O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição seletiva de células pancreáticas produtoras de insulina. Existem diversas formas de tratamento do DM1, tais como administração de insulina, imunossupressores, transplantes de pâncreas ou de ilhotas pancreáticas, porém todos se mostram ineficientes em algum aspecto. Recentemente, uma nova classe de medicamentos, os inibidores da enzima dipeptidil peptidase 4 (iDPP-4), demonstrou eficiência terapêutica e segurança no tratamento de pacientes com diabetes mellitus do tipo 2 devido ao aumento do hormônio peptídeo-1 semelhante ao glucagon (GLP-1, do inglês glucagon-like peptide-1). Além disso, o uso de inibidores de DPP-4 em modelos experimentais de DM1 demonstrou proteção das células pancreáticas contra apoptose, estimulação de neogênese de ilhotas pancreáticas e melhora do controle homeostático da glicose. Esse presente projeto teve como objetivo avaliar o perfil imunológico e endocrinológico da administração do inibidor de DPP-4 (MK0431) em DM1 experimental quimicamente induzido por estreptozotocina em camundongos C57Bl/6. Os animais diabéticos foram tratados com ração controle ou ração contendo inibidor de DPP-4 (4g MK0431/Kg de ração) ad libitum durante 30 e 90 dias. Durante o tratamento os animais tiveram glicemia, peso e teste de tolerância oral à glicose avaliados. Ao final do tratamento, os animais foram eutanasiados e o sangue, baço, timo, linfonodos pancreáticos e pâncreas foram coletados. Após 30 dias de tratamento com inibidor, foi observado um aumento do hormônio GLP-1 no soro, além de um padrão imunológico favorável. Dentre os mecanismos imunológicos, foi possível observar um aumento de células T reguladoras (CD4+CD25+Foxp3+) no baço e uma diminuição da citocina IFN- no homogenato pancreático. Após 90 dias de tratamento com inibidor, também foi detectado um aumento de insulina e GLP-1 séricos e uma diminuição nos níveis glicêmicos dos animais tratados. Observou-se uma redução no padrão inflamatório no microambiente pancreático, caracterizado pela diminuição das citocinas TNF- e IFN- no homogenato pancreático e por uma redução da freqüência de macrófagos CD11b+ nos linfonodos pancreáticos. Os resultados obtidos neste projeto contribuíram para validar a eficácia terapêutica da administração de inibidor de DPP-4 no tratamento do DM1 experimental, bem como os mecanismos imunológicos e endocrinológicos envolvidos. Sem a ocorrência de efeitos tóxicos relevantes, o uso de inibidores de DPP-4 pode se tornar uma alternativa terapêutica para o tratamento do DM1 em humanos, que constitui uma doença crônica associada à baixa qualidade de vida em longo prazo e necessidade de tratamento de alto custo. / Davanso, M.R. Immunoendocrinological analyses after administration of dipeptidyl-peptidase-4 inhibitor on experimental type 1 diabetes. 2012. 105p. Thesis (Masters Degree) School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2012. Type 1 Diabetes Mellitus (DM1) is an autoimmune disease characterized by the selective destruction of the insulin-producing pancreatic cells. Several forms of treatment for DM1 are current known such as insulin administration, immunosuppressors, pancreas or pancreatic islets transplantation, however, they all are inefficient in some aspect. Recently, a new class of drugs, the dipeptidyl-peptidase-4 inhibitors (iDPP-4) showed therapeutic efficacy and safety in the treatment with type 2 diabetes mellitus patients due to an increase in the glucagon-like peptide-1 (GLP-1). In addition, the use of DPP-4 inhibitors in experimental models of DM1 has demonstrated a protection of pancreatic cells against apoptosis, stimulation of pancreatic islets neogenesis and improvement in the glucose homeostatic control. This project evaluated the immunological and endocrinological profile of the DPP-4 (MK0431) inhibitor administration in experimental chemically induced DM1 by streptozotocin in C57BI/6 mice. The diabetic animals were treated with either a normal chow diet or diet containing DPP-4 inhibitor (4g MK0431/Kg of diet) ad libitum during 30 and 90 days. During the treatment the animals were evaluated regarding glycemia, weight, and oral glucose tolerance test. At the end of the treatment, the animals were killed and the blood, spleen, thymus, pancreatic lymph nodes and pancreas were collected. After 30 days of treatment with inhibitor, it was observed an increase in the hormone GLP-1 in the serum, besides a favorable immunological pattern. Among the immunologic mechanisms, it was possible to observe an increase in the regulator T cells (CD4+CD25+Foxp3+) of the spleen and a decrease in the cytokine IFN- in the pancreatic homogenate. After 90 days of treatment with inhibitor, it was also noticed an increase in the insulin and serum GLP-1 levels as well as a decrease in the glycemic levels in the treated animals. It was observed a reduction in the inflammatory pattern in the pancreatic microenvironment characterized by a decrease in the cytokines TNF- and IFN- in the pancreatic homogenate and by a reduction in the frequency of CD11b+ macrophages in the pancreatic lymph nodes. The results obtained in this project contributed to validate the therapeutic efficacy of the DPP-4 inhibitor administration in the treatment of experimental DM1, as well as the immunological and endocrinological mechanisms involved. Without the occurrence of relevant toxic effects, the use of DPP-4 inhibitors may become a therapeutic alternative for the treatment of DM1 in humans, which constitutes a chronic disease associated to low life quality and need for high cost treatment.

