Estudo da resposta imune celular à infecção experimental por micobactérias de diferentes virulências : desenvolvimento de novas estratégias preventivas

Tese de doutoramento em Ciências da Saúde – Ciências Biológicas e Biomédicas.Mycobacterium ulcerans is the etiological agent of Buruli ulcer (BU), a serious tropical, necrotizing skin disease that can cause terrible deformities and disabilities if not treated at early stages. This pathogen was first identified in 1948 by MacCallum and colleagues in Australia; however, until the last decade, BU has received little attention from the scientific community. BU affects mainly rural and poor communities in Africa. Different control measures have been suggested, however, associated with BU, there is a strong social stigma, and BU is often seen as a curse rather than a disease. Thus, the development of a specific vaccine would be the most satisfactory strategy to control BU. It is therefore critical to advance the knowledge on the host/parasite interactions concerning this enigmatic disease. The pathology of BU is closely associated with mycolactone, a polyketide exotoxin produced by M. ulcerans and which is the major virulence factor of the agent of BU. It is known that cell wall-associated mycolactone or free toxin that diffuses across the tissues, induces apoptosis and necrosis of cells, and, ultimately, the formation of ulcers. Indeed, tissue necrosis and extracellular clumps of M. ulcerans bacilli are the histopathological hallmark of BU. The extensiveness of necrotic areas devoid of cells, which expand, possibly due to mycolactone diffusion, beyond areas where M. ulcerans bacilli can be found, has lead to the assumption that M. ulcerans induces low or absent cellular inflammatory responses. Additionally, the presence of large clumps of free bacteria in the central acellular areas directed early investigators to classify M. ulcerans as an extracellular pathogen, an exception among pathogenic mycobacteria. In contrast, many reports have described the occurrence of cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) responses in BU patients, strongly suggesting that macrophages play an effector role in the control of infection. The use of an animal model of infection, in which it is possible to analyze the entire lesion at very early time points, is essential to understand the interaction of M. ulcerans with the host immune cells, particularly phagocytes. Previous work from our laboratory in the mouse footpad model of infection has shown that inoculation of M. ulcerans in the subcutaneous tissue induces an inflammatory cellular response with the involvement of macrophages and neutrophils. Moreover, our group has shown that even in advanced lesions, inflammatory cells are consistently present, although restricted to the periphery of the necrotic infectious center. The constant availability of phagocytes to interact with M. ulcerans, at the persistent areas of inflammatory cellular infiltration, directed me to reevaluate the interaction of this pathogenic mycobacterium with the host macrophages, in the context of my PhD thesis, both in human and in experimental infections. In experimental murine infections, we showed that M. ulcerans bacilli were phagocytosed in the subcutaneous tissue early after infection. At later time points, as previously described, we found free M. ulcerans organisms concentrated in central necrotic acellular areas. Surrounding these necrotic areas, where inflammatory infiltrates could be seen, M. ulcerans bacilli were however found mainly within macrophages. A histological evaluation of serial sections of BU tissue samples from 15 African patients with inflammatory infiltrates, confirmed the occurrence in 8 patients of intracellular bacilli within phagocytes infiltrating the peripheral areas. In vitro, at low multiplicities of infection (MOI), we found that M. ulcerans was efficiently phagocytosed by macrophages, at similar rates as those for the intracellular pathogens Mycobacterium tuberculosis and Mycobacterium bovis BCG. Within macrophages, M. ulcerans was found surrounded by the phagosomal membrane in tight or spacious phagosomes. We also demonstrated that both virulent, mycolactone-producing, as well as mutant, nonproducing strains grew inside cultured macrophages when low MOIs were used to prevent mycolactoneassociated cytotoxicity. After this initial phase of intracellular residence and proliferation, mycolactone-producing strains were found to lyse the macrophage, becoming extracellular. These results suggest that M. ulcerans is an intracellular pathogen like the other virulent mycobacteria, which explains the reported occurrence of CMI and DTH in model and human cases of M. ulcerans infection. Following the demonstration of an intramacrophage growth phase for the agent of BU, our work focused on the role played by cytokines that are associated with the host resistance to intracellular pathogens. Tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) are two cytokines with a key role in the activation of macrophages, required to the control of intracellular mycobacteria. A previous report showed that a lipidic fraction of M. ulcerans culture filtrates containing mycolactone suppressed the production of TNF by cultured human monocytes. On the other hand, the expression of TNF has been reported in BU lesions. Therefore, we considered pertinent to study the effect of the infection with different strains of M. ulcerans on the production of this cytokine by macrophages, as well as to assess in vivo the role played by TNF in infections by M. ulcerans. We confirmed previous observations of other authors by showing that mycolactone inhibits TNF production, in a dose-dependent manner, and we have expanded these observations by using a model of primary mouse macrophages infected with live M. ulcerans bacilli producing different types of mycolactone and displaying different degrees of virulence. Our work shows that macrophages infected with highly virulent M. ulcerans produce much lower levels of TNF, as compared with macrophages infected with intermediate or nonvirulent strains. The reduced production of TNF, including at low MOIs, was not due to an early death of infected macrophages, since the production of the inflammatory chemokine macrophage inflammatory protein-2 (MIP-2) was not inhibited in macrophages infected with the highly virulent strain. Finally, we showed that TNF induced during M. ulcerans infection plays a protective role, since TNF-p55-defficient mice were more susceptible to nonvirulent and intermediate virulent strains of M. ulcerans when compared to wild-type mice . IFN-γ is a key cytokine in the activation of the macrophage’s microbicidal mechanisms required for the control of intracellular pathogens. We showed that IFN-γ-deficient mice displayed an increased susceptibility to infection by nonvirulent and intermediate virulent strains of M. ulcerans, but not to a highly virulent strain. In vitro, IFN-γ-activated macrophages control the proliferation of the nonvirulent strain as well as the intermediate virulent strain at a low MOI. For the nonvirulent strain, the intramacrophage mechanism of control was nitric oxide (NO) dependent, since with the inhibition of NO production, macrophages were no longer able to control the proliferation of this strain. Contrarily, NO production was not detected in macrophages infected with intermediate and highly virulent strains. However, macrophages infected with the intermediate virulent, as well as with the nonvirulent strain, expressed LRG-47, being this indicative of phagosome maturation. In summary, we have shown that M. ulcerans, like the other pathogenic mycobacteria, has a phase of intramacrophage residence and multiplication. Additionally, we demonstrated that M. ulcerans induces the production of TNF by infected macrophages and that this cytokine plays a protective role in experimental BU; however, mycolactone, the toxin produced by M. ulcerans, inhibits the production of this cytokine. We also demonstrate that IFN-γ is required to the induction of NO and phagosome maturation which are the macrophage’s microbicidal mechanisms required to control intracellularly M. ulcerans proliferation. Taken together, the results here presented are in accordance with the data in the literature showing that resistance to M. ulcerans infections are associated with CMI and DTH. The peculiarity of the immunology of M. ulcerans infections lies in the association of the mycobacterial nature of this microorganism with its unique capacity to secrete a potent cytotoxic exotoxin which influences CMI. These findings have potential relevance for the development of a vaccine against BU targeting cellular immune mechanisms associated with the activation of the macrophage effector functions.A bactéria Mycobacterium ulcerans é o agente etiológico da úlcera do Buruli (BU), uma doença tropical necrotizante que afecta a pele, causando deformidades terríveis se não for tratada numa fase precoce. Este agente patogénico foi