras certas nas horas certas e, sobretudo, pela confiança. Obrigada por tudo!
À minha coorientadora, Dra. Ana Magalhães, por ter aceite fazer parte da equipa de orientação e, acima de tudo, por ajudar a ampliar os meus conhecimentos acerca da glicobiologia. Obrigada pela partilha científica, pela total disponibilidade e apoio.
Ao meu coorientador, o Professor Pedro Rodrigues pelas discussões e constante partilha científica.
Agradeço ao meu coorientador Dr. José Carlos Machado também pela disponibilidade e discussões científicas.
Quero agradecer à direção do programa de Pós-Graduação Ciência para o Desenvolvimento (PGCD), em especial à Joana Sá, pela oportunidade de fazer parte deste excelente projeto. O PGCD foi o trampolim para esta jornada. À família PGCD um muito obrigada!
Ao Dr. Celso Reis agradeço pela excelente colaboração que resultou no meu trabalho de tese. Ao Professor Rui Appelgerg e ao Professor Gil Castro por participarem na maturação do trabalho científico resultante da tese e por todo o apoio.
A special thanks to Dr Cristina Vilaplana and her team for receiving me in the Experimental Tuberculosis Unit at the IGTP (Barcelona), to do part of my PhD work. Thank you for everything!
I want to thank Dr Anne O'Garra for the great scientific discussion and valuable input in the research article resulting from this thesis.
Agradeço o apoio dos membros das Plataformas Científicas do i3S, em especial os serviços de Microscopia de Luz Avançada (ALM), de Histologia e Microscopia Eletrônica (HEMS), membros da infraestrutura nacional PPBI - Plataforma Portuguesa de
Um obrigada à Rita Matos do Glycobiology in Cancer Group por toda a ajuda necessária ao longo da colaboração e do projeto, e à Professora Fátima Gärtner por toda ajuda na análise de patologias.
Aos colegas do Immune Regulation Group, vocês são fantásticos! Um especial obrigada à Ana e à Deisy.
Ao Jeremy por me ter ensinado praticamente tudo o que sei fazer no P3, pelas infinitas horas que passamos a trabalhar no P3, por toda ajuda ao longo deste projecto, pela confiança (às vezes) e por ser um ótimo companheiro de congressos. Obrigada Jeremias!
Ao Baltazar, nha ermon di Guiné, um muito obrigada por partilhar esta jornada comigo, pela paciência e por estar sempre 100% disponível. As palavras não são suficientes para expressar a minha gratidão.
À Catarina pela excelente pessoa que é, pelas conversas ciêntificas e não ciêntificas, por ser uma ótima conselheira, por estar sempre disponível para ajudar e pela amiga que te tornaste.
À Raquel por toda ajuda ao longo da tese, principalmente por ser fulcral em tudo que diz respeito à citometria desta tese. Obrigada pela paciência e por tudo que aprendi contigo.
À Luísa e ao Leandro, não só por terem tido um contributo muito importante neste trabalho, mas pelas pessoas fantásticas que são e por agora fazerem parte do meu círculo de amizades. Leandro de fato és o melhor partner de sempre no P3!
Aos colegas do III, em especial à Carolina, à Tânia e à Rita por estarem sempre disponíveis para ajudar, mas especialmente pelos conselhos, cuidados e pela constante preocupação.
Ao Porto’s Gang pelos momentos de descontração durante este período, pelas longas conversas, pela paciência em ouvir as histórias dos dias menos bons e por estarem sempre prontos para celebrar as vitórias. Um obrigada à Deisy pelo apoio dentro e fora do laboratório, à Irina por ter sempre algo bom para adoçar a alma, à Julie por ser a melhor companheira de dança de todos os tempos, e ao Danilo por todas as “sessões de terapia”. Fizeram com que tudo fosse mais fácil.
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Às minhas little sisters, Annais e Carlisa, por cada palavra de encorajamento, cada abraço ao chegar a casa e cada wake up call. Vocês foram fundamentais nesta fase da minha vida.
À Illyane, minha companheira de caminhada, pela amizade e pelo apoio incondicional. À Eliane e à Taise por serem as melhores amigas do mundo, por estarem sempre presentes, pela compreensão e paciência inesgotáveis, apesar da distância. À Sofia e à Nayara pela amizade e pelo colo magnífico que recebi sempre que precisei. À Djelissa por ter sempre um ombro amigo à minha disposição.
Agradeço ainda a todos os meus amigos (aos que sempre estiveram presentes e aos que ganhei ao longo desta caminhada) e familiares pela amizade, força e companheirismo.
Por último, agradeço ao meu irmão, JC, e aos meus pais, Edna e João, pelo amor e apoio incondicional. Dedico-vos esta tese.
Na impossibilidade de nomear todos aqueles que contribuíram para a realização deste trabalho, deixo um agradecimento geral.
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T
ABLE OF
C
ONTENTS
Acknowledgments ... vii
List of Figures ... xiii
List of Tables ... xv
Abbreviations ... xvii
Resumo ... xix
Abstract ... xxi
Chapter 1 - General introduction ... 1
1. Tuberculosis – a global threat ... 3
1.1. Prevention, diagnosis and treatment of TB ... 4
1.1.1. TB vaccine ... 4
1.1.2. TB diagnosis ... 6
1.1.2.1. Active TB infection ... 6
1.1.2.2. Latent TB infection ... 7
1.1.3. TB antbiotherapy ... 7
1.2. The spectrum of TB infection ... 8
1.3. M. tuberculosis – The causative agent of TB ... 10
1.3.1. MTBC Lineage 2 (Beijing) ... 11
2. The immune response in TB ... 13
2.1. The early immune response to tb ... 13
2.2. The adaptive immune response in TB ... 16
2.3. Host- immune mediators ... 17
2.3.1. Tumour necrosis factor ... 17
2.3.2. IL-1 family ... 18 2.3.3. Type I IFN ... 19 2.3.4. IL-12 ... 20 2.3.5. IFN- ... 21 2.3.6. IL-17/IL-23 axis ... 21 2.3.7. IL-10 ... 21
2.4. The Immune pathology in TB ... 22
2.4.1. The many faces of TB granuloma ... 23
3.1. Protein glycosylation ... 26
3.1.1. O-glycosylation ... 27
3.2. Glycosylation and Disease ... 28
3.2.1. The “sweet” regulation of the immune system ... 29
3.2.2. Glycosylation as a possible new player in M. tuberculosis infection ... 31
4. Experimental models for the study TB ... 31
Chapter 2 - Aims ... 47
Chapter 3 - Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils ... 51
Chapter 4 - General discussion and Conclusion ... 109
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L
IST OF
F
IGURES
Chapter 1 - General introduction
Figure 1 – Global estimated TB cases in 2017……….. 3
Figure 2 – The spectrum of TB infection………. 9
Figure 3 – MTBC phylogeographical distribution………... 11
Figure 4 – Lineage 2 phylogenetic structure………. 12
Figure 5 – The host protective immune response during M. tuberculosis infection…... 15
Figure 6 – The cellular composition of the granuloma lesion………. 23
Figure 7 – Major classes of glycan structures………... 26
Figure 8 – The O-glycosylation biosynthetic pathway………. 28
Chapter 3 – Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils. Figure 1 – Deficiency in Gcnt1 associates with increased susceptibility to M. tuberculosis infection .……….…... 78
Figure 2 – Exacerbated neutrophilia drives increased susceptibility of Gcnt1-/- mice to M. tuberculosis infection ……….…….. 80
Figure 3 – Gcnt1 modulates granulopoiesis and CXCL2 expression ...…... 82
Figure 4 – Blood neutrophilia of Gcnt1-/- mice is promoted by deficiency of this enzyme in hematopoietic cells, but increased susceptibility to M. tuberculosis infection also requires the non-hematopoietic compartment .………...… 84
Figure 5 – M. tuberculosis infection impacts the expression of several glycosyltransferase-encoding genes in humans ……....…….……… 86
Supplemental Figure 1 – Lung pathology in C57BL/6 vs Gcnt1-/- mice upon aerosol infection with M. tuberculosis……….. 88
Supplemental Figure 2 – Overall characterization of lung immune cell populations, immunofluorescence detection of MPO in mice lungs and characterization of neutrophils response to in vitro M. tuberculosis infection………...………..90
Supplemental Figure 3 – Gating strategies and percentage of lymphoid cell populations in the BM of uninfected C57BL/6 or Gcnt1-/- .………...…... 92
Supplemental Figure 4 – Global characterization of the cellular content of BM reconstituted mice, pre- and post-infection with M. tuberculosis …...………... 94
xv
L
IST OF
T
ABLES
Chapter 1 – General introduction
Table I – TB vaccine candidates currently in clinical trials…….………...……….5 Chapter 3 – Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils.