Poly(ADP-ribose) polymerases belong to a family of proteins with 17 members in human beings. PARP1, the founding member of the family is a protein that synthesizes linear or branched polymers of ADP-ribose on itself or on target proteins. Different members of this family, that do not all possess ADP-ribosyl polymerase activity, are involved in the regulation of various cellular mechanisms. Some members of the family are particularly involved in the positive or negative control of the immune response. PARP1 is a key player in the regulation of inflammation, through its positive control of cell death and of proinflammatory cytokine production. On the other hand, the tankyrases (PARP5a and PARP5b) and PARP14 seem to regulate inflammatory responses in a negative fashion. PARP12 is a poorly characterized member of the family, whose expression is greatly increase following stimulation with type-I interferons, cytokines mainly involved in antiviral defences.<p>PARP12 is a protein that possesses three main domains: A putative RNA binding N-terminal domain composed of tandem CCCH zinc-fingers, a central WWE domain and a C-terminal PARP catalytic domain. In this work, we have shown that the expression of PARP12 is strictly-dependent on type-I interferons, that it possesses ADP-ribosyl transferase activity and that in can regulate the translation of messenger RNA into proteins. PARP12 can be found in stress granules, sites of storage of untranslated mRNAs, and is capable of directly inhibiting the translation of a reporter mRNA when tethered to it, in a manner dependent on its catalytic activity. Furthermore overexpression of wild-type PARP12, in contrast to overexpression of a mutant with no detectable catalytic activity (PARP12-G575W), leads to a general arrest of most cellular translation.<p>On the other hand, we have shown that PARP12 can activate the transcription of genes under the control of an NFκB-dependent promoter, especially when its zinc-fingers are deleted or mutated (PARP12ΔZnF). PARP12ΔZnF is located in structures that can enclose TRIF, RIP1, NEMO, p62/SQSTM1 and ubiquitin. These proteins have all possess an important role in the activation of NFκB signalling cascades. Moreover, we have shown that endogenous PARP12 is situated in ALIS (Aggresome-Like Induced Structures) in LPS-stimulated macrophages. These structures have a possible role in the presentation of antigens on class I major histocompatibility complexes, implying that PARP12 may be involved in the regulation of antigen presentation. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les cellules Natural Killer (NK) sont des effecteurs de l’immunité innée et constituent de véritables sentinelles dans l’immuno-surveillance contre les virus et les processus tumoraux. Pour échapper à la reconnaissance NK, les virus ainsi que les cellules tumorales utilisent de nombreux subterfuges. Au cours de ce travail, nous nous sommes intéressés à deux facteurs régulateurs de la fonction des cellules NK, la molécule PD-1 et la prostaglandine E2 (PGE2). Programmed death 1 (PD-1) est une molécule régulatrice exprimée sur les lymphocytes T et B activés. L’engagement de PD-1 par ses ligands inhibe leurs fonctions effectrices et prolifératives. Au cours de certaines infections virales chroniques ou de tumeurs, l’expression de PD-1 est associée à l'épuisement fonctionnel des lymphocytes T effecteurs. Le blocage de l’axe PD-1/PD-1 ligands restaure les fonctions effectrices des lymphocytes et représente ainsi une approche thérapeutique prometteuse. Nous avons mis en évidence de manière fortuite une expression inhabituelle du récepteur PD-1 sur les cellules NK au cours de certaines infections virales chroniques (HHV8, VIH ou VHC). L’expression de PD-1 sur les cellules NK caractérise une population récemment activée (CD69++, CD25 + et Nkp44 +), susceptible à l’apoptose (Annexin V+), exprimant une moindre quantité du récepteur de cytotoxicité naturelle NKp46. L’analyse fonctionnelle montre que les cellules NK PD-1+ ont des capacités de cytotoxicité (dégranulation CD107a) et de production de cytokines (IFNγ) réduites en comparaison avec leurs homologues PD-1-. De façon intéressante, l’IL-2 et l’IL-15 peuvent restaurer les fonctions effectrices des cellules NK PD-1+. Contrairement aux cellules T dont l’expression de PD-1 est induite par divers stimuli, seule la stimulation des récepteurs NKp46 et NKp30, en synergie avec l’action de l’IL-15 ou l’IL-2, induit in