identificado pela primeira vez por MacCallum et al. em 1948 na Austrália; no entanto, até à última década, a BU passou, praticamente, despercebida à comunidade científica. A BU afecta essencialmente comunidades rurais, pobres, de Africa. Diferentes medidas têm sido sugeridas para controlar a BU, no entanto, associado a esta doença existe um estigma social muito forte, sendo que, frequentemente, a BU é vista como uma maldição e não como uma doença. Assim sendo, o desenvolvimento de uma vacina contra a BU seria a estratégia mais adequada para a controlar a doença. Para tal, é essencial aumentar o reduzido conhecimento existente, no que respeita à interacção hospedeiro/agente infeccioso. A patologia da BU está intimamente associada com a micolactona, uma exotoxina produzida pelo M. ulcerans, sendo este o factor de virulência mais importante do agente da BU. É sabido que a micolactona associada à parede celular, ou livre, que se difunde pelos tecidos, induz apoptose e necrose das células do hospedeiro, levando à formação de úlceras. De facto, a necrose dos tecidos e a localização extracelular do M. ulcerans são as características histopatológicas distintivas da BU. A extensão das lesões necróticas desprovidas de células que, possivelmente devido à difusão da toxina, se expandem para além das áreas onde o bacilo se encontra, levou à interpretação que o M. ulcerans induzia respostas inflamatórias celulares reduzidas ou ausentes. Adicionalmente, a presença de grumos de bacilos livres na área central, acelular, do foco da infecção, levou à caracterização do M. ulcerans como uma micobactéria extracelular, uma excepção entre o grupo das micobactérias patogénicas. No entanto, diversos estudos descreveram a ocorrência de imunidade mediada por células (CMI), assim como de hipersensibilidade do tipo retardado (DTH), em doentes com BU, o que sugere um papel efector do macrófago no controlo desta infecção. A utilização de um modelo animal de infecção, que possibilite a análise completa da lesão em tempos precoces, é essencial para que seja possível entender a interacção de M. ulcerans com as células do sistema imunológico, nomeadamente com os fagócitos. Trabalho prévio do nosso laboratório mostrou que, no modelo murino de infecção da almofada plantar, a infecção subcutânea com M. ulcerans induz uma resposta inflamatória celular com a presença de macrófagos e neutrófilos. Adicionalmente, o nosso grupo mostrou que, mesmo em estadios avançados da lesão, as células inflamatórias se encontravam constantemente presentes na lesão, restritas, no entanto, à periferia do centro necrótico acelular. A constante disponibilidade de fagócitos para interagir com o M. ulcerans, nas zonas de infiltrado à periferia da lesão, direccionou o meu trabalho de doutoramento no sentido de reavaliar a interacção deste agente patogénico com os fagócitos do hospedeiro, quer em lesões humanas, quer em infecções experimentais. Em infecções experimentais no modelo murino, mostrámos que o M. ulcerans era fagocitado no tecido subcutâneo em tempos curtos após a infecção. Mais tardiamente, conforme anteriormente descrito, o M. ulcerans encontrava-se essencialmente na área necrótica, acelular da lesão. À periferia desta área central necrótica, e nos locais onde encontrava o infiltrado inflamatório, o M. ulcerans era observado essencialmente no interior de macrófagos. A análise histopatológica de cortes seriados de 15 amostras de lesões de BU, contendo infiltrados inflamatórios e colhidas de doentes Africanos, confirmou, em 8 amostras, a localização intracelular do bacilo em fagócitos presentes nas áreas periféricas da lesão. In vitro, a baixas multiplicidades de infecção (MOI), mostrámos que o M. ulcerans é eficientemente fagocitado por macrófagos, a níveis similares aos encontrados para o Mycobacterium tuberculosis e Mycobacterium bovis BCG. No interior dos macrófagos, o M. ulcerans foi encontrado rodeado pela membrana do fagossoma. Mostrámos, também, que, quer estirpes virulentas, produtoras de micolactona, quer estirpes mutantes, deficientes em micolactona, crescem dentro de macrófagos, quando baixas MOIs são usadas, prevenindo-se, assim, a citotoxicidade associada à produção de micolactona. Após esta fase inicial de residência e multiplicação intracelular, as estirpes produtoras de micolactona provocam a lise dos macrófagos, tornando-se extracelulares. Estes resultados sugerem que o M. ulcerans é um agente patogénico intracelular, tal como as outras micobactérias patogénicas, o que explica a ocorrência de CMI e