Table I – Lung histopathology analysis of M. tuberculosis infected mice ………...57 Table II – Overall characteristics of the participants originating lung samples ..…..…….62 Table S1 – Log fold change and significance values for differential expression of
glycosyltransferase coding genes in Berry London, Berry Leicester and Berry South Africa datasets ……….………...………..98
Table S2 – Oligonucleotide sequences used in this study ………...………106 Table S3 – Antibodies used in this study .……...107
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BBREVIATIONS -/- Deficient AM Alveolar macrophages BCG Bacillus Calmette-Guérin BM Bone marrow C2GnTs Core 2 β1-6 N-acetylglucosaminyltransferases CCL Chemokine (C-C motif) ligandCD Cluster of differentiation C-type Calcium-dependent
CXCL Chemokine (C-X-C motif) ligand CXCR Chemokine (C-X-C motif) receptor DC Dendritic cell
dLN Draining lymph node DR Drug resistant
ER Endoplasmic reticulum
ESAT-6 Early secretory antigen target-6 Fuc Fucose
FUT Fucosyltransferase Gal Galactose
GalNAc N-acetylgalactosamine
Gcnt1 Glucosaminyl (N-acetyl) transferase 1 GH Glycoside Hydrolase
GlcNAc N-acetylglucosamine
GT Glycosyltransferase
HIV Human immunodeficiency virus H&E Haematoxylin and eosin
IFN Interferon
IFNAR Interferon alpha receptor
IGRA Interferon-gamma release assay IL Interleukin
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Le Lewis x
Ley Lewis y
LTBI Latent tuberculosis infection MDR Multidrug-resistant
MSMD Mendelian susceptibility to mycobacterial diseases MTBC Mycobacterium tuberculosis complex
PGE2 Eicosanoid prostaglandin E2 PPD Purified protein derivative RNA-seq RNA sequencing
SA Sialic acid sLea Sialyl-Lewis A sLex Sialyl-Lewis X TB Tuberculosis Th T helper TLR Toll-like receptor TNF Tumour necrosis factor TST Tuberculin skin test WHO World health organization WT Wild-type
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R
ESUMOA tuberculose (TB) constitui um dos principais desafios ao nível da saúde mundial, encontrando-se entre as dez doenças mais fatais e sendo a principal causa de morte por um único agente infeccioso. Estima-se que mais de 1,7 bilhões de pessoas estejam infectadas com Mycobacterium tuberculosis, o agente patogénico reponsável pela TB, o que significa que cerca de um quarto da população mundial possui uma TB latente. O curso da infecção por M. tuberculosis em humanos depende da interação entre os processos de activação e supressão imunológica. Para além disso depende da capacidade do hospedeiro desenvolver uma resposta imune eficiente para eliminação da infecção, ao mesmo tempo que previne patologias, tais como a destruição de tecidos e a própria transmissão da bactéria. Estudos recentes têm revelado o papel crucial de novas moléculas e vias biosintéticas na regulação e modulação da resposta imune.
Nos últimos anos, diferentes estruturas glicosiladas têm sido reconhecidas como importantes agentes imunológicos, desempenhando um papel central em mecanismos de controlo imune, reconhecimento de agentes patogénicos e tropismo, e por fim na regulação da resposta imune. Adicionalmente, a grande maioria das células efetoras do sistema imunológico possuem receptores de estruturas glicosiladas na sua superfície. Embora existam várias evidêcias que suportam as implicações funcionais da glicosilação em diferentes doenças infecciosas, há um substancial desconhecimento acerca da modulação dos mecanismos de glicosilação no contexto da infeção por M.
tuberculosis e das suas consequências funcionais na regulação da resposta imune.
Esta tese teve como objectivo principal avaliar o impacto dos processos de biossíntese de O-glicanos no contexto de infeção por M. tuberculosis e, desta forma, identificar a função de potenciais novos reguladores imunes do hospedeiro na TB. Para desvendar essa questão, utilizamos murganhos deficientes para Gcnt1 (-/-), uma enzima
crucial na biossíntese de estruturas core 2 em O-glicanos. A deficiência no gene da
Gcnt1 levou ao aumento da suscetibilidade à infecção por M. tuberculosis, sendo esta
mais pronunciada em infecções com doses bacterianas mais elevadas. Mecanisticamente, demonstramos que tanto o compartimento estromal como o hematopoiético contribuem para o fenótipo observado, sendo a suscetibilidade dos murganhos deficientes para Gcnt1 associada a uma patologia pulmonar exacerbada, causada por uma neutrofilia pulmonar desregulada. Este fenótipo foi posteriormente associado a uma aumento da expressão da quimiocina CXCL2 no pulmão. Em seguida, investigamos se a infecção por M. tuberculosis afeta a via biossintética de estruturas Lewis X sialiladas, amplamente em neutrófilos humanos. A análise de dados de sequenciação de RNA (RNA-Seq) de sangue total demonstraram alterações nos níveis
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Lewis X sialiladas foram detectadas em pulmões de pacientes com TB.