vitro de façon reproductible l’expression de PD-1 sur les cellules NK de témoins sains. Pour pouvoir disposer d’un modèle in vitro de cellules NK PD-1+, nous avons généré des cellules exprimant PD-1 de manière stable par transduction lentivirale de la lignée NKL. En comparaison avec les cellules transduites par le vecteur vide, les cellules NKL PD-1+ ont des capacités cytotoxiques réduites, confirmant nos résultats chez les patients. Nous avons aussi étudié les mécanismes par lesquels la PGE2, une autre molécule immuno-modularice, régule les fonctions des cellules NK. Nos résultats suggèrent que la PGE2 agit à travers ses récepteurs EP2 et EP4 pour inhiber l’expression de NKG2D et de l’IL-15Rγ induite par l’IL-15 sur les cellules NK. Ce travail doit nous permettre de mieux comprendre comment la PGE2 s’oppose aux effets activateurs de l’IL-15 sur les cellules NK, et représente un mécanisme de rétrocontrôle de l’inflammation. En conclusion, nos résultats montrent que l’expression de PD-1 sur les cellules NK représente un mécanisme supplémentaire d’échappement viral à la réponse immune. La suite de l’étude sur la PGE2 devrait nous permettre d’évaluer l’intérêt de l’utilisation d’antagonistes spécifiques dans l’immunothérapie anti-tumorale par l’IL-15. Le lien entre la PGE2 et l’expression de PD-1 sur les cellules NK est en cours d’investigation. / Natural Killer (NK) cells are effectors of the innate immune system, and play a crucial role in virus and cancer immunesurveillance. To escape NK-cell mediated elimination of infected or transformed cells, viruses and tumors have developed multiple strategies to interfere with NK-cell functions. In the present study, we investigated the role of two regulatory molecules, Programmed Death-1 (PD-1) and Prostaglandine E2 (PGE2), in controlling NK cell activation and effector functions. PD-1 is a key immune checkpoint receptor expressed by activated T and B lymphocytes. Upon interaction with its cognate ligands, PD-1 inhibits lymphocyte proliferation and functions. During cancer or chronic viral infections, PD-1 expression is associated with functional exhaustion of effector T cells. Blockade of PD-1 signaling restores T-cell functions, and represents a promising therapeutic tool. We fortuitously observed unusual expression of PD-1 on a subset of CD56dim NK cells in some patients with persistent viral infection (HHV8, HIV or HCV). We show that PD-1 expression on NK cells characterizes a subpopulation of recently activated cells (CD69+, CD25+, Nkp44+) that are sensitive to apoptosis (Annexin V+) and not senescent (CD57-). NKp46 expression was also markedly decreased on PD-1+ NK cells. In vitro functional experiments showed that PD-1+ NK cells had impaired cytotoxic capacity (CD107a degranulation) and reduced IFN-γ production compared to their PD-1- counterpart, suggesting that they might represent functionally exhausted NK cells. Interestingly, exogenous IL-2 and IL-15 could restore PD-1+ NK cell effector functions. While strong non-specific stimulation by PMA/ionomycin transiently induced PD-1 on control NK cells, only activation through NKp46 or NKp30 receptors in the presence of IL-15 could reproducibly induce stable PD-1 expression. To investigate the effect of PD-1 expression on NK cells in the absence of any confounding factor related to the underlying disease, we generated NK cells stably expressing PD-1 after lentiviral transduction. Compared to NK cells transduced with the control vector, PD-1+ cells showed a constitutively decreased CD107a degranulation, thus confirming our findings in NK cells from infected patients. In the second part of this study, we investigated the immunosuppressive role of PGE2 in the control of IL-15-mediated NK cell activation. Our results suggest that PGE2 acts through EP2 and EP4 receptors to inhibit IL-15 induced NKG2D and IL-15Rγ expression on NK cells. These findings allow a better comprehension of PGE2/IL-15 antagonism in the regulation of NK cell responses. In conclusion, our results indicate that PD-1 expression on NK cells could represent a supplementary mechanism of immune evasion strategy, and allow introducing the concept of exhausted NK cells, similar to exhausted PD-1+ T cells. Our results also demonstrate that PGE2 exerts a negative feedback on IL-15-mediated effects on NK cells. Blocking PGE2 or its receptors could be of interest in IL-15 tumor immunotherapy to potentiate IL-15-induced cytotoxic functions of NK cells.

