Final Report Summary - BNOX (The role of reactive oxygen species in B cell tolerization and immune memory)
The main scientific objective of the project was to identify how reactive oxygen species (ROS) impact on B cell function in the context of inflammation. Furthermore, the other main objective of the current grant was to allow the grantee to attain scientific independence and improve her leadership in research. In order to clearly define how these two goals were completed, the present report will be divided in two sections, one referring to the scientific results, the other to the career development.
1. Scientific achievements
Project 1: Immune response in Ncf1-deficient mice with DSS-induced colitis
CGD is a genetically heterogeneous immunodeficiency disorder caused by deficiency in oxidative burst, essential for the clearance of phagocytised micro-organisms causing increased susceptibility to severe bacterial and fungal infections. CGD is caused by mutations in the phagocyte NADPH oxidase complex, the enzyme that generates microbicidal oxygen radicals. One of the more common clinical complications in CGD is chronic intestinal inflammation. Ncf1 mutation leads to a lack of ROS in mice which are described as susceptible to autoimmune diseases and hyperresponsiveness to some pathogens. We used these mutant mice to study how this lack of ROS influences a DSS-induced colitis immune response, the recovery and a second DSS induction.
We observed that the Ncf1 mice present worst clinical scores during an acute colitis induction and indications of more enhanced and aberrant immune Th1/Th17 response, with a weaker recovery during the rest period and signs of a chronic evolution when subject to a subsequent DSS induction. Thus, we suggest that ROS deficiency in these mice leads to abnormalities in the immune response of the intestinal mucosa, driving aberrant immune responses with sever clinical outcome and a poor resolution evolving into a chronic inflammation.
Project 2: B cells against Candida albicans - The role of reactive oxygen species in response to infection
Classically, patients with CGD suffer from recurrent infections due to the incapacity of their immune system to fight off disease-causing organisms, such as bacteria (e.g. Staphylococcus aureus) and fungi (e.g. Aspergillus fumigatus and Candida albicans), due to a deficient production of ROS. In this study, the potential involvement of B cells in the immune response against in vitro C. albicans infection was investigated in Ncf1-deficient and in control mice.
We observed that the B cells are chemoattracted to the live C. albicans which was accompanied by an increased expression of the early-activation marker CD69 and a time-dependent increase of CD205 which is responsible for the fungal PAMPS recognition. Moreover, programmed cell death and the release of ROS were the two strategies used by the B cells to overcome the infection. In fact the apoptosis was observed not only in the B cells, probably to elude the C. albicans attack, but, more interesting, was induced also in the fungal cells; proving in this way an antifungal response by the B cells. After reculture of C. albicans, the fungi have shown a decrease of viability. Additionally, we could show that, despite both control and Ncf1 B cells increase ROS production, the Ncf1 B cells produced less ROS than the control counterparts. Moreover, after the first hours of fungal contact we could see that the expression of four important genes linked with B cell maturation, proliferation and differentiation, was significantly reduced in the Ncf1-deficient B cells when compared to the wild-type ones. Thus, suggesting that the weaker anti-fungal response in Ncf1-deficient B cells is partially due to an impaired activation and differentiation of B cells as consequence of ROS deficiency.
Overall, we have proved a hitherto unknown role for B cells in anti-fungal responses that should be explored in designing new therapeutic strategies. Moreover, by proving that a deficient production of ROS impairs the anti-fungal B cell response, we propose that the recurrent fungal infections observed in CGD patients, are not solely due to a deficient phagocytic capacity by macrophages and neutrophils, but also due to insufficient B cell activation and differentiation.
Project 3: Comparative analysis of chronic granulomatous disease and its mouse model reveals upregulated interferon signature and lymphocyte antigen receptor signalling pathways
CGD is a rare primary immunodeficiency characterised with recurrent life threatening infections associated with hyperinflammatory and autoimmune manifestations. Samples from CGD patients and its Ncf1 mutated mouse model were subjected to genome wide gene expression analysis. Mice were studied as naïve and after induction of arthritis to better study hyperinflammation in these otherwise infection free mice. Hypothesis free data-analysis was complemented with more detailed manual examination of the differentially regulated genes. We found a pronounced downstream type I IFN receptor response in both CGD patients and its mouse model. The type I IFN signature expanded in mouse upon development of autoimmune arthritis and was accompanied by type II IFN induced transcripts in arthritic mice.
Secondly, CGD patients' transcriptional signature displayed upregulation of B cell activation genes. In mouse, the importance of lymphocyte signalling in NOX2 complex deficient hyperinflammation was reflected in downregulation of both B and T antigen receptor signalling transcripts in spleen upon development of arthritis. In conclusion, our results validated the Ncf1 mutated mouse as a proper tool to study CGD and ROS deficient hyperinflammation and autoimmunity and indicate the major pathways modified by a lack of oxidative burst in vivo.
2. Career development
During the whole period covered by the fellowship, the grantee established her independent team at the Centro de Neurociências e Biologia Celular, which includes national and international researchers (EU Member States - Italy, Holland - and partner countries - Brazil, India). She was the principal supervisor and co-supervisor of national and international bachelor (two); MSc (six); and PhD (two) thesis, from which have been concluded: 1 bachelor (Portugal); four MSc (one in Italy; one in India; two in Portugal); and from the remaining ones, four will be fully finished by January 2012 (one PhD, Sweden; one MSc, Portugal; one MSc, Holland; one BSc, Brazil). Furthermore, she is currently a member of the council of the International Union for Immunological Societies. She has twice coordinated the immunology module of the in-house PhD programme. She has organised hands-on courses on flow cytometry at the University of Fayoum (Egypt). More importantly, the reintegration grant served as a seed to secure further competitive financing from national and international agencies for her research projects.