Este trabalho demonstra que a modulação das estruturas core 2 em O-glicanos durante uma infecção por M. tuberculosis constitui um mecanismo protetivo do hospedeiro, ressaltando ainda novos fatores associados à regulação da resposta imune em TB.
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BSTRACTTuberculosis (TB) is one of the world’s top health challenges being among the ten most fatal diseases worldwide and the number one cause of death by a single infectious agent. It is estimated that more than 1.7 billion people are infected with Mycobacterium
tuberculosis, the TB causative pathogen, meaning that around one fourth of the global
human population harbours latent TB. The outcome of human infection with M.
tuberculosis depends on the interplay between immune activation and suppression and
the host capacity of mounting an immune response that is able to clear the infection but prevents immune-driven pathology such as tissue destruction and bacteria transmission. Recent studies have been revealing novel regulatory molecules and pathways critical for immune response modulation.
Glycan structures have been recognized, during the last years, as central immunological players, with important roles in immune surveillance, pathogen recognition and tropism, and regulation of the immune response. Moreover, the large majority of the immune system effector cells present surface receptors for glycosylated structures. Although there is paramount data demonstrating the functional implications of glycosylation in different infectious disease contexts, there is a substantial lack of knowledge on the modulation of the host cell glycosylation machinery during M.
tuberculosis infection and its functional consequences in immune response regulation.
In this thesis we aimed to evaluate the impact of O-glycosylation biosynthetic processes during M. tuberculosis infection and therefore shed light on new possible regulators of the host immunity during TB. To unveil this question, we resorted to Gcnt1 deficient (-/-) mice, a crucial enzyme in the biosynthesis of functional core-2- O-glycan
structures. Gcnt1 deficiency led to increased susceptibility to M. tuberculosis infection, which was more pronounced upon infection with high bacterial doses. Mechanistically, both the stromal and the hematopoietic compartments were involved, being the increased susceptibility of Gcnt1-/- mice associated with extensive lung pathology, due
to deregulated neutrophilia. This was further associated with increased lung expression of the neutrophil chemoattractant CXCL2. We next investigated whether M. tuberculosis infection impacts the biosynthetic pathway of sLex core 2-O-glycan, widely present in
human neutrophils. The analysis of available whole blood RNA sequencing (RNA-Seq) datasets showed altered expression of several glycosyltransferases in active patients when compared to control or latently infected individuals. Moreover, sLex was detected
in the lung of TB patients. Our findings support the modulation of core 2-O-glycans during
M. tuberculosis infection as a mechanism associated with host protection, which shed
2 Part of this chapter has been published in:
Fonseca KL, Rodrigues PNS, Olsson IAS, Saraiva M (2017) Experimental study of tuberculosis: From animal models to complex cell systems and organoids. PLoS Pathogens 13(8): e1006421. https://doi.org/10.1371/journal.ppat.1006421
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1. T
UBERCULOSIS–
A GLOBAL THREATTuberculosis (TB) disease, one of the oldest diseases in human history, is still a global health problem (Figure 1) (1). In 2017, an estimated 10 million people were diagnosed as new TB cases or relapses. Moreover, it is estimated that one-fourth of the world population bears a latent tuberculosis infection (LTBI) (2). Additionally, almost 40% of all TB cases (more than 4.0 million TB patients per year) are estimated to be under-reported or undiagnosed, which constitutes a big challenge for TB control (1, 3-5). The 30 TB most affected countries are mainly low and middle-income countries in South-East Asia, Western Pacific and African regions, that comprises 8.7 million (87%) of total estimated cases, as shown in Figure 1 (1).
According to the World Health Organization (WHO), TB disease is one of the top 10 causes of death in the world. Despite the measures implemented in the last decades to tackle TB, this disease claims nearly 1.3 million lives annually among human immunodeficiency virus (HIV)-negative people (3). Also, a total of 0.3 million (around 25%) individuals co-infected with HIV, die of TB (1, 5).
Figure 1 – Global estimated TB cases in 2017. Estimated TB incidence rates, as reported by
the WHO. Reproduced from (1).
Recent years have seen huge progress in TB control, with the implementation of many strategies and policies to tackle TB, such as the directly observed therapy, short course (DOTS), the STOP TB and the End TB strategies (6-8). In addition to these strategies, multiple advances have been made in the epidemiological field, the understanding of the immunological mechanisms and pathophysiology of TB disease, and through improvements in therapeutic and diagnostic tools (3). As a result, between 2000 – 2017
4
an estimated 54.0 million lives were saved and the TB mortality rates are now decreasing at 5% per year (1, 3). Nevertheless, the estimated numbers for new TB cases are falling slowly with an average decline of 1.8% between 2016-2017, which needs to accelerate to a 4 to 5% annual decline, to reach the WHO’s 2030 milestones to End TB (1, 7).
1.1. P
REVENTION,
DIAGNOSIS AND TREATMENT OFTB
The available means to fight TB include a 100-year-old vaccine (Mycobacterium bovis Bacillus Calmette-Guérin; BCG), which efficacy varies from 0-80% (9) and a complex, lengthy and costly antibiotic regimen (10). On the other hand, the emergence of drug-resistant TB (DR-TB) constitutes a big threat for the control of this deadliest infection. Approximately half a million per year of all TB cases were multidrug-resistant (MDR), with around 8.5% of these being extensively drug-resistant (XDR), threatening a return to an era of untreatable disease (3). Therefore, considering the morbidity and mortality rates caused by TB and consequently the economic burden associated, controlling the spread of this disease is critical.
1.1.1. TB
VACCINEOne of the major problems associated to TB control is the lack of an effective vaccine. The most effective and available vaccine is BCG, a live attenuated strain of M. bovis, which lacks the region of difference 1 (RD1) in the genome (11). The RD1 region contains genes that encode for a number of virulence factors present in Mycobacterium
tuberculosis, M. bovis and other virulent strains (12).
The efficacy of BCG is variable among adults with pulmonary TB (13), the most common form of TB and that leading to transmission of the bacilli. However, BCG is protective against disseminated forms of the disease, including meningitis and miliary TB, which are the main causes of mortality and morbidity in infants and young children (14). Although intensive research is dedicated to the development of novel TB vaccines, so far none has been shown to be more efficient than BCG (1). Nevertheless, TB vaccine candidates are being developed and several are already in clinical trial, as listed in table I. Understanding the bases of the protective immune response to TB is acknowledged as a key determinant for the successful development of novel vaccines.