La survenue de la maladie du greffon contre l’hôte aiguë (GVHa) après l’allogreffe de cellules souches hématopoïétiques (CSH) reste une source majeure de mortalité et morbidité pour laquelle des avancées thérapeutiques restent indispensables. Les lymphocytes NKT invariants (iNKT) possèdent de multiples propriétés à potentiel immuno-régulateur et anti-tumoral, mais peu de données existent chez l’homme dans le cadre de l’allogreffe de CSH. Nous avons récemment montré qu’une bonne reconstitution en iNKT post greffe ainsi qu’une bonne richesse et capacité d’expansion des iNKT du greffon, notamment du sous type CD4-, étaient corrélés à une moindre survenue de GVHa chez les patients. Ce travail avait pour objectif de déterminer quel sous type de iNKT humain pouvait réguler la réponse allo-immune et ses mécanismes d’action. Dans le modèle murin humanisé de xéno-GVH, chez la souris NSG, nous avons mis en évidence que le sous-type iNKT CD4- humain ,contrairement au sous type CD4+, permettait de diminuer la survenue de GVH et d’améliorer la survie des souris NSG greffées avec des PBMC humains enrichis ou non en lymphocytes iNKT. Nous avons montré de façon concordante, in vivo et in vitro, que les iNKT CD4- humains diminuent le profil Th1 et Th17 des lymphocytes T conventionnels et leur marqueurs d’activation après stimulation allo- et xéno-génique. Par ailleurs, les iNKT CD4- humains induisent la mortalité des cellules dendritiques in vitro et in vivo, contrairement au sous-type iNKT CD4+ qui induit leur maturation. Par contre, les iNKT CD4- n’ont pas d’impact sur les lymphocytes T régulateurs, ni sur la prolifération des T conventionnels. Ils n’altèrent pas non plus la qualité de la prise de greffe des cellules humaines dans la souris NSG. Nous concluons, que contrairement aux iNKT murins, le sous-type iNKT CD4- humain, et non pas CD4+, est capable de diminuer la survenue de xéno-GVH dans le modèle murin NSG par une régulation directe de la réponse immune allogénique. Ces résultats obtenus dans ce modèle préclinique ouvrent la possibilité d’une nouvelle thérapie cellulaire utilisant ces cellules chez l’homme pour prévenir la survenue de GVH aiguë. / The occurrence of acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (allo-SCT) is a major source of mortality and morbidity and new therapeutic advances are urgently needed. Despite the numerous immunomodulatory properties depicted for the iNKT lymphocytes, there are few studies of these lymphocytes in human allo-SCT and GVHD setting. We previously reported that a higher iNKT reconstitution in patients after allo-SCT and higher graft CD4- iNKT expansion rates, were associated with reduced risk of aGVHD in patients. In this study we aimed to assess a direct immune regulatory role of CD4+ or CD4- iNKT subsets and their mechanisms of action during the allo-immune response. We demonstrated that the human iNKT CD4- subset, but not the CD4+ subset, was associated with reduced xeno-GVHD and prolonged survival of humanized NSG mice infused with human PBSC with or without iNKT cells. We also concomitantly showed, both in vitro and in vivo, that human iNKT CD4- could dampen Th1 and Th17 conventional T cell allo and xeno-responses as well as T cell activation markers. In addition, whereas iNKT CD4+ could stimulate DC maturation, iNKT CD4- induced their apoptosis, in vitro and in vivo. We did not observe any impact on regulatory T cells and conventional T cell proliferation. iNKT CD4- did not impact the engraftment of human cells in NSG mice. We conclude that, by contrast with mice, in humans, the CD4- subset and not their CD4+ counterpart can directly down-regulate the allo-immune response. These results obtained in a robust humanized preclinical xeno-GVH mice model could open new strategies of cellular therapy in the prevention of acute GVHD.