DTH, descritas em infecções humanas e em infecções experimentais. Após a demonstração da existência de uma fase intracelular no ciclo de vida de M. ulcerans, o trabalho focou-se no papel das citocinas que estão associadas à resistência do hospedeiro a agentes patogénicos intracelulares. O factor de necrose tumoral (TNF) e interferão-gama (IFN-γ) são duas citocinas com um papel chave na activação dos mecanismos microbicidas do macrófago, necessários para o controlo intracelular das micobactérias. Resultados anteriormente publicados mostram que uma fracção lipídica, contendo micolactona, suprime a produção de TNF por monócitos humanos cultivados in vitro. No entanto, a expressão de TNF foi reportada em lesões humanas da BU. Tendo estes aspectos em consideração, considerou-se pertinente estudar o efeito da infecção, por diferentes estirpes de M. ulcerans, na produção desta citocina por macrófagos infectados, assim como a relevância biológica do TNF em infecções por M. ulcerans. O nosso trabalho confirmou resultados previamente publicados, mostrando que a micolactona inibe a produção de TNF, de uma forma dependente da dose, e expandiu essas observações, usando um modelo de macrófagos primários infectados com estirpes de M. ulcerans que produzem diferentes tipos de micolactona e que apresentam diferentes níveis de virulência. Verificámos, de facto, que macrófagos infectados com estirpes virulentas de M. ulcerans produzem menores quantidades de TNF, comparativamente com macrófagos infectados com estirpes avirulentas ou de virulência intermédia. A reduzida produção de TNF, incluindo a baixas MOIs, não se deveu a uma morte prematura dos macrófagos infectados, uma vez que e a produção de proteína inflamatória de macrófagos-2 (MIP-2) não era inibida pelas estirpes virulentas. Finalmente, mostrámos que o TNF induzido após infecção tem um papel protector, uma vez que ratinhos deficientes no receptor P55 do TNF são mais susceptíveis à infecção por estirpes de M. ulcerans avirulentas ou de virulência intermédia, quando comparados com ratinhos selvagem. O IFN-γ é uma citocina chave na activação dos mecanismos microbicidas do macrófago, necessários para o controlo da proliferação de agentes patogénicos intracelulares. Neste trabalho, mostrámos que ratinhos deficientes na produção de IFN-γ são mais susceptíveis à infecção por estirpes de M. ulcerans avirulentas ou de virulência intermédia, o mesmo não se verificando com uma estirpe de elevada virulência. In vitro, verificámos que macrófagos activados com IFN-γ controlam a proliferação de estirpes de M. ulcerans avirulentas e, também, de virulência intermédia, quando foi testada uma baixa MOI. Para a estirpe avirulenta, os mecanismos de controlo intramacrofágico mostraram ser dependentes da produção de óxido nítrico (NO), uma vez que, inibindo-se a produção de NO, os macrófagos activados deixam de controlar a sua proliferação. Contrariamente à infecção pela estirpe avirulenta, a produção de NO não foi detectada para as outras estirpes testadas. No entanto, macrófagos infectados com a estirpe de M. ulcerans de virulência intermédia expressavam LRG-47, o que sugere a ocorrência de maturação do fagossoma. Em resumo, mostrámos que o M. ulcerans, tal como as outras micobactérias patogénicas, tem uma fase de residência e multiplicação intracelular. Mostrámos, também, que o M. ulcerans induz a produção de TNF por macrófagos infectados, tendo esta citocina um papel chave na resposta protectora contra infecções experimentais por M. ulcerans; no entanto, a micolactona, a toxina produzida por M. ulcerans, inibe a produção desta citocina. Demonstrámos ainda, que o IFN-γ é necessário para a indução da produção de NO e para a maturação do fagosoma, sendo estes os mecanismos microbicidas necessários para controlar a proliferação intramacrofágica de M. ulcerans. No seu conjunto, os resultados aqui apresentados estão em acordo com resultados previamente publicados que mostram que a resistência à infecção por M. ulcerans está associada com CMI e DTH. A peculiaridade da resposta imune ao M. ulcerans encontra-se na associação entre a natureza micobacteriana deste microorganismo e a sua capacidade de produzir uma exotoxina citotóxica, influenciando assim a resposta imune celular. Estes resultados têm uma relevância potencial para o desenvolvimento de uma vacina contra a BU, tendo como alvo mecanismos imunes mediados por células, associados com a activação dos mecanismos efectores do macrófago.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/9757/2003

Immune-evasion strategies of mycobacteria and their implications for the protective immune response

Mycobacteria are intracellular pathogens that have macrophages as their main host cells. However, macrophages are also the primary line of defense against invading microorganisms. To survive in the intracellular compartment, virulent mycobacteria have developed several strategies to modulate the activation and the effector functions of macrophages. Despite this, antigen-specific T cells develop during infection. While T cell responses are critical for protection they can also contribute to the success of mycobacteria as human pathogens, as immunopathology associated with these responses facilitates transmission. Here, we provide a brief overview of different immune-evasion strategies of mycobacteria and their impact on the protective immune response. This understanding will further our knowledge in host-pathogen interactions and may provide critical insights for the development of novel host-specific therapies.Our work is funded by the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER); Infect-ERA grant BU_SPONT_HEAL; and the Fundação para a Ciência e Tecnologia (FCT) through the FCT investigator grant IF/01390/2014 to E.T. and the postdoctoral grant SFRH/BPD/112903/2015 to A.G.F.info:eu-repo/semantics/publishedVersio

Antibacterial bioadhesive layer-by-layer coatings for orthopedic applications

In this study, thin LbL films were produced by combining the adhesive properties of the hyaluronic acidâ dopamine conjugate with the bioactivity and bactericidal properties of silver doped bioactive glass nanoparticles. The build-up of these films was investigated by quartz crystal microbalance with dissipation monitoring. LbL coatings were then constructed on a glass substrate for further characterization. We found that these antimicrobial bioinspired films display enhanced adhesive strength. In vitro bioactivity tests were performed by immersing them in simulated body fluid solution for 14 days where the constructed films promoted the formation of a bone-like apatite layer. From microbiological assays, it was found that coatings containing silver doped nanoparticles exhibited a remarkable antibacterial effect against Staphylococcus aureus and Escherichia coli cultures. Finally, in vitro cellular behavior tests showed enhanced cell adhesion, proliferation and viability for these antibacterial bioadhesive films. Therefore, the constructed thin films showed promising properties and evidenced great potential to be used as coatings for orthopedic implants.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) and the European program FEDER/COMPETE for the financial support through project BioSeaGlue: EXPL/CTM-BIO/0646/2013 (FCOMP-01-0124-FEDER041105). This work was co-funded by ‘‘Programa Operacional Regional do Norte’’ (ON.2-O Novo Norte) under the ‘‘Quadro de Referência Estratégico Nacional’’ (QREN), through the ‘‘Fundo Europeu de Desenvolvimento Regional’’ (FEDER). E. T. also thanks the FCT investigator grant (IF/01390/2014)

Increased CD3+, CD8+, or FoxP3+ T lymphocyte infiltrations are associated with the pathogenesis of colorectal cancer but not with the overall survival of patients

Tumor-infiltrating lymphocytes include heterogeneous populations of T lymphocytes that play crucial roles in the tumor immune response; importantly, their presence in the tumor tissue may predict clinical outcomes. Therefore, we herein studied the prognostic significance of the presence and location of CD3+, CD8+, and FoxP3+ T lymphocytes in colorectal cancer samples. In the intratumor analysis, our data did not reveal any association between lymphocyte infiltrations with clinical or pathological data. However, in the tumor margins, we found that the presence of high infiltrations of CD3+, CD8+, or FoxP3+ T lymphocytes were associated with TNM stages I-II (p = 0.021, p = 0.022, and p = 0.012, respectively) and absence of lymph node metastases (p = 0.010, p = 0.003, and p = 0.004, respectively). Despite these associations with good prognostic indicators, we were not able to find any statistically significant alterations in the overall survival of the patients, even though high infiltrations of FoxP3+ T lymphocytes in the tumor margins resulted in an increased overall survival of 14 months. Taken together, these data show that the presence of CD3+, CD8+, or FoxP3+T lymphocyte infiltrates in the tumor margins are associated with the pathogenesis of CRC, but only high Foxp3+ T lymphocyte infiltrations in the tumor invasive margins are inclined to indicate favorable prognosis.This work was supported by National funds through the Foundation for Science and Technology (FCT)—projects PTDC/MED-ONC/28658/2017, UIDB/50026/2020, and UIDP/50026/2020; and by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023. AMB was supported by the FCT fellowship SFRH/BD/120371/2016 and ET by the FCT investigator grant IF/01390/2014 and Estímulo Individual ao Emprego Científico CEECIND/03070/2020