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Table I - TB vaccine candidates currently in clinical trials. Adapted from (1, 12, 15)
Vaccine candidate Description Vaccine strategy Current phase
MTBVAC (16-18) Live attenuated M. tuberculosis Considered as post-exposure vaccine Phase II
VPM1002 (19, 20) Recombinant BCG* Considered as post-exposure vaccine Phase III
RUTI® (21, 22) Detoxified, fragmented M. tuberculosis For treating latent TB post chemotherapy Phase II completed Vaccae™ (23, 24) Heat-inactivated, whole-cell Mycobacterium vaccae Chemotherapy adjunct in HIV-infected individuals Phase III
DAR-901 (25) Whole-cell, inactivated non-tuberculous mycobacterium Prophylactic, post-exposure and therapeutic Phase II; phase IIb
MIP (26, 27) Whole cell, heat-inactivated Mycobacterium indicus pranii Therapeutic Phase III
M72 /AS01E (28-30) Adjuvanted recombinant protein expressing M. tuberculosis antigens 32A and 39A
Boost response to BCG, also considered as
post-exposure vaccine Phase IIb
H4:IC31(31) Adjuvanted recombinant protein expressing M. tuberculosis
antigens Ag85B and TB10.4 Boost response to BCG Phase IIb
H56:IC31(32)
Adjuvanted recombinant protein expressing M. tuberculosis antigens Ag85B, early secretory antigen target-6 (ESAT-6) [H1]; or Ag85B, ESAT-6, Rv2660c [H56]
Boost response to BCG, also considered as
post-exposure vaccine, and a BCG replacement Phase II
ID93 + GLA-SE (33, 34) Adjuvanted recombinant protein expressing M. tuberculosis antigens Rv3619, Rv3620, Rv1813 and Rv2608
Boost response to BCG, also considered as
post-exposure vaccine Phase II
MVA85A (35-38) Viral vector (modified vaccinia virus Ankara) intradermal followed by aerosol; prime–boost vaccine Boost response to BCG, also considered as
post-exposure vaccine Phase II
Ad5 Ag85A (39) Viral vector (human adenovirus 5) expressing M.
tuberculosis antigen Ag85A
Boost response to BCG, also considered as
post-exposure vaccine, and a BCG replacement Phase I/II * BCG, Bacillus Calmette-Guérin; HIV, Human immunodeficiency virus; TB, tuberculosis.
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1.1.2. TB
DIAGNOSISA range of different technologies, such as imaging, microscopy, culture-based, immunological and, more recently, molecular techniques are used for TB diagnosis (40), as detailed below.
1.1.2.1. A
CTIVETB
INFECTIONActive pulmonary TB diagnosis is commonly based on the patient clinical history and must be confirmed by the presence of M. tuberculosis in sputum smear for acid-fast bacilli (AFB) or culture for M. tuberculosis (41). Despite numerous advances in TB diagnostics, microbiological techniques are still the most reliable methods to an unequivocal diagnosis of active TB disease (41). The screen for AFB is rapid, inexpensive and ideal for high-burden settings (42), what justifies its use mostly in low and middle-income countries. In spite of that, acid-fast stains detect other mycobacteria species, are unable to screen for extra-pulmonary TB, and their sensitivity varies between 22–80% since the detection is dependent on the acid-fast concentration in the sputum (43). Moreover, radiography tools as for example chest-X-ray or positron emission tomography–computed tomography (PET-CT) scans are used to complement the diagnostic processes (44).
A new era has come with the introduction of molecular testing in TB diagnosis (45). The ability to detect M. tuberculosis genetic material directly from patient’s specimen or microbial cultures allowed not only the detection of M. tuberculosis, but also the screening for mutations responsible for antibiotics resistance. Real time PCR-based techniques such as Xpert MTB/RIF, Xpert MTB/RIF Ultra and Xpert XDR tests (Cepheid Inc) yield faster results and allow for detection of resistance to the main first line TB drugs, rifampicin and isoniazid, and other second line TB drugs (46). Most importantly, these technologies showed higher sensitivity and reliability, when compared to sputum smear microscopy, although having higher costs (46). Thus, the development of new diagnostic tools needs to combine increased sensitivity and specificity, shorter turnaround time, robust point-of-care platforms and drug resistance screening (14).
Another molecular tool with possible impact on the diagnosis of TB is based on the detection of host immune signatures associated with active TB in the whole blood (47). The initial report of a whole blood transcriptional interferon (IFN)-based signature distinguishing active TB patients from latently infected individuals and from healthy controls (48) has been confirmed in several subsequent studies performed in different settings and using different techniques (47). Furthermore, this signature has been shown
7
to also identify response to treatment (49). This platform holds promise as a tool to improve TB diagnosis in the future.
1.1.2.2. L
ATENTTB
INFECTIONLTBI is characterized by the absence of clinical symptoms and defined by the evidence of host immunological sensitization to M. tuberculosis antigens, what makes difficult the distinction between a latent and an active infection (50). The diagnosis of LTBI is indirectly established by measuring host T cell memory responses primed by mycobacterial antigens, resorting to two main immunological tests (5).
The tuberculin skin test (TST) consists of an intradermal injection of tuberculin purified protein derivative (PPD) mix, which leads to a delayed-type hypersensitivity skin reaction by individuals previously exposed to mycobacterial antigens (51). TST is widely used for infection diagnosis due to the low reagent and equipment costs, and no need for skilled labour, which is mainly relevant in high TB burden settings (51). However, the limitations associated with this test relate to a poor specificity, since cross-reactivity to the BCG vaccine or exposure to non-tuberculous mycobacteria (NTM) can lead to false-positive results. Also, false negative results can occur in HIV co-infected patients, due to host- immunosuppression (51).
The IFN-γ release assay (IGRA) blood tests have been introduced as an alternative to TST, presenting higher sensitivity and specificity (52). IGRAs detect the ex vivo release of IFN-γ from T cells, a key host-cytokine produced against M. tuberculosis. T cell responses are in this case specific against the M. tuberculosis early secretory ESAT-6 and culture filtrate protein-10 (CFP-10) antigens (53). As compared to TST, IGRAs cross-reactivity with BCG and NTM do not constitute a problem, due to ESAT-6 and CFP-10 antigens absence in these bacteria (52). Nonetheless, IGRA test present some limitations, such as the higher costs associated, laboratory requirement, and false-negative results in co-infected with HIV (53).
1.1.3. TB
ANTBIOTHERAPYGenerally, drug-susceptible TB disease is treated with a 6 months-drug regimen with isoniazid, rifampicin, pyrazinamide, and ethambutol for 2 months (intensive phase), followed by isoniazid and rifampicin administered for an additional 4 months (continuation or sterilizing phase) (54). Patients compliance with the whole treatment process leads to a success rate above 80% (55). The length of the current TB treatment, together with the adverse effects (for example, liver and renal dysfunction, and
8
gastrointestinal disorders) are major drawbacks in achieving compliance (54). The long treatment period often causes a lack of treatment adherence, which has many consequences as treatment failure, relapse cases and selection of drug resistant M.
tuberculosis strains (54).
The emergence of DR-TB constitutes a major health concern and barrier to disease prevention, diagnosis and treatment. MDR-TB is defined as resistance to at least two of the main front-line drugs, i.e. isoniazid and rifampicin (1). Additionally, the resistance to second-line drugs as fluoroquinolones and any other injectable drugs, together with isoniazid and rifampicin, is leading to a more threatening form of TB disease – the XDR-TB (54). According to the WHO guidelines, DR-XDR-TB treatment regimens vary in length (9– 24 months of therapy) (5) and drug combination, and are normally individual- specific, depending on the drug to which the M. tuberculosis strain is resistant to (55, 56).