Es ist davon auszugehen, dass Fehlfunktionen im Immunsystem mit der Ausprä-gung des Krankheitsbildes der aggressiven Parodontitis im Zusammenhang ste-hen. In dieser Arbeit sollen anhand klinischer, immunologischer und mikrobiologischer Untersuchungen ein immunologisches Risikoprofil bei Patienten mit aggressiver Parodontitis erschlossen, gegebenenfalls Unterschiede zur moderaten chronischen Parodontitis beleuchtet und explorativ Zusammenhänge zwischen immunologischen und mikrobiologischen Befunden eruiert werden. Es wurden geeignete Patienten und gesunde Probanden laut Ethikvotum rekrutiert. Die immunologischen Untersuchungen erfolgten an PBMCs unter Verwendung durchflusszytometrischer Methoden und mittels ELISpot-Assay. Mikrobiologische Untersuchungen subgingivaler Plaque wurden als klassische Kultur und 16S rRNA-Sequenzierung durchgeführt. Immundefekterkrankungen konnten bei allen Individuen ausgeschlossen werden. Im Gruppenvergleich wurde eine erhöhte Stimulierbarkeit der PBMCs von Patienten mit moderater chronischer Parodontitis bezüglich ihrer IL-1β-Freisetzung bei Inkubation mit LPS festgestellt. Des Weiteren wies diese Patientengruppe einen vergleichsweise höheren Anteil an Gedächtnis-B-Zellen auf. In der mikrobiologischen Untersuchung konnten bekannte parodontopathogene Spezies nachgewiesen und Prevotella denticola als bislang nicht explizit erwähntes Pathogen mit aggressiver Parodontitis assoziiert werden. Unsere Untersuchung weisen auf Zusammenhänge zwischen immunologischen und mikrobiologischen Befunde bezüglich einiger parodontopathogener Bakterien, wie Prevotella oralis, und Stimulierbarkeit der IL-1β-Freisetzung, B-Zelldifferenzierung und T-Zellverhältnis hin.

The intestinal epithelium represents one of the first lines of defense against pathogenic bacteria. Immune regulation at this critical barrier is necessary to maintain organismal fitness, and mis-regulation here has been linked to numerous debilitating diseases. Functional relationships between the nervous system and immune system have been found to be critical in the proper coordination of immune defenses at barrier surfaces, however the precise mechanisms underlying theses interactions remains unclear. Through conducting a forward genetic screen utilizing the model organism Caenorhabditis elegans, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen and resistance to bacterial infection. However, derepression of the p38 MAPK PMK-1 pathway also results in severe developmental and reproductive defects, demonstrating the critical function of OLRN-1 to both prime C. elegans intestinal epithelial cells for the induction of anti-pathogen responses, and to limit the deleterious effects of immune hyper-activation. These data reveal an unexpected connection between olfactory receptor development and innate immunity, as well as demonstrate how neuronal regulation of immune responses within the intestinal epithelium is critical for both reproductive and developmental fitness.

