In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis

On the components of adaptive immune response involved in the control of a primary and secondary infection by Brucella melitensis

Brucellosis is a bacterial zoonose caused by organisms belonging to the genus Brucella. These bacteria are facultative intracellular pathogens that cause abortion in domestic (cattle, goats, sheeps, etc.) and wild (deers, bisons, etc.) animals and a febrile chronic illness in humans. The disease exists worldwide and continues to have a great health significance and economic importance in developing countries. Despite past and current efforts to eradicate brucellosis by vaccination and culling within cattle and herds, as many as 500 000 new human cases are reported annually worldwide. Vaccines against brucellosis were initially developed on an empirical basis. The current studies are moving towards a more rational design but are hindered by an incomplete knowledge of the in vivo life style of Brucella and the immune mechanisms involved in the establishment of a protective memory. Despite progresses, several fields of host adaptive immunity remain highly controversial such as the implication of B cells, CD4+ and CD8+ T cells. While it is commonly accepted that Brucella infection induces a Th1 immune response characterized by the production of IFN-γ that activates bactericidal mechanisms of macrophages, little has been described with regards to a potential implication of the Th2 response or the recently described Th17 response. Objectives of this thesis were therefore to determinate the implication of lymphocyte subsets and signaling pathways after primary infection with Brucella melitensis and characterize their roles in the development of a protective secondary immune response in the murine experimental model. In the first part of this study we clearly confirm the central role of MHC-II-dependent antigen presentation to CD4+ T cells and the IFN-γ-mediated Th1 response in the control of B. melitensis primary infection. We also report that the absence of B cells, MHC-I-dependent antigen presentation, Th2 and Th17 responses appears to have no important positive or negative impact on the course of infection. In the second part of this work, we show that Brucella is able to persist several weeks in the blood of infected mice. Surprisingly, we found that bacteria are initially localized extracellularly and then infect erythrocytes where they are already detectable after 24h. Finally, the last part demonstrates that humoral immunity and CD4+ Th1 cells are both necessary and complementary for a sterilizing immune response upon a secondary infection with B. melitensis. Circulating specific antibodies and IFN-γ production by CD4+ T cells activated at the site of infection after the re-call infection appear as key immunological markers of protection in the murine experimental model of Brucella infection. In conclusion, this work improves our understanding of the nature of murine immune response developed following B. melitensis infection and tries to provide correlates of protection that could help to define rational strategies for designing new vaccines against brucellosis. This study also reveals for the first time that erythrocytes constitute an unexpected niche for Brucella, and maybe a currently undescribed way of transmission by blood-sucking arthropods.La brucellose est une zoonose bactérienne causée par des organismes appartenant au genre Brucella. Ces bactéries sont des pathogènes intracellulaires facultatifs qui causent l’avortement tant chez les animaux domestiques (bovins, chèvres, moutons, etc.) que sauvages (cerfs, bisons, etc.) et une maladie chronique chez l’homme. La brucellose est présente dans le monde entier et constitue un problème majeur de santé publique qui affecte également l’économie dans les pays en voie de développement. Malgré les efforts passés et présents visant à éradiquer la brucellose, notamment par la vaccination et l’abattage systématique des troupeaux infectés, plus de 500 000 nouveaux cas humains sont signalés chaque année dans le monde. À l’origine, les vaccins contre la brucellose ont été développés empiriquement. Les études vaccinales actuelles s’orientent vers une conception plus rationnelle, mais sont freinées par une méconnaissance du cycle infectieux de Brucella in vivo et des mécanismes immunitaires impliqués dans l’établissement d’une mémoire immunitaire protectrice. Malgré de récents progrès, plusieurs domaines de la réponse adaptative développée en modèle murin suite à l’infection par Brucella sont encore fort controversés, comme l’importance de l’implication des lymphocytes B, T CD4+ et T CD8+. Bien qu’il soit communément admis que l’infection par Brucella induit une réponse immunitaire de type Th1, caractérisée par la production d’IFN-γ qui active les mécanismes bactéricides des macrophages, peu de choses ont été décrites concernant une implication potentielle des réponses Th2 ou Th17. En conséquence, les objectifs de cette thèse étaient de déterminer l’implication des différentes populations de lymphocytes et des voies de signalisation induites après infection par Brucella melitensis en modèle expérimental murin et de caractériser leurs rôles dans le développement d’une réponse immunitaire protectrice lors d’une ré-infection. Dans la première partie de ce travail, nous avons clairement confirmé le rôle central de la voie de présentation des antigènes dépendante du MHC-II aux lymphocytes T CD4+ ainsi que la nécessité de la production d’IFN-γ par les lymphocytes Th1 dans le contrôle d’une infection primaire par B. melitensis. Nous avons également démontré que l’absence de lymphocytes B, de la voie de présentation dépendante du MHC-I ou des réponses Th2 et Th17 n’a pas d’impact majeur sur le déroulement de l’infection. Dans la seconde partie de ce travail, nous avons montré que Brucella est capable de persister plusieurs semaines dans le sang de souris infectées. De manière surprenante, nous avons constaté que, dans un premier temps, les bactéries étaient libres dans le sang et qu’elles infectaient ensuite les globules rouges où elles sont déjà détectables après 24h. Enfin, la dernière partie de ce travail démontre que les réponses humorale et cellulaire sont toutes deux complémentaires et nécessaires à l’établissement d’une réponse immune stérilisante lors d’une ré-infection par B. melitensis. Les anticorps circulants et la production d’IFN-γ par les lymphocytes T CD4+ activés sur le site d’infection lors d’une réponse secondaire apparaissent comme des effecteurs clés de la protection en modèle murin. En conclusion, ce travail améliore notre compréhension de la réponse immunitaire induite par B. melitensis en modèle murin. Il essaye également de proposer des marqueurs immunologiques de protection qui pourraient aider à définir des stratégies plus rationnelles pour la conception de nouveaux vaccins contre la brucellose. Enfin, cette étude révèle pour la première fois que les globules rouges constituent une niche d’infection inattendue qui pourrait assurer la transmission de la brucellose par des insectes suceurs de sang.(DOCSC03) -- FUNDP, 201

