Exploring NAD(+) metabolism in host-pathogen interactions

Nicotinamide adenine dinucleotide (NAD+) is a vital molecule found in all living cells. NAD+ intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD+ metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD+ metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD+ homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD+ or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD+ metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.JG was supported by PD/BD/106053/2015. BV was supported by IRD (Institut de Recherche pour le Développement) institutional funding. JE was supported by a European Community’s Seventh Framework Program under grant agreement No. 602773 (Project KINDRED), an ANR grant (LEISH-APO, France) and a Partenariat Hubert Curien (PHC) (program Volubilis, MA/11/262). JE also thanks the Canada Research Chair program for his support. RS thank FCT—Foundation for Science and Technology—for their Investigator FCT Grant (IF/00021/2014)info:eu-repo/semantics/publishedVersio

The influence of surface modified poly(L-lactic acid) films on the differentiation of human monocytes into macrophages

Macrophages play a crucial role in the biological performance of biomaterials, as key factors in defining the optimal inflammation-healing balance towards tissue regeneration and implant integration. Here, we investigate how different surface modifications performed on poly(L-lactic acid) (PLLA) films would influence the differentiation of human monocytes into macrophages. We tested PLLA films without modification, surface-modified by plasma treatment (pPLLA) or by combining plasma treatment with different coating materials, namely poly(L-lysine) and a series of proteins from the extracellular matrix: collagen I, fibronectin, vitronectin, laminin and albumin. While all the tested films are non-cytotoxic, differences in cell adhesion and morphology are observed. Monocyte-derived macrophages (MDM) present a more rounded shape in non-modified films, while a more elongated phenotype is observed containing filopodia-like and podosome-like structures in all modified films. No major differences are found for the expression of HLA-DR+/CD80(+) and CD206(+)/CD163(+) surface markers, as well as for the ability of MDM to phagocytize. Interestingly, MDM differentiated on pPLLA present the highest expression of MMP9. Upon differentiation, MDM in all surface modified films present lower amounts of IL-6 and IL-10 compared to non-modified films. After stimulating MDM with the potent pro-inflammatory agent LPS, pPLLA and poly(L-lysine) and fibronectin-modified films reveal a significant reduction in IL-6 secretion, while the opposite effect is observed with IL-10. Of note, in comparison to non-modified films, all surface modified films induce a significant reduction of the IL-6/IL-10 ratio, a valuable prognosticator of the pro-versus anti-inflammatory balance. These findings provide important insights into MDM-biomaterial interactions, while strengthening the need for designing immune-informed biomaterials.project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER). C. R. Correia and J. F. Mano acknowledge the funding from the European Research Council for project ATLAS with the grant agreement number ERC-2014-ADG-669858. J. Gaifem, M.B. Oliveira and R. Silvestre acknowledge the Portuguese Foundation for Science and Technology (FCT) for the doctoral (PD/BD/106053/2015), post-doctoral (SFRH/BPD/111354/2015) and FCT Investigator (IF/00021/2014) grants, respectively. The authors also acknowledge Hospital de Braga for providing the buffy coatsThis work was developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER). C. R. Correia and J. F. Mano acknowledge the funding from the European Research Council for project ATLAS with the grant agreement number ERC-2014-ADG-669858. J. Gaifem, M.B. Oliveira and R. Silvestre acknowledge the Portuguese Foundation for Science and Technology (FCT) for the doctoral (PD/BD/106053/2015), post-doctoral (SFRH/BPD/111354/2015) and FCT Investigator (IF/00021/2014) grants, respectively. The authors also acknowledge Hospital de Braga for providing the buffy coats.info:eu-repo/semantics/publishedVersio

Definition of the anti-inflammatory oligosaccharides derived from the galactosaminogalactan (GAG) from Aspergillus fumigatus