1. Scientific achievements
Project 1: Immune response in Ncf1-deficient mice with DSS-induced colitis
CGD is a genetically heterogeneous immunodeficiency disorder caused by deficiency in oxidative burst, essential for the clearance of phagocytised micro-organisms causing increased susceptibility to severe bacterial and fungal infections. CGD is caused by mutations in the phagocyte NADPH oxidase complex, the enzyme that generates microbicidal oxygen radicals. One of the more common clinical complications in CGD is chronic intestinal inflammation. Ncf1 mutation leads to a lack of ROS in mice which are described as susceptible to autoimmune diseases and hyperresponsiveness to some pathogens. We used these mutant mice to study how this lack of ROS influences a DSS-induced colitis immune response, the recovery and a second DSS induction.
We observed that the Ncf1 mice present worst clinical scores during an acute colitis induction and indications of more enhanced and aberrant immune Th1/Th17 response, with a weaker recovery during the rest period and signs of a chronic evolution when subject to a subsequent DSS induction. Thus, we suggest that ROS deficiency in these mice leads to abnormalities in the immune response of the intestinal mucosa, driving aberrant immune responses with sever clinical outcome and a poor resolution evolving into a chronic inflammation.
Project 2: B cells against Candida albicans - The role of reactive oxygen species in response to infection
Classically, patients with CGD suffer from recurrent infections due to the incapacity of their immune system to fight off disease-causing organisms, such as bacteria (e.g. Staphylococcus aureus) and fungi (e.g. Aspergillus fumigatus and Candida albicans), due to a deficient production of ROS. In this study, the potential involvement of B cells in the immune response against in vitro C. albicans infection was investigated in Ncf1-deficient and in control mice.
We observed that the B cells are chemoattracted to the live C. albicans which was accompanied by an increased expression of the early-activation marker CD69 and a time-dependent increase of CD205 which is responsible for the fungal PAMPS recognition. Moreover, programmed cell death and the release of ROS were the two strategies used by the B cells to overcome the infection. In fact the apoptosis was observed not only in the B cells, probably to elude the C. albicans attack, but, more interesting, was induced also in the fungal cells; proving in this way an antifungal response by the B cells. After reculture of C. albicans, the fungi have shown a decrease of viability. Additionally, we could show that, despite both control and Ncf1 B cells increase ROS production, the Ncf1 B cells produced less ROS than the control counterparts. Moreover, after the first hours of fungal contact we could see that the expression of four important genes linked with B cell maturation, proliferation and differentiation, was significantly reduced in the Ncf1-deficient B cells when compared to the wild-type ones. Thus, suggesting that the weaker anti-fungal response in Ncf1-deficient B cells is partially due to an impaired activation and differentiation of B cells as consequence of ROS deficiency.
Overall, we have proved a hitherto unknown role for B cells in anti-fungal responses that should be explored in designing new therapeutic strategies. Moreover, by proving that a deficient production of ROS impairs the anti-fungal B cell response, we propose that the recurrent fungal infections observed in CGD patients, are not solely due to a deficient phagocytic capacity by macrophages and neutrophils, but also due to insufficient B cell activation and differentiation.
Project 3: Comparative analysis of chronic granulomatous disease and its mouse model reveals upregulated interferon signature and lymphocyte antigen receptor signalling pathways
CGD is a rare primary immunodeficiency characterised with recurrent life threatening infections associated with hyperinflammatory and autoimmune manifestations. Samples from CGD patients and its Ncf1 mutated mouse model were subjected to genome wide gene expression analysis. Mice were studied as naïve and after induction of arthritis to better study hyperinflammation in these otherwise infection free mice. Hypothesis free data-analysis was complemented with more detailed manual examination of the differentially regulated genes. We found a pronounced downstream type I IFN receptor response in both CGD patients and its mouse model. The type I IFN signature expanded in mouse upon development of autoimmune arthritis and was accompanied by type II IFN induced transcripts in arthritic mice.
Secondly, CGD patients' transcriptional signature displayed upregulation of B cell activation genes. In mouse, the importance of lymphocyte signalling in NOX2 complex deficient hyperinflammation was reflected in downregulation of both B and T antigen receptor signalling transcripts in spleen upon development of arthritis. In conclusion, our results validated the Ncf1 mutated mouse as a proper tool to study CGD and ROS deficient hyperinflammation and autoimmunity and indicate the major pathways modified by a lack of oxidative burst in vivo.
2. Career development
During the whole period covered by the fellowship, the grantee established her independent team at the Centro de Neurociências e Biologia Celular, which includes national and international researchers (EU Member States - Italy, Holland - and partner countries - Brazil, India). She was the principal supervisor and co-supervisor of national and international bachelor (two); MSc (six); and PhD (two) thesis, from which have been concluded: 1 bachelor (Portugal); four MSc (one in Italy; one in India; two in Portugal); and from the remaining ones, four will be fully finished by January 2012 (one PhD, Sweden; one MSc, Portugal; one MSc, Holland; one BSc, Brazil). Furthermore, she is currently a member of the council of the International Union for Immunological Societies. She has twice coordinated the immunology module of the in-house PhD programme. She has organised hands-on courses on flow cytometry at the University of Fayoum (Egypt). More importantly, the reintegration grant served as a seed to secure further competitive financing from national and international agencies for her research projects.