Crucial role of gamma interferon-producing CD4+ Th1 cells but dispensable function of CD8+ T cell, B cell, Th2, and Th17 responses in the control of Brucella melitensis infection in mice.

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4(+) T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8(+) T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Brucella melitensis invades murine erythrocytes during infection.

Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Humoral Immunity and CD4+ Th1 Cells Are Both Necessary for a Fully Protective Immune Response upon Secondary Infection with Brucella melitensis.

Brucella spp are intracellular bacteria that cause brucellosis, one of the most common zoonoses in the world. Given the serious medical consequences of this disease, a safe and effective human vaccine is urgently needed. Efforts to develop this vaccine have been hampered by our lack of understanding of what constitutes a protective memory response against Brucella. In this study, we characterize the cells and signaling pathways implicated in the generation of a protective immune memory response following priming by the injection of heat-killed or live Brucella melitensis 16M. Using a panel of gene-deficient mice, we demonstrated that during a secondary recall response, both the Brucella-specific humoral response and CD4(+) Th1 cells must act together to confer protective immunity in the spleen to B. melitensis infection. Humoral protective immunity is induced by the inoculation of both heat-killed and live bacteria, and its development does not require T cells, MyD88/IL-12p35 signaling pathways, or an activation-induced deaminase-mediated isotype switch. In striking contrast, the presence of memory IFN-γ-producing CD4(+) Th1 cells requires the administration of live bacteria and functional MyD88/IL-12p35 pathways. In summary, our work identifies several immune markers closely associated with protective immune memory and could help to define a rational strategy to obtain an effective human vaccine against brucellosis.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Phenotypical characterization of <i>B. melitensis</i> infected cells in the spleen.

<p>Wild-type C57BL/6 and BALB/c mice were injected i.p. with PBS or 10⁸ CFU of mCherry-Br. Mice were sacrificed at selected times and spleens were collected and examined by immunohistofluorescence. Data indicate the percentage of mCherry-Br that colocalizes with (<b>A</b>) CD90.2-, B220-, Ly6G-, MOMA-1-, F4/80-, CD11c-, (<b>B</b>) MHCII-, iNOS-, CD11b-expressing cells at the selected time in C57BL/7 mice. (<b>C</b>) Comparison at 120 h p.i. between C57BL/6 and BALB/c mice. The bars are the mean±SD from at least 3 spleen sections per spleen from 5 mice.</p

CD11b⁺ granulomas in the spleen and liver of wild type and MyD88-deficient infected mice.

<p>Mice were injected i.p. with PBS (control) or 10⁸ CFU of mCherry-Br. The figure presents the localization by immunohistofluorescence of CD11b^hi and iNOS expressing cells in spleen (<b>A</b>) and liver (<b>B</b>) from control and 120 h mCherry-Br infected wild-type and MyD88^−/− C57BL/6. Panels are color-coded with the text for the antigen or mCherry-Br examined. Scale bar = 200 µm, as indicated. r.p.: red pulp; w.p.: white pulp. Data are representative of at least 3 independent experiments.</p

Phenotypical analysis of granuloma in the liver of wild type and MyD88-deficient infected mice.

<p>Colocalization by immunohistofluorescence of CD11c (<b>A</b>), F4/80 (<b>B</b>), Ly-6G (<b>C</b>) and iNOS expressing cells with mCherry-Br signal in liver from 120 h mCherry-Br infected wild-type and MyD88^−/− C57BL/6. Mice were injected i.p. with 10⁸ CFU of mCherry-Br. Panels are color-coded with the text for the antigen or mCherry-Br examined. Scale bar = 50 µm, as indicated. Data are representative of at least 3 independent experiments.</p

Phenotypical analysis of granuloma in the liver and spleen of wild type and RAG-deficient infected mice.

<p>Localization by immunohistofluorescence of (<b>A</b>) F4/80, (<b>B</b>) CD11b, (<b>C</b>) CD11c and iNOS expressing cells in liver and spleen from 120 h mCherry-Br infected wild-type and RAG^−/− C57BL/6. Mice were injected i.p. with 10⁸ CFU of mCherry-Br. Images represent a single granuloma. Panels are color-coded with the text for the antigen or mCherry-Br examined. Scale bar = 50 µm, as indicated. Data are representative of at least 3 independent experiments.</p