Galactosaminogalactan (GAG) is an insoluble aminosugar polymer produced by Aspergillus fumigatus and has anti-inflammatory properties. Here, the minimum glycosidic sequences required for the induction of IL-1Ra by peripheral blood mononuclear cells (PBMCs) was investigated. Using chemical degradation of native GAG to isolate soluble oligomers, we have found that the de-N-acetylation of galactosamine residues and the size of oligomer are critical for the in vitro immune response. A minimal oligomer size of 20 galactosamine residues is required for the anti-inflammatory response but the presence of galactose residues is not necessary. In a Dextran sulfate induced colitis mouse model, a fraction of de-N-acetylated oligomers of 13 < dp < 20 rescue inflammatory damage like the native GAG polymer in an IL-1Ra dependent pathway. Our results demonstrate the therapeutic suitability of water-soluble GAG oligosaccharides in IL-1 mediated hyper-inflammatory diseases and suggest that α-1,4-galactosamine oligomers chemically synthesized could represent new anti-inflammatory glycodrugs.Aviesan project Aspergillus, the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (Grant No ANR-10-LABX-62-IBEID), la Fondation pour la Recherche Médicale (DEQ20150331722 LATGE Equipe FRM 2015). RS thanks Fundação para a Ciência e Tecnologia (FCT) contract IF/00021/201

SARS-CoV-2 Infection Drives a Glycan Switch of Peripheral T Cells at Diagnosis

COVID-19 is a highly selective disease in which SARS-CoV-2 infection can result in different clinical manifestations ranging from asymptomatic/mild to severe disease that requires hospitalization. In this study, we demonstrated that SARS-CoV-2 infection results in a glycosylation reprogramming of circulating lymphocytes at diagnosis. We identified a specific glycosignature of T cells, defined upon SARS-CoV-2 infection and apparently triggered by a serological factor. This specific glycan switch of T cells is detected at diagnosis being more pronounced in asymptomatic patients. We further demonstrated that asymptomatic patients display an increased expression of a viral-sensing receptor through the upregulation of DC-SIGN in monocytes. We showed that higher levels of DC-SIGN in monocytes at diagnosis correlates with better COVID-19 prognosis. This new evidence pave the way to the identification of a novel glycan-based response in T cells that may confer protection against SARS-CoV-2 infection in asymptomatic patients, highlighting a novel prognostic biomarker and potential therapeutic target.info:eu-repo/semantics/publishedVersio

Mathematical modelling using predictive biomarkers for the outcome of canine Leishmaniasis upon chemotherapy

Prediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient’s prognosis.This work was funded by the Brazilian agencies Bahia Research Foundation—FAPESB (Grant nº PRONEM 498/2011-PNE 0002/2011 to S.M.B-M), National Council for Scientific and Technological Development —CNPq (PQ scholarship nº 307813/2018-5 to SMBM, and nº 303621/2015-0 to HG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior —CAPES (PDSE scholarship nº 88881.189587/2018-01 to R.S.G; Finance Code 001 and PV scholarship nº 23066.033859/2018-73 to R.S.). This work was supported by grants from CESPU (TramTap-CESPU-2016, Chronic-TramTap_CESPU_2017 and TraTapMDMA-CESPU-2018), from the 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), funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and the Fundação para a Ciência e Tecnologia (FCT) (contract IF/00021/2014 to R.S.), Infect-Era (project INLEISH to R.S.) and Proyecto SNIP N◦ 292900 “Creación del Servicio de Laboratorio de Enfermedades Infecciosas y Parasitarias de Animales Domésticos de la Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas.Instituto de Investigación en Ganadería y Biotecnología-IGBI. Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas

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

IL-10 overexpression predisposes to invasive aspergillosis by suppressing antifungal immunity

© 2017 American Academy of Allergy, Asthma & ImmunologyProinflammatory immune responses are critically required for antimicrobial host defenses; however, excessive inflammation has the potential to damage host tissues thereby paradoxically contributing to the progression of infection. A central negative regulator of inflammatory responses is IL-10, an immunosuppressive cytokine with a wide variety of functions across multiple cell types. Although the role of IL-10 during infection appears to vary for different microorganisms, a largely detrimental role has been attributed to this cytokine during fungal disease. Given the variable risk of infection and its outcome among patients with comparable predisposing factors, susceptibility to invasive aspergillosis (IA) is thought to rely largely on genetic predisposition. The initial investigation of genetic variability at the IL10 locus led to the identification of single nucleotide polymorphisms (SNPs) influencing its transcriptional activity; thus, IL-10 may be a reasonable candidate for the genetic regulation of susceptibility to IA in high-risk patients.Supported by the 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), the Fundação para a Ciência e Tecnologia (FCT) (contracts IF/00735/2014 to A.C., IF/01390/2014 to E.T., IF/00021/2014 to R.S., and SFRH/BPD/96176/2013 to C.C.), the Conselho de Reitores das Universidades Portuguesas (CRUP), Portugal (Ações Integradas Luso-Alemãs A-43/16), the Deutscher Akademischer Austauschdienst (DAAD) (project-ID 57212690), the Fondo de Investigaciones Sanitarias (Madrid, Spain) (grant #PI12/02688) and the ERA-NET PathoGenoMics (grant #0315900A).info:eu-repo/semantics/publishedVersio

