Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4(+) T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4(+) T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose- 1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α 2-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.Fil: Santangelo, María de la Paz. State University of Colorado - Fort Collins; Estados Unidos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gest, Petra M.. State University of Colorado - Fort Collins; Estados UnidosFil: Guerin, Marcelo E.. Universidad del País Vasco; EspañaFil: Coinçon, Mathieu. University of Montreal; CanadáFil: Pham, Ha. State University of Colorado - Fort Collins; Estados UnidosFil: Ryan, Gavin. State University of Colorado - Fort Collins; Estados UnidosFil: Puckett, Susan E.. Cornell University; Estados UnidosFil: Spencer, John S.. State University of Colorado - Fort Collins; Estados UnidosFil: Gonzalez Juarrero, Mercedes. State University of Colorado - Fort Collins; Estados UnidosFil: Daher, Racha. Universite de Paris XI. Institut de Chimie Moléculaire et des Matériaux d'Orsay; FranciaFil: Lenaerts, Anne J.. State University of Colorado - Fort Collins; Estados UnidosFil: Schnappinger, Dirk. Cornell University; Estados UnidosFil: Therisod, Michel. Universite de Paris XI. