RESUMO
In this study we explored the immunomodulatory properties of highly purified free galactan, the soluble exopolysaccharide secreted by Mycoplasma mycoides subsp. mycoides (Mmm). Galactan was shown to bind to TLR2 but not TLR4 using HEK293 reporter cells and to induce the production of the anti-inflammatory cytokine IL-10 in bovine macrophages, whereas low IL-12p40 and no TNF-α, both pro-inflammatory cytokines, were induced in these cells. In addition, pre-treatment of macrophages with galactan substantially reduced lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines TNF- and IL-12p40 while increasing LPS-induced secretion of immunosuppressive IL-10. Also, galactan did not activate naïve lymphocytes and induced only low production of the Th1 cytokine IFN-γ in Mmm-experienced lymphocytes. Finally, galactan triggered weak recall proliferation of CD4+ T lymphocytes from contagious bovine pleuropneumonia-infected animals despite having a positive effect on the expression of co-stimulatory molecules on macrophages. All together, these results suggest that galactan possesses anti-inflammatory properties and potentially provides Mmm with a mechanism to evade host innate and adaptive cell-mediated immune responses.
Assuntos
Imunidade Adaptativa , Doenças dos Bovinos/microbiologia , Galactanos/metabolismo , Imunidade Inata , Macrófagos/imunologia , Mycoplasma mycoides/fisiologia , Pleuropneumonia Contagiosa/microbiologia , Animais , Bovinos , Células HEK293 , Humanos , Interleucina-10/metabolismo , Polissacarídeos BacterianosRESUMO
We investigated the interactions of unopsonized and opsonized Mycoplasma mycoides subsp. mycoides (Mmm) with bovine macrophages in vitro. Mmm survived and proliferated extracellularly on bovine macrophage cell layers in the absence of Mmm-specific antisera. Bovine complement used at non-bactericidal concentrations did neither have opsonizing effect nor promoted intracellular survival, whereas Mmm-specific antisera substantially increased phagocytosis and Mmm killing. A phagocytosis-independent uptake of Mmm by macrophages occurred at a high multiplicity of infection, also found to induce the production of TNF, and both responses were unaffected by non-bactericidal doses of bovine complement. Bovine complement used at higher doses killed Mmm in cell-free cultures and completely abrogated TNF responses by macrophages. These results provide a framework to identify Mmm antigens involved in interactions with macrophages and targeted by potentially protective antibodies and point towards a pivotal role of complement in the control of inflammatory responses in contagious bovine pleuropneumonia.
Assuntos
Macrófagos , Fagocitose , Animais , Bovinos , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Mycoplasma/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Pleuropneumonia Contagiosa/microbiologia , Pleuropneumonia Contagiosa/imunologia , Mycoplasma mycoides/imunologiaRESUMO
Peste des petits ruminants (PPR) is an acute transboundary infectious viral disease of small ruminants, mainly sheep and goats. Host susceptibility varies considerably depending on the PPR virus (PPRV) strain, the host species and breed. The effect of strains with different levels of virulence on the modulation of the immune system has not been thoroughly compared in an experimental setting so far. In this study, we used a multi-omics approach to investigate the host cellular factors involved in different infection phenotypes. Peripheral blood mononuclear cells (PBMCs) from Saanen goats were activated with a T-cell mitogen and infected with PPRV strains of different virulence: Morocco 2008 (high virulence), Ivory Coast 1989 (low virulence) and Nigeria 75/1 (live attenuated vaccine strain). Our results showed that the highly virulent strain replicated better than the other two in PBMCs and rapidly induced cell death and a stronger inhibition of lymphocyte proliferation. However, all the strains affected lymphocyte proliferation and induced upregulation of key antiviral genes and proteins, meaning a classical antiviral response is orchestrated regardless of the virulence of the PPRV strain. On the other hand, the highly virulent strain induced stronger inflammatory responses and activated more genes related to lymphocyte migration and recruitment, and inflammatory processes. Both transcriptomic and proteomic approaches were successful in detecting viral and antiviral effectors under all conditions. The present work identified key immunological factors related to PPRV virulence in vitro.
Assuntos
Cabras/imunologia , Leucócitos Mononucleares/imunologia , Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste dos Pequenos Ruminantes/patogenicidade , Virulência/imunologia , Animais , Perfilação da Expressão Gênica , Cabras/virologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , ProteômicaRESUMO
Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Imagem Molecular/métodos , Mycoplasma/química , Mycoplasma/citologia , Espectrometria de Fluorescência/métodos , Animais , Bovinos , Células Cultivadas , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mycoplasma/metabolismo , Mycoplasma/patogenicidade , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/fisiopatologia , Fagócitos/citologia , Fagócitos/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP.
Assuntos
Adenovírus Humanos/genética , Vacinas Bacterianas/imunologia , Portadores de Fármacos , Vetores Genéticos , Lipoproteína(a)/imunologia , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Citocinas/metabolismo , Feminino , Humanos , Lipoproteína(a)/biossíntese , Lipoproteína(a)/genética , Camundongos Endogâmicos BALB C , Mycoplasma mycoides/genéticaRESUMO
IFN-gamma is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-gamma inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14-39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23-29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15-19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Ehrlichia ruminantium/imunologia , Doenças das Cabras/microbiologia , Hidropericárdio/imunologia , Interferon gama/biossíntese , Animais , Antígenos de Protozoários/imunologia , Concanavalina A/imunologia , Ehrlichia ruminantium/metabolismo , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/sangue , Cabras , Hidropericárdio/sangue , Hidropericárdio/prevenção & controle , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Peso Molecular , Vacinação/veterináriaRESUMO
Interferon gamma (IFN-gamma) is considered as a key mediator of protective cell-mediated immunity against intracellular pathogens in general, and against Ehrlichia ruminantium, the causative agent of tick-borne heartwater disease of ruminants, in particular. However, the source of this important cytokine in animals immunized against E. ruminantium remains largely unknown. We have analyzed in goats protected by vaccination with a killed E. ruminantium vaccine, the potential of individual, genuine (i.e., non-cloned), T cell subsets to produce IFN-gamma after antigenic recall in vitro. In all vaccinated but none control animals, E. ruminantium-induced IFN-gamma secretion was observed in 24 h stimulated blood. Flow cytometric analysis of stimulated peripheral blood mononuclear cells (PBMCs) collected after each vaccine inoculation indicated that immune CD4+ and CD8+ T cells contribute to the same extent to the production of IFN-gamma, while WC1+ T cells are less important. This was confirmed by blocking the secretion of IFN-gamma with anti-classes I and II major histocompatibility complex antibodies. Blocking experiments also suggest that CD8+ need the help of CD4+ T cells in order to produce IFN-gamma. Thus, this work underlines the key role of CD4+ T cells in the production of IFN-gamma by immune goat PBMC. It also describes, for the first time in ruminants, E. ruminantium-specific CD8+ effector T cells. Since CD4+ and CD8+ T cells collectively contribute to the production of IFN-gamma in most vaccinated animals, and since these responses are associated with protection, it may be that a recombinant vaccine will need to incorporate E. ruminantium antigens capable of driving both responses.
Assuntos
Vacinas Bacterianas/farmacologia , Ehrlichia ruminantium/imunologia , Doenças das Cabras/imunologia , Doenças das Cabras/prevenção & controle , Cabras/imunologia , Hidropericárdio/imunologia , Hidropericárdio/prevenção & controle , Interferon gama/biossíntese , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Isoanticorpos/administração & dosagem , CinéticaRESUMO
A better understanding of how T1 vaccination confers immunity would facilitate the rational design of improved vaccines against contagious bovine pleuropneumonia (CBPP). We show here that mycoplasmas-induced recall proliferation and IFN-γ responses are detected in cattle that received multiple shots of T1 vaccines. These anamnestic responses were under the strict control of CD4(+) T lymphocytes. Moreover, CD62L expression indicated that both CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes are elicited in these animals. Comparative analysis with data from cattle that completely recovered from CBPP infection revealed similar anamnestic T-cell responses albeit at a lower magnitude for T1-vaccinated animals, particularly in the Tcm compartment. In conclusion, we discuss how our current understanding of T-cell responses will contribute to ongoing efforts for the improvement of future CBPP vaccines.
Assuntos
Vacinas Bacterianas/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Doenças dos Bovinos/prevenção & controle , Memória Imunológica , Pleuropneumonia Contagiosa/prevenção & controle , Animais , Vacinas Bacterianas/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Pleuropneumonia Contagiosa/imunologiaRESUMO
Central memory T cells (Tcm) have not previously been characterized in cattle and any other ruminant species. Here we described two phenotypically and functionally different subsets of pathogen-specific memory CD4(+) T cells in cattle that survived infection with Mycoplasma mycoides subsp. mycoides small colony (MmmSC). The first subset is CD45RO(+)CD45R(-)CD62L(-) and comprises two thirds of IFN-gamma producing CD4(+) T cells after MmmSC recall stimulation. The second is CD45RO(+)CD45R(-)CD62L(+) and represents the majority of proliferating CD4(+) T cells after 7 days of stimulation. Cell sorting experiments confirmed that both CD4(+)CD62L(+) and CD4(+)CD62L(-) subsets are present in vivo and proliferate independently in recall responses to MmmSC. In addition, MmmSC stimulation strongly decreased CCR7 and increased CCR5 transcripts levels in CD4(+)CD62L(-) cells whereas CD4(+)CD62L(+) were only slightly affected. High levels of recall proliferation but low IFN-gamma production, together with the capacity to preferentially migrate through the lymph nodes (i.e., expression of CD62L and CCR7), are characteristics of Tcm, in humans and mice. Tcm are associated with long-term protective immunity and a privileged target for vaccine development. Our results demonstrate the existence of Tcm in cattle and suggest that CD62L may serve as a marker to monitor Tcm in infections and vaccine development studies in ruminant.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Bovinos/imunologia , Memória Imunológica , Selectina L/imunologia , Mycoplasma mycoides/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Proliferação de Células , Células Cultivadas , Interferon gama/biossíntese , Interferon gama/imunologiaRESUMO
Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Regulação Bacteriana da Expressão Gênica/imunologia , Lipoproteínas/metabolismo , Mycoplasma mycoides/metabolismo , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Proliferação de Células , Lipoproteínas/genética , Linfonodos/citologia , Pleuropneumonia Contagiosa/imunologia , Pleuropneumonia Contagiosa/microbiologiaRESUMO
Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Doenças dos Bovinos/imunologia , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/imunologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/microbiologia , Proliferação de Células , Mapeamento de Epitopos , Memória Imunológica , Interferon gama/metabolismo , Mycoplasma mycoides/genética , Mycoplasma mycoides/crescimento & desenvolvimento , Pleuropneumonia Contagiosa/microbiologiaRESUMO
A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.
Assuntos
Doenças dos Bovinos/prevenção & controle , Imunidade Celular , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/prevenção & controle , Vacinação/veterinária , Animais , Técnicas de Tipagem Bacteriana/veterinária , Bovinos , Citocinas/biossíntese , Feminino , Citometria de Fluxo/veterinária , Linfonodos/citologia , Linfonodos/imunologia , MasculinoRESUMO
Mass production of Ehrlichia ruminantium variants from different regions of sub-Saharan Africa is one of the difficulties that must be overcome in producing a heartwater vaccine. Vaccine productivity can be limited by endogenous induction of interferon (IFN), which inhibits the propagation of Ehrlichia ruminantium (ER) in cell culture. Different kinds of endothelial cells, in which ER multiply efficiently, could be grown in a scalable way in VueLife Teflon bags on Cytodex 3 microcarriers where bead-to-bead transfer of cells occurs. The digital holographic microscope designed at the Université Libre de Bruxelles allows detection of the most appropriate time to harvest intracellular microorganisms for vaccine production.
Assuntos
Vacinas Bacterianas/uso terapêutico , Ehrlichiose/prevenção & controle , Interferons/metabolismo , Animais , Bovinos , Humanos , Células HíbridasRESUMO
BACKGROUND: Flow cytometry (FCM) provides an alternative to radioactive methods for the analysis of T-cell responses. However, a comparative study of common FCM assays in an outbred ruminant model is lacking, which motivated this work. METHODS: Goats immunized with the obligate intracellular bacterium Cowdria ruminantium, inactivated and emulsified in oil-based adjuvants, were used as a model to study T-cell recall responses in vitro. FCM-based methods to measure Cowdria-induced lymphoblastogenesis, DNA synthesis, and interleukin-2 receptor (IL-2R) expression by T-cell subsets were compared. RESULTS: IL-2R expression was the most sensitive and reliable method provided that the number of molecules per cell was analyzed and not simply the percentage of positive cells of a given phenotype. Despite high background due to adjuvant and low proliferation, this method could detect antigen-specific activation of immune CD4(+) and CD8(+) T cells. CONCLUSIONS: FCM-based measurement of lymphoblastogenesis and DNA synthesis are not the most appropriate methods to analyze T-lymphocyte activation during vaccination of outbred animals. On several occasions, analysis of IL-2R expression was the only assay capable of discriminating between vaccinated and naive animals in this model.