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

Acknowledgements This work was supported by the 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), the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/136814/2018 to S.M.G., SFRH/BD/141127/2018 to C.D.O., PD/BD/137680/2018 to D.A., IF/00474/2014 to N.S.O., IF/01390/2014 to E.T., IF/00959/2014 to S.C., IF/00021/2014 to R.S., PTDC/SAU-SER/29635/2017 and CEECIND/04601/2017 to C.C., and CEECIND/03628/2017 to A.C.), the Institut Mérieux (Mérieux Research Grant 2017 to C.C.), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to A.C.). M.G.N. was supported by a Spinoza grant of the Netherlands Organization for Scientific Research. A.A.B. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center/Transregio TR124 FungiNet (project A1). G.D.B. was funded by the Wellcome Trust (102705), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD

Deciphering the microbiome and metabolic factors contributing to protection against ulcerative colitis

Tese de Doutoramento (Especialidade Envelhecimento e Doenças Crónicas)Inflammatory bowel disease (IBD) is a complex set of inflammatory disorders from the gastrointestinal tract, comprising Crohn’s disease (CD) and ulcerative colitis (UC). Although the precise etiology of this disease remains unclear, the pathogenesis of IBD has been defined as an immune-mediated condition with a strong genetic predisposition, triggered by environmental factors that affect the mucosal barrier and the balance of the gut microbiota. In this work, we aimed to identify relevant microbiota species that may influence the susceptibility or protection against colitis development and how the modulation of dietary nutrients impacts the outcome of disease. While analyzing dextran sulfate sodium (DSS)-induced colitis in genetically similar C57BL/6 mice housed in two different animal facilities, we serendipitously observed a group of animals with a remarkable protection to disease development. The two groups of mice display distinct microbiota and metabolic profiles, clustering separately in multivariate data analysis. This can be at the genesis of the resistant phenotype, since fecal microbiota transplant (FMT) from resistant to susceptible mice was able to reverse colitis susceptibility. We identified Akkermansia muciniphila and Parabacteroides distasonis as metagenomic top hits, being enriched in resistant mice, and in susceptible mice after receiving FMT from the resistant group. Resistant mice also evidenced significantly increased levels of IL-10, IL-17 and IL-22, as well as increased expression of claudin- and mucin-encoding genes in homeostatic conditions when compared to the susceptible group, suggesting that the protective phenotype is associated with an intestinal epithelial barrier more prone to sustaining an inflammatory insult. Indeed, by analyzing the effect of these bacterial candidates on colonic epithelial cells using a biochip-based human gut model, an increased expression of E-cadherin was found when cells were incubated with A. muciniphila, recapitulating the findings observed in vivo. We are currently dissecting in the gut-on-a-chip model how these candidates modulate epithelial barrier integrity to increase protection against an inflammatory event, to further validate these results in an in vivo susceptible model of colitis. In parallel, we were focused in evaluating how dietary supplementation can contribute to the protection against the development of IBD or, at least, for a better resolution of the disease. It is known that the metabolic environment in the gut is altered during pathology. Therefore, we analyzed the colon metabolic profile of mice during colitis development, in order to identify possible altered nutrients that might be used as promising prophylactic or therapeutic approaches. We observed that mice treated with DSS display a twofold decrease in threonine levels in the intestine. Knowing this, threonine supplementation was evaluated as possible therapy by treating mice on the beginning of colitis development or when the inflammation was already established. We observed that threonine has a detrimental effect when administered during colitis onset, with mice evidencing a delayed remission of the disease when compared to control group or mice treated with threonine only during inflammation. This detrimental effect is associated with a reduction in the number of goblet cells per crypt and also in decreased amounts of IL-22 in the gut. Overall, the findings presented in this thesis contribute to a better understanding of the different factors that can modulate protection and/or susceptibility to IBD development, providing novel insights to possible strategies of tackling IBD.A doença inflamatória intestinal (DII) é um conjunto de distúrbios inflamatórios do trato gastrointestinal que engloba a doença de Crohn e a colite ulcerosa. Embora a etiologia desta doença ainda não seja clara, a patogénese da DII é uma condição mediada por respostas imunes, com uma forte predisposição genética, desencadeada por fatores ambientais que afetam a barreira da mucosa e o equilíbrio do microbiota intestinal. Este trabalho tem como objetivos a identificação de espécies do microbiota que possam influenciar a suscetibilidade ou proteção contra o desenvolvimento de colite e de que forma é que a modulação de nutrientes presentes na dieta afeta o decurso da doença. Ao analisar o perfil de colite induzida por dextrano sulfato de sódio (DSS) em murganhos C57BL/6 geneticamente semelhantes e alojados em diferentes biotérios, observamos por acaso que um dos grupos de animais apresentava uma elevada proteção contra o desenvolvimento da doença. Os dois grupos de murganhos apresentam perfis metabólicos de microbiota distintos, agrupando-se isoladamente na análise de dados multivariada. Estes perfis podem estar na base do fenótipo de proteção, visto que o transplante de microbiota fecal (FMT) de animais resistentes para suscetíveis foi capaz de reverter a suscetibilidade à colite. Através de uma análise de metagenómica, identificamos que as espécies Akkermansia muciniphila e Parabacteroides distasonis encontram-se significativamente enriquecidas em murganhos resistentes ou em animais suscetíveis após FMT proveniente de murganhos resistentes. Estes animais também apresentaram níveis significativamente elevados de IL-10, IL-17 e IL-22, bem como um aumento na expressão de genes que codificam as proteínas claudinas e mucinas, em condições de homeostasia quando comparados ao grupo suscetível, o que sugere que o fenótipo protetor está associado à existência de uma barreira epitelial intestinal mais propensa a tolerar um insulto inflamatório. Aliás, através da análise das bactérias candidatas em células epiteliais do cólon usando um modelo de intestino humano em formato biochip, foi detetada uma maior expressão de E-caderina nas células incubadas com A. muciniphila, recapitulando as observações feitas in vivo. Neste momento estamos a explorar no modelo “gut-on-a-chip” de que forma esses candidatos modulam a integridade da barreira epitelial para aumentar a proteção contra um evento inflamatório, para validar posteriormente esses resultados num modelo in vivo de suscetibilidade à colite. Em paralelo avaliamos de que forma a suplementação na dieta contribui para a proteção contra o desenvolvimento de DII ou, pelo menos, para uma melhor resolução da doença. O ambiente metabólico no intestino é alterado durante a patologia. Como tal, analisamos o perfil metabólico do cólon de murganhos durante o desenvolvimento de colite, com vista a identificar possíveis nutrientes alterados que possam ser utilizados em estratégias de profilaxia ou terapia. Murganhos tratados com DSS exibiram uma diminuição de duas vezes nos níveis de treonina no intestino. Como tal, a suplementação com treonina foi avaliada como possível terapia, sendo administrado este nutriente a murganhos no início do desenvolvimento de colite ou quando a inflamação já estava estabelecida. Descobrimos que a treonina tem um efeito prejudicial quando administrada durante o início da colite, com os animais a evidenciar uma recuperação mais lenta da doença quando comparados com o grupo controlo ou com murganhos que receberam treonina apenas durante a inflamação. Simultaneamente a este efeito prejudicial, encontrou-se uma redução no número de células caliciformes por cripta intestinal e níveis mais baixos de IL- 22 no intestino. Em conclusão, os resultados apresentados nesta tese contribuem para uma melhor compreensão dos diferentes fatores que podem modular a proteção e/ou suscetibilidade ao desenvolvimento de DII, dando novas perspetivas sobre as possíveis estratégias para combater esta doença.Foundation for Science and Technology (FCT) through the PhD fellowship PD/BD/106053/2015 via Inter-University Doctoral Programme in Ageing and Chronic Diseases – PhDOC. FEDER funds through the Operational Programme Competitiveness Factors – COMPETE and Nacional Funds through FCT – Foundation for Science and Technology under the project POCI- 01-0145-FEDER-007038; and by the project NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

" Role Of In Endoplasmic Reticulum Stress Response In Sacccharoromyces cerevisiae ".

Mestrado em Contaminação e Toxicologia AmbientaisMaster of Environmental Contamination and Toxicolog