TRPM4, un canal cationique non-sélective activé par le Ca2+ intracellulaire, est un acteur moléculaire important impliqué de la régulation du signal calcique et l’activation des lymphocytes T conventionnels mais son rôle dans la fonction des lymphocytes T régulateurs (Tregs Foxp3+) reste inconnu. Dans un modèle de souris transgéniques dans lequel le gène Trpm4 a été sélectivement invalidé dans la population des Tregs Foxp3+ (souris Foxp3(YFP)Cre+Trpm4flox/flox), nous avons démontré dans différents modèles in vivo d’inflammation aiguë et chronique que TRPM4 contrôle la fonction suppressive et la mort de ces cellules. Dans le modèle de fibrosarcome induit par le méthylcholanthrène (3-MCA) ou implanté (modèle MCA205), dans lequel le rôle des Tregs est documenté, l’absence de fonction de TRPM4 induit une diminution significative de l’incidence et de la croissance tumorale. Dans l’environnent inflammatoire chronique et hypoxique de ces tumeurs, l’expression de TRPM4 protège les Tregs infiltrant la tumeur de la mort cellulaire induit par l’ATP extracellulaire et stimule ainsi le développent et la progression tumorale. L’absence d’expression de TRPM4 dans les Tregs stimule la réponse anti-tumorale médiée par l’IFNg et induit la régression des tumeurs. En conclusion, en inhibant l’entrée de Ca2+ extracellulaire, TRPM4 régule négativement les fonctions suppressives des Tregs et protège ces cellules de la mort cellulaire induite par l’activation. / TRPM4, a Ca2+-activated non-selective cation ion channel is an important regulator of Ca2+ signaling and cell activation in conventional T cells, but its role in Foxp3+ Tregs function remains unknown. Using a model in which Trpm4 gene was selectively invalidated in Foxp3+ Tregs population (Foxp3(YFP)Cre+Trpm4flox/flox mice) we have shown in different in vivo models of acute and chronic inflammation that TRPM4 is an important regulator of Tregs functions and survival. In a model of primary carcinogenesis induced by methylcholantrene (3-MCA) or implanted fibrosarcoma (MCA205 model), in which Tregs role has been documented, lack of TRPM4 expression and function induced significantly decreased incidence and tumor growth. We found that within chronic inflammatory and hypoxic tumor microenvironment, TRPM4 protected Tregs from ATP-induced cell death and therefore promoted tumor initiation and progression. In contrast, TRPM4 deficiency in Tregs favored IFN-g-mediated spontaneous anti-tumor immune response. Thus, through inhibiting Ca2+ influx, TRPM4 acts as a negative modulator of Tregs suppressive functions and protects Tregs from activation-induced cell death.

Bordetella pertussis e Bordetella parapertussis são os principais agentes causadores da coqueluche no homem. O óxido nítrico é fundamental para o controle de diversos processos fisiopatológicos. Neste trabalho analisamos sinais moleculares envolvidos na produção de NO em macrófagos derivados de medula óssea murina (BMDMO) infectadas por Bpertussis e Bparapertussis. Nossos resultados mostraram que BMDMO de C57BL/6 estimulados com Bpertussis não sintetizaram níveis significativos de nitrito, ao contrário da infecção com Bparapertussis. BMDMO de C57BL/6 infectados por Bpertussis e Bparapertussis produziram níveis elevados de arginase e de TGFb e esta produção foi dependente de TLR4, porém a produção de NO pelos BMDMO de C3H/HeJ infectados com Bparapertussis foi independente deste receptor. A adição exógena de PT em BMDMO infectados com Bparapertussis reduziu a quantidade de NO sintetizada. Concluímos que TGFb e arginase contribuem para o controle da produção de NO durante a infecção in vitro de BMDMO com Bpertussis e este mecanismo depende de LPS envolvendo TLR4 e PT. / Bordetella pertussis and Bordetella parapertussis are the main etiologic causes of human whooping cough. Nitric oxide (NO) is crucial for several physiopathologic events. Herein we analyzed the molecular signals required for NO production by murine bone marrow-derived macrophages (BMDM) infected with Bpertussis or Bparapertussis. Our data show that BMDM obtained from C57Bl/6 mice was not able to produce measurable levels of nitrite when stimulated with Bpertussis while infection of these cells with Bparapertussis induced high levels of nitrite. Arginase and TLR4-dependent TGF-b were produced in response to infection with either Bpertussis or Bparapertussis. NO production by BMDM obtained from C3H/HeJ mice occurred after Bparapertussis infection in the absence of TLR4. Addition of pertussis toxin to the C57Bl/6 BMDM cultures infected with Bparapertussis decreased NO levels. In conclusion, TGF-b and arginase play a role controlling NO production by BMDM during in vitro infection by Bpertussis. This effect depends on the presence of LPS-TLR4 and PT signaling pathways.

<p>IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated.</p><p>IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.</p>

IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated. IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.