Exploring inhalable polymeric dry powders for anti-tuberculosis drug delivery

The growing interest on polymeric delivery systems for pulmonary administration of drugs anticipates a more direct and efficient treatment of diseases such as tuberculosis (TB) that uses the pulmonary route as the natural route of infection. Polymeric microparticles or nano-in-microparticles offer target delivery of drugs to the lungs and the potential to control and sustain drug release within TB infected macrophages improving the efficiency of the anti-TB treatment and reducing side effects. In a dry powder form these inhalable delivery systems have increased stability and prolonged storage time without requiring refrigeration, besides being cost-effective and patient convenient. Thus, this review aims to compile the recent innovations of inhalable polymeric dry powder systems for the delivery of anti-TB drugs exploring the methods of production, aerodynamic characterization and the efficacy of targeted drug delivery systems using in vitro and in vivo models of the disease. Advanced knowledge and promising outcomes of these systems are anticipated to simplify and revolutionize the pulmonary drug delivery and to contribute towards more effective anti-TB treatments.The authors wish to acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) for the postdoctoral grant of M.S.M (SFRH/BPD/110868/2015), M.T.R (SFRH/BPD/111729/2015) and R.M.A.D (SFRH/BPD/112459/2015), FCT grant of E.T. (IF/01390/2014) and Recognize project (UTAP-ICDT/ CTM-BIO/0023/2014). This article is also a result of the project “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechonologies, marine-derived biomaterials and stem cells”, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR.info:eu-repo/semantics/publishedVersio

The impact of IL-10 dynamic modulation on host immune response against visceral leishmaniasis

Leishmaniasis is a vector-borne disease caused by protozoan parasites from the genus Leishmania. The most severe form of disease is visceral leishmaniasis (VL), which is fatal if left untreated. It has been demonstrated that interleukin (IL)-10, is associated with disease progression and susceptibility. In this work, we took advantage of a transgenic mouse model that expresses high levels of IL-10 upon zinc sulfate administration (pMT-10). We addressed the role of IL-10 during the initial stages of L. donovani infection by analyzing the parasite burden in the spleen and liver of the infected pMT-10 and WT mice as well as the histopathological alterations upon IL-10 induction. Furthermore, the profile of cytokines expressed by T cells was assessed. Our results demonstrate that an increase in IL-10 production has an impact early but not later after infection. This specific temporal role for IL-10-mediated susceptibility to VL is of interest.Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contracts SFRH/BD/120127/2016 to IM, PD/BDE/127830/2016 to CF, SFRH/BD/120371/2016 to AMB, IF/01147/2013 to RDO, IF/01390/2014 to ET, IF/00735/2014 to AC, SFRH/BPD/96176/2013 to CC and IF/00021/2014 to RS), and Infect-Era (project INLEISH). JE also thanks the Canada Research Chair program for financial assistanceinfo:eu-repo/semantics/publishedVersio

Type I IFN inhibits alternative macrophage activation during mycobacterium tuberculosis infection and leads to enhanced protection in the absence of IFN-gamma signaling

Supplementary material: http://www.jimmunol.org/content/suppl/2016/11/12/jimmunol.1600584.DCSupplementalTuberculosis causes ∼1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in tuberculosis pathogenesis, likely by interfering with IFN-γ–dependent immunity. In this article, we reveal a novel mechanism by which type I IFN may confer protection against Mycobacterium tuberculosis infection in the absence of IFN-γ signaling. We show that production of type I IFN by M. tuberculosis–infected macrophages induced NO synthase 2 and inhibited arginase 1 gene expression. In vivo, absence of both type I and type II IFN receptors led to strikingly increased levels of arginase 1 gene expression and protein activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased lung bacterial burden and pathology and decreased survival compared with mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-γ signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated phenotype to a more permissive alternatively activated phenotype. Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-γ signaling, contributes to host protection.This work was supported by the Fundação para a Ciência e Tecnologia, Portugal, cofunded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), Quadro de Referência Estratégico Nacional, through the Fundo Europeu de Desenvolvimento Regional (PTDC/SAU-MII/101977/2008 and PTDC/BIA-BCM/102776/2008); by the Francis Crick Institute, which receives its core funding from Cancer Research U.K. (FC001126), the U.K. Medical Research Council (FC001126), and the Wellcome Trust (FC001126); by the U.K. Medical Research Council (MR/U117565642/1); and by the European Research Council (294682-TB-PATH). This work was also supported by Research Grant 2015 from the European Society of Clinical Microbiology and Infectious Diseases (to M.S.). L.M.-T. was funded by the Fundação para a Ciência e Tecnologia (SFRH/BPD/77399/2011) and the European Research Council (294682-TB-PATH). The M.S. laboratory was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia, Portugal, in the framework of the Institute for Research and Innovation in Health Sciences project (POCI-01-0145-FEDER-007274). M.S. is a Fundação para a Ciência e Tecnologia Associate Investigator. E.T. is a Fundação para a Ciência e Tecnologia Auxiliary Investigator