1.2. T
HE SPECTRUM OFTB
INFECTIONTB is an airborne disease, transmitted by exposure to infectious aerosol droplets carrying M. tuberculosis particles, from an active patient with pulmonary TB to an otherwise healthy individual (42). Once infected, some individuals appear to clear infection, others become latently infected and, a few develop active disease (Figure 2) (57). Active TB patients (red dash in Figure 2) normally present symptoms (like chronic cough, fever, night sweats and weight loss), with a positive TST or IGRA, chest-X-ray with TB-associated pathology, and bacilli growth on sputum culture (58). Although M.
tuberculosis infection affects preferentially the lung, it can also disseminate to other
lymphoid and non-lymphoid organs and cause extra-pulmonary TB disease (59). A small group of individuals is capable to clear the bacilli (TB negative; green dash in Figure 2) through the innate or adaptive immune responses, showing absence of symptoms, negative sputum culture, and normal chest x-ray (5), although with or without a positive TST or IGRA test (58). Nonetheless, most people infected with M. tuberculosis can control the infection, without eliminating the pathogen (LTBI; yellow dash on Figure 2). These individuals are categorized as having a LTBI and are estimated in almost 2 billion of the entire world population (2). From the clinical point of view, latently infected individuals show no symptoms of TB and are thought to be unable to transmit disease (60). Moreover, LTBI people are usually screened based on immunologic tests, having positive TST or IGRA but normal chest x-ray (50).
Transitions between the different TB status occur. Indeed, around 10% of LTBI individuals are estimated to be in risk to reactivate, developing active TB and thus
9
become able to transmit the bacilli. There are several identified risks associated with the development of active TB, namely through reactivation of LTBI (Figure 2). Among these, HIV co-infection remains the most important risk factor associated with TB, increasing the risk by almost 30 times (1). Other risks include alcoholism, diabetes mellitus, smoking, malnutrition, age and sex (61-63). Moreover, anti-tumour necrosis factor (TNF) therapy used to treat autoimmune diseases also constitutes a risk for LTBI transition to active TB disease (64). On the other hand, some LTBI individuals are thought to clear the bacteria upon infection (Figure 2), by yet not well-understood mechanisms. In all, immune imbalances are key determinants in defining the TB outcome.
Figure 2 – The spectrum of TB infection. Upon contact with an active TB case and transmission
of M. tuberculosis, several outcomes are possible, from clearance of infection to severe active TB disease. This spectrum of outcomes can at the moment be distinguished by differences in symptoms, IGRA/PPD results, M. tuberculosis presence in the sputum and CXR characteristics. Transitions between the different outcomes of TB are possible, being immune imbalances determinant for progression from LTBI to active disease. CXR, chest-X-ray; IGRA, IFN-γ release assay; LTBI, latent tuberculosis infection; PPD, purified protein derivative; TB, tuberculosis. Adapted from (58).
Noteworthy, recent evidence shows that the outcome of human infection with M.
tuberculosis is in fact a continuous spectrum of disease, rather than a binary
classification (58). This is because among individuals classically classified as LTBI, a group presenting subclinical forms of disease exists, who will progress to active TB (65). Thus, a better stratification of LTBI spectrum is important for a good prediction on people that are at risk for progression into an active TB (50). This will help in controlling disease transmission (66) and possibly understand which individuals would benefit from preventive therapy programs (5). Furthermore, a spectrum of TB severity is also seen in active TB patients depending on the clinical manifestation of disease.
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1.3. M.
TUBERCULOSIS–
T
HE CAUSATIVE AGENT OFTB
The etiological agent of TB – M. tuberculosis - was firstly reported in 1882, by Robert Koch (67). M. tuberculosis sensu stricto is an acid-fast, aerobic and facultative intracellular bacillus, with a thick and impermeable cellular wall, mainly composed by peptidoglycan and a complex layer of lipids (68). This rod-shaped bacterium is a slow-grower and has a range size from 0.65 to >7 µm (68, 69). Human and animal TB are mainly caused by members of the M. tuberculosis complex (MTBC), a group of genetically closely related elements from the Mycobacterium genus (70). The MTBC comprises the human-adapted members, M. tuberculosis and Mycobacterium africanum, and other mammalian-associated pathogens such as Mycobacterium bovis,
Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium origys, Mycobacterium mungi and Mycobacterium suricattae (71).
Sequencing of the first complete M. tuberculosis strain (H37Rv) genome, revealed a genome of 4,411,529 base pairs, with around 4,000 genes, and a very high guanine and cytosine content (72). More recently, the access to mycobacterial whole-genome sequencing (WGS) brought new insights in the genetic diversity within the MTBC, which is presently known to comprise seven human-adapted phylogenetic lineages (L1 to L7) (Figure 3A), each of them further divided into multiple sublineages (73, 74). These different lineages are clearly associated to a different geographical distribution, reflecting the parallel evolution with the human population (Figure 3B, C, D) (73). Despite this, some lineages, as L2 East- Asian and L4 Euro-American are geographically more spread, and so called generalists (Figure 3B) (71, 73). In contrast, L5 and L6 are geographically restricted to the West-Africa region and as such are considered specialists. These two lineages comprise M. africanum strains (Figure 3D) (71).
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Figure 3 – MTBC phylogeographical distribution. A) The phylogeny of the MTBC. B)
Distribution of L2 East- Asian and L4 Euro-American isolates; C) Distribution of L1 Indo-Oceanic and L3 East-African-Indian isolates and D) Distribution of L5 and L6 West-Africa isolates. Reproduced from (73).
In recent years, the importance of the MTBC diversity surpasses the phylogeographic distribution of the TB-causing bacteria. Indeed, diversity among the isolates of the different lineages has been shown to impact disease presentation, transmission, drug resistance acquisition and immune responses (73, 75, 76). In the context of experimental TB research, L4 M. tuberculosis strain H37Rv has been mostly used. However, with the continuous understanding of MTBC diversity, more studies are starting to use clinical isolates of M. tuberculosis, such as for example L2 M. tuberculosis strain HN878. As this strain has been used in the context of this thesis, an overview of L2 characteristics is provided below.
1.3.1. MTBC
L
INEAGE2
(B
EIJING)
The Beijing family of M. tuberculosis strain was firstly reported in 1995 and found to dominate the East-Asian geographical region (Figure 3B) (77). More recently, molecular epidemiological studies have shown that the Beijing strains have intercontinental distribution (Figure 4) and are rapidly spreading around the globe, suggesting a higher strain-associated virulence and higher rates of transmission (74, 78). The Beijing genotype is also strongly associated to a rapid progression to disease upon infection,
12
severe forms of TB disease, treatment failure and with MDR-TB and XDR-TB outbreak occurrence, that can be explained by their hypermutability, transmissibility, pathogenicity and virulence (71, 79-81). Nevertheless, the mechanisms underlying this increased ability to acquire drug resistance remain unclear.