Development of Inhalable Superparamagnetic Iron Oxide Nanoparticles (SPIONs) in microparticulate system for antituberculosis drug delivery

Tuberculosis (TB) is an infectious disease which affects millions of people worldwide. Inhalable polymeric dry powders are promising alternatives as anti-TB drug carriers to the alveoli milieu and infected macrophages, with potential to significantly improve the therapeutics efficiency. Here, the development of a magnetically responsive microparticulate system for pulmonary delivery of an anti-TB drug candidate (P3) is reported. Microparticles (MPs) are developed based on a cast method using calcium carbonate sacrificial templates and incorporate superparamagnetic iron oxide nanoparticles to concentrate MPs in alveoli and enable drug on demand release upon actuation of an external alternate magnetic field (AMF). The MPs are shown to be suitable for P3 delivery to the lower airways and for alveolar macrophage phagocytosis. The developed MPs reveal unique and promising features to be used as an inhalable dry powder allowing the AMF control over dosage and frequency of drug delivery anticipating improved TB treatments.The authors wish to acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) for the postdoctoral grant of M.S.M. (SFRH/BPD/110868/2015) and R.M.A.D (SFRH/BPD/112459/2015), FCT grant of E.T. (IF/01390/2014) and Recognize project (UTAP-ICDT/CTM-BIO/0023/2014). This article is also a result of the project “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine-derived biomaterials and stem cells,” supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant Agreement No. 739572 – The Discoveries CTR.info:eu-repo/semantics/publishedVersio

Pathological role of interleukin 17 in mice subjected to repeated BCG vaccination after infection with Mycobacterium tuberculosis

Infection usually leads to the development of acquired immune responses associated with clearance or control of the infecting organism. However, if not adequately regulated, immune-mediated pathology can result. Tuberculosis is a worldwide threat, and development of an effective vaccine requires that the protective immune response to Mycobacterium tuberculosis (Mtb) be dissected from the pathological immune response. This distinction is particularly important if new vaccines are to be delivered to Mtb-exposed individuals, as repeated antigenic exposure can lead to pathological complications. Using a model wherein mice are vaccinated with bacille Calmette-Guérin after Mtb infection, we show that repeated vaccination results in increased IL-17, tumor necrosis factor, IL-6, and MIP-2 expression, influx of granulocytes/neutrophils, and lung tissue damage. This pathological response is abrogated in mice deficient in the gene encoding IL-23p19 or in the presence of IL-17–blocking antibody. This finding that repeated exposure to mycobacterial antigen promotes enhanced IL-17–dependent pathological consequences has important implications for the design of effective vaccines against Mtb

First Cultivation and Characterization of Mycobacterium ulcerans from the Environment

Mycobacterium ulcerans infection, or Buruli ulcer, is the third most common mycobacteriosis of humans worldwide, after tuberculosis and leprosy. Buruli ulcer is a neglected, devastating, necrotizing disease, sometimes producing massive, disfiguring ulcers, with huge social impact. Buruli ulcer occurs predominantly in impoverished, humid, tropical, rural areas of Africa, where the incidence has been increasing, surpassing tuberculosis and leprosy in some regions. Besides being a disease of the poor, Buruli ulcer is a poverty-promoting chronic infectious disease. There is strong evidence that M. ulcerans is not transmitted person to person but is an environmental pathogen transmitted to humans from its aquatic niches. However, until now M. ulcerans has not been isolated in pure culture from environmental sources. This manuscript describes the first isolation, to our knowledge, of M. ulcerans in pure culture from an environmental source. This strain, which is highly virulent for mice, has microbiological features typical of African strains of M. ulcerans and was isolated from an aquatic insect from a Buruli ulcer–endemic area in Benin, West Africa. Our findings support the concept that M. ulcerans is a pathogen of humans with an aquatic environmental niche and will have positive consequences for the control of this neglected and socially important tropical disease