Figure 4 – Lineage 2 phylogenetic structure. Phylogeny and worldwide distribution of the
so-called “modern” lineage 2 of the MTBC, that includes the Beijing family of strains. MDR-TB, multidrug-resistant tuberculosis. Adapted from (74).
Experimental studies using both murine and cellular models have shown that Beijing
M. tuberculosis strains are likely to be more virulent (82, 83). M. tuberculosis HN878
strain (member of the Beijing family), firstly isolated during an outbreak in Texas (USA), was found to be hypervirulent when infecting immunocompetent mice, leading to decreased survival when compared to mice infected with different clinical isolates, including the laboratory strain H37Rv (84). The suggested mechanisms underlying this increased susceptibility to HN878 have been clarified with time. Studies conducted by Kaplan and colleagues suggested an increased susceptibility to HN878 linked to a failure in inducing the development of protective T helper (Th) 1 cellular responses, accompanied by enhanced expression of type I IFNs in infected mice (84, 85). In line with this, in a different study, M. tuberculosis HN878-infected mice displayed faster bacterial growth in the lungs and noticeable lung damage as compared to those infected with other strains (86). The higher virulence of HN878 has been attributed in part to the presence of an unusual cell wall phenolic glycolipid molecule (PGL) (87). Interestingly, more recent studies, affirm the detrimental role of type I IFN in both human TB and experimental models of M. tuberculosis infection (88).
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2. T
HE IMMUNE RESPONSE INTB
The immune response in TB is complex (Figure 5). Studies conducted in both man and experimental models have shown the critical role of specific molecular and cellular mediators for protective responses and for the subsequent pathogenesis of TB (89). The following sections provide a general overview of the major events that compose the innate and adaptive immune responses to TB and of the involved mediators.
2.1. T
HE EARLY IMMUNE RESPONSE TO TBM. tuberculosis is transmitted from an individual to another via aerosol. Upon
inhalation, the droplet nuclei carrying the bacteria enters the lung and the infection is established in the alveolar space (90). Once in the alveoli, M. tuberculosis interacts with lung resident macrophages – the alveolar macrophages (AMs) - that are believed to be the primary cellular niche of the bacteria (91). AMs develop from fetal liver monocytes during embryogenesis, and are capable of self-renewal at steady state (92). During M.
tuberculosis infection, AMs uptake the bacilli and mediate its dissemination from the
alveoli to the lung interstitium, to further initiate the host immune response (Figure 5A and B) (93).
In addition to AM, during the initial steps of infection, M. tuberculosis was recently shown to interact with other lung cells (69, 90). Specifically, a role for lung epithelial cells in sensing M. tuberculosis when entering the lung was shown (69, 90). Specialized epithelial cells known as microfold cells (M cells) have been demonstrated to be infected by M. tuberculosis, translocated to draining lymph nodes (dLN) and recognized by various components of mucosa-associated lymphatic tissue (69). These non-classical immune cells are believed to activate non-conventional T cells – the mucosal associated invariant T (MAIT) cells – with a further contribution for the immune response against TB (94-96). However, the role of epithelial cells in M. tuberculosis infection and TB pathogenesis remains to be fully elucidated.
M. tuberculosis interacts with the host innate immune cells through a variety of
pattern-recognition receptors (PRRs), including toll-like receptors (TLRs), calcium-dependent (C-type) lectins receptors (CLRs) and Nod-like receptors (NLRs). which are crucial for the recognition of pathogen-associated molecular patterns (PAMPs) and bacteria uptake (89). In the context of M. tuberculosis infection, TLR2, TLR4 and TLR9 are the most studied TLRs in macrophages and dendritic cells (DCs) (97-100).
To activate and recruit other immune cells and to limit the infection, AMs express several pro-inflammatory mediators such as TNF, interleukin (IL)-12, IL-6, and IL-1 and
14
macrophage inflammatory protein-1α (MIP-1α/CCL3) (101). Therefore, after entering the lung parenchyma, M. tuberculosis encounters other immune cells, including recruited macrophages, neutrophils and DCs (Figure 5B) (89). Recruited macrophages arise from blood monocytes at steady state and during M. tuberculosis infection are attracted to the lung to control the bacilli growth, mainly through the chemokine (C-C motif) ligand 2 (CCL2) production (102). Recently, M. tuberculosis-infected recruited macrophages have been demonstrated to hold a different transcriptional profile and distinct metabolic states, as compared to AM (103). A study conducted by Russel and collaborators demonstrated a highly glycolytic metabolic state and a more restrictive phenotype to M.
tuberculosis replication in recruited macrophages, while AMs presented a metabolic shift
towards β-oxidation and fatty acid uptake, being more permissive to replication and survival of the bacilli (103). Together with macrophages, during infection, neutrophils are attracted to the lung, playing an important role for the immune response (104), as detailed below (section 2.4.2). These initial events are important to shape the immune response to M. tuberculosis. The activated innate immune cells also take up the bacteria, attempt at destroying it and initiate the adaptive immune response (Figure 5B) (91). The bridge between the innate and adaptive immunity in TB is established by DCs, so called-antigen presenting cells (APCs). Upon bacteria engulfment, DCs migrate to the dLN where they will promote the differentiation of the adaptive immune response (105), as developed in the next section.
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Figure 5 – The host protective immune response during M. tuberculosis infection. Upon inhalation via aerosol, M. tuberculosis infects phagocytic cells,
including lung alveolar macrophages, neutrophils and dendritic cells (DCs) (A and B), leading to the production of pro-inflammatory cytokines, chemokines and antimicrobial peptides (B). M. tuberculosis-activated DCs migrate to the draining lymph node (dLN) (9-11 days after infection in the mouse model), in a cytokine and chemokine-dependent process (C). Once in the dLN, DCs trigger the differentiation of naïve T cells into T helper (Th)1, Th17 and T regulatory (Treg) effector cells (C). T effector cells migrate back to the lung (18-21 days post-infection in the mouse model), to promote macrophage activation through Th1-IFN-γ production, neutrophil recruitment via Th-17/IL-17/22 axis and inflammation control via IL-10 (D). These events prime cytokine production, microbicidal mechanisms activation (D) and culminate in granuloma formation (E) for M. tuberculosis infection control. CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; TGF-β, transforming growth factor beta; TNF, tumour necrosis factor. Adapted from (95, 106).
16
2.2. T
HE ADAPTIVE IMMUNE RESPONSE INTB
The migration of M. tuberculosis from the primary site of infection – the lung – to the dLN is assured by DCs that play a central role in naïve T cell activation for the development of an effector T cell response (Figure 5B and C) (107-109). In the case of
M. tuberculosis infection Th1 and Th17 cell responses are differentiated (110, 111). The
Th1 differentiation depend on the expression of IL-12p70 by M. tuberculosis-activated DCs (Figure 5 C) (108). The hallmark cytokine associated to Th1 cell responses is IFN-γ (Figure 5D) (112), a highly protective cytokine in TB, as detailed below. On the other hand, the differentiation of Th17 requires the presence of IL-6 and IL-1β in the micro-environment, as well as of IL-23, which sustains Th17 responses (Figure 5C) (113). Th17 cells are important sources of IL-17, which protective role during M. tuberculosis infection seems not to be as important as that of IFN-γ (111). The orchestrated differentiation of Th1 and Th17 is however important as a cross regulatory mechanism to ensure minimal tissue damage (Figure 5D) (114, 115).
Importantly, Th cell differentiation in the context of M. tuberculosis infection is well accepted to be a delayed process at least in the mouse model, where it starts 9–11 days post-infection (Figure 5B) (107, 116). It is also generally accepted that anticipation of specific Th cell responses would have a protective effect on TB. After 18-21 days of exposure to M. tuberculosis, the antigen-specific Th1 and Th17 cells migrate back to the lung through chemokine-dependent mechanisms (Figure 5C) (95). Once at the site of infection (lung), T cells are critical for activating macrophages and curtail mycobacterial growth (Figure 5D) (117). As mentioned above, Th1 cells are the major source of IFN-γ which is essential for the activation of several microbicidal mechanisms in the macrophage (89). IFN-γ acting on macrophages leads to the expression of the inducible nitric oxide synthase (iNOS), which promotes the release of nitric oxide (118). Additionally, activation of the macrophage IFN-γ receptor enhances phagolysosome fusion (119) and autophagy (120). Together, these mechanisms culminate with M.
tuberculosis containment (110). On the other hand, Th17 effector cells secrete IL-17 and
IL-22 and are involved in efficient neutrophil recruitment and tissue repair (111). In addition to effector T cells, regulatory T cells (Treg) are also recruited to the site of infection where they play an important role in controlling the inflammatory response and lung damage containment (121, 122), presumably via IL-10 production (123).
The role of cluster differentiation (CD)4+ T cells is thus well established in TB. In the
mouse model, lack of this subset of T cells leads to an uncontrolled bacterial growth and a premature death of the infected host (110). In humans, the increased susceptibility of HIV-infected individuals to M. tuberculosis has been associated to the lack of CD4+ T
17
cells (124). Also, mutations in the IL-12/IFN-γ axis, that compromise the development or action of Th1 cells via IFN-γ, are associated with Mendelian susceptibility to mycobacterial disease (MSMD) (125). M. tuberculosis infection also primes the CD8+ T
cells, which are able to produce IL-2, IFN-γ, and TNF-α to activate macrophages to control the infection, and also via granule-mediated function to kill the bacilli (126). The role of CD8+ T cells is however much less prominent than that of CD4+ cells. In addition
to T cells, B cells are recruited to the lung during M. tuberculosis infection (89). Although the role of B cells in the immune response to M. tuberculosis remains elusive, the presence of activated B cells has been observed in the granuloma of non-human primates (127), hinting at a possible role of humoral response in TB. Overall, the recruitment of different myeloid and lymphoid immune cells culminates with the granuloma structure formation (Figure 5E), for host defence and M. tuberculosis infection containment, as explained below (section 2.4).
It is important to mention that despite the host protective response mounted against the bacteria, M. tuberculosis developed diverse mechanisms to evade the immune system (128). M. tuberculosis is able to modulate the host immune response through the avoidance of phagosome-lysosome fusion by inhibiting its maturation and acidification, and consequently bacillus elimination (128). Also, the bacteria inhibit the production free-radical mechanisms (such as reactive oxygen species - ROS), the apoptosis, phagocytosis and inflammasome activation systems, that prevents mycobacterial growth and the induction of the acquired immune response (128).
2.3. H
OST-
IMMUNE MEDIATORSThe cellular immunity mounted against M. tuberculosis is fine-tuned through the production of diverse chemical factors such as cytokines, chemokines and other host soluble factors. Among others, the following inflammatory factors are well described as fundamental for cellular recruitment to the site of infection, pathogen containment and pathology control during M. tuberculosis infection.
2.3.1. T
UMOUR NECROSIS FACTORTNF-α has a primordial function in the control of M. tuberculosis infection. This soluble factor binds to TNF receptors (TNF-R) and induces the activation of pro-inflammatory responses (101). Although macrophages are the main source of this pro-inflammatory cytokine, TNF- α can be produced by a range of other immune cells, such as neutrophils, DCs and T cells (89). The main functions of TNF-α rely on the intracellular M.
18
tuberculosis killing after phagocytosis, macrophages and leukocytes trafficking, and
granuloma maintenance (129). The critical role of TNF-α in controlling M. tuberculosis infection has been well addressed by several studies using mice lacking the 55-kDa TNF receptor gene, which showed inability to contain the bacteria, with increased bacteria burden and tissue pathology and further translated in increased susceptibility to infection (112, 130). Moreover, latently infected humans submitted to anti-TNF treatment for inflammatory autoimmune diseases, as rheumatoid arthritis and Crohn's disease, showed high rates of TB reactivation (131).
2.3.2. IL-1
FAMILYThe IL-1α and IL-1β pro-inflammatory cytokines are members of the IL-1 family and are primarily produced by monocytes, macrophages, and DCs upon encountering the bacteria. During M. tuberculosis infection, IL-1 cytokines signal via the IL-1 receptor (IL-1R) present in most cells, leading to the recruitment and activation of downstream regulator molecules, and consequently to the activation of a highly inflammatory host protective response (132). In the context of TB, both IL-1α and IL-1β are known to be required for host resistance against the infection (132). Indeed, mice deficient for both IL-1α and IL-1β, or for the IL1-R showed an increased susceptibility to infection and an increased pulmonary bacterial burden (133, 134). Recent studies showed that the role of IL-1β during M. tuberculosis infection surpasses the control of bacterial burden. Indeed, IL-1β plays a role in early protection against M. tuberculosis via group 3 innate lymphoid cells (ILC3) (135) and in the bacilli dissemination from the alveolar space to lung interstitium (93). Finally, a role for IL-1R signalling in limiting the proportion of infected cells and dissemination and thus affording protection has been recently revealed (136). Importantly, excessive IL-1β production may also be detrimental to the host and has been associated with more severe TB disease and increased lung damage (118). It is therefore not surprising that several mechanisms are in place to control IL-1β production, such as the production of type I IFN and of nitric oxide (118, 137).
In humans, genetic studies demonstrate an association of polymorphisms in the IL-1 or IL-1R genes with altered disease progression and susceptibility (138). For all these reasons, IL-1 has emerged as a candidate for host-directed therapies for TB (139), by placing this cytokine in a cross-regulatory network together with type I IFN and the production of the eicosanoid prostaglandin E2 (PGE2). Indeed, IL-1 induces PGE2, which enhances the antimicrobial activity of M. tuberculosis-infected macrophages. Additionally, type I IFN was showed to inhibit IL-1 production and in turn to be inhibited by PGE2. The IL-1–PGE2–IFN pathway was also engaged in active TB disease,
19
correlating with disease severity (139). In this context, M. tuberculosis-infected mice presenting high IFN levels showed reduce disease severity when supplemented with PGE2 (139). Importantly, these findings were validated in TB patients.
2.3.3. T
YPEI
IFN
Type I IFN belong to a family of a large group of structurally similar cytokines, in which IFN-α (represented by several partially homologous genes) and IFN-β (represented by a single gene) are the best characterized ones (140). All type I IFN share a ubiquitously expressed heterodimeric transmembrane receptor named the IFN-α receptor (IFNAR), composed of IFNAR1 and IFNAR2 subunits, which signal trough signal transducer and activator of transcription (STAT)1 and STAT2 to activate IFN-related genes (141). During
M. tuberculosis infection, type I IFNs are mainly produced by monocytes/macrophages,
neutrophils and DCs, and are commonly associated to a pathogenic role in disease by supporting the infection (88). Mice deficient for IFNAR have been demonstrated to be more protected against M. tuberculosis infection, showing decreased bacterial loads in the lung and/or improved survival, as compared to control groups (85, 86, 142, 143), although this protection has not been universally observed (144-147). Moreover, M.
tuberculosis-infected mice intranasally treated with the type I IFN inducer polyIC, exhibit
increased lung bacillary loads and widespread pulmonary immunopathology in wild-type (WT) mice, but not in Ifnar deficient (-/-) ones (144). Similarly, prior exposure of mice to
influenza A virus, another well-known inducer of type I IFN, has been shown to worsen the M. tuberculosis infection outcome via IFNAR signalling (148). On the other hand, type I IFN can display protective features under specific conditions. Experimental studies conducted by our laboratory and other laboratories have demonstrated the importance of type I IFN in protecting the host during M. tuberculosis infection when in the absence of the IFN- receptor gene (Ifngr) (145, 146). Accordingly, mice deficient for both Ifnar and Ifngr displayed increased lung bacterial burden, increased pathology and consequently decreased survival, when compared with single Ifngr -/- mice (145, 146).
In human TB, type I IFN-inducible transcriptional blood signature was reported in active TB patients, which was associated with the extent of disease and decreased responses to treatment (48). The overexpression of IFN-inducible gene patterns was distinct as compared to other pulmonary diseases (48, 49, 149), suggesting a TB-specific signature. In addition, transcriptional studies resorting to cohorts of patients with active disease and latently infected or healthy individuals suggested a differential peripheral activation of the type I IFN response among the spectrum of TB infection, which can be used as an indicator risk of TB disease progression (48, 149).
20
Although in both human and mouse models of TB, the detrimental role of excessive production of type I IFN family of cytokines has been reported, the underlying mechanisms are not fully understood. However, experimental studies implicating type I IFN in important cross-regulatory mechanisms can help unveil this conundrum (150, 151). In the presence of exogenous IFN-β, M. tuberculosis-infected bone marrow (BM) derived macrophages was found to dampen the production of host-protective cytokines such as TNF-α, IL-12, and IL-1β, by upregulating the production of the IL-10 cytokine (detailed hereafter) (150). Thus, type I IFN may act by tempering the protective pro-inflammatory immune response. Nevertheless, basal levels of type I IFN were shown to be required to maximize the secretion of IL-12 and TNF-α by macrophages in response to M. tuberculosis infection (150). Furthermore, studies by our group show a role for type I IFN is preventing the alternative activation of macrophages during TB (146). Overall, these studies suggest a dual role on type I IFN in the host immune response to M.
tuberculosis infection.
2.3.4. IL-12
As previously mentioned, IL-12 plays a fundamental role as a bridge between innate and adaptive immune responses, mediating the early T-cell activation. The bioactive IL-12 (IL-IL-12p70) heterodimeric cytokine is composed by two different subunits, the p35 and p40, being primarily produced by M. tuberculosis- activated macrophages and DCs (152). The critical role of IL-12p40 in preventing TB in humans has been demonstrated by an inherent predisposition to M. tuberculosis infection in people harbouring IL-12- related MSMD, and also an increased susceptibility to the infection, even to BCG vaccination (101, 125, 153). Experimental studies resorting to mice models lacking the IL-12p40 subunit reported an impairment to control the bacterial infection and failure to sustain IFN-γ Th1 cellular responses (89, 101, 154). Moreover, treatment of IL-12p40-deficient DCs with IL-12p40 homodimer (IL-12(p40)(2)) restored the M. tuberculosis-activated DC migration and their ability to activate T cell (108).
21
2.3.5. IFN-
The type II IFN group includes only one cytokine – the IFN-γ – which is fundamental for the host protective immunity in TB (138). Despite being secreted by other lymphoid cells, such as CD8+ T cells, γδ T cells and natural killer (NK) cells, the production IFN-γ
is mainly mediated by CD4+ Th1 cells (89). The major functions of this cytokine relate to
macrophage activation, cytokine production with further induction effector of mechanisms to bacterial control, such as iNOS induction (89, 101, 138). In both mice and man, it is well established that IFN-γ production is essential for survival following M.
tuberculosis infection. Indeed, mice lacking IFN-γ production showed impaired
production of antimicrobial products, extensive tissue destruction and necrosis and fails in controlling M. tuberculosis replication, presenting high bacterial loads in the lungs (155-157). Also, IFN-γ has been demonstrated to inhibit pathogenic neutrophil accumulation, by regulating Th17 responses (114, 115, 157). In humans, genetic deficiencies in the IFN-γ pathway has been associated to MSMD (158).
2.3.6. IL-17/IL-23
AXISAlthough Th17 effector cells are conventionally recognized as the main producers of IL-17 (101), during mycobacterial infection this pro-inflammatory cytokine can also be produced by other lymphoid cellular subsets, mainly by lung resident γδ T cells (159). IL-17 plays an important role in the early host immunity to M. tuberculosis since it is required for granulocytes/neutrophil recruitment to the lung, but also acts in the chronic phase of infection, mainly for granuloma formation, maintenance, and immunopathology prevention (111). However, this cytokine is also associated to immune-mediated pathology, as without a balanced Th1/Th17 response immune pathology develops, as observed in animal experimental models (89, 115).
2.3.7. IL-10
A well-orchestrated immune response is essential to avoid excessive inflammation and ultimately, tissue destruction and pathology. The role of IL-10 in regulating the inflammatory processes during TB has been addressed in both human and experimental studies (123). Experimental studies resorting to mice lacking IL-10 signalling revealed earlier DCs migration to the dLNs, an enhanced T cell influx to the lung, accompanied by earlier cytokines production and consequently decreased bacterial loads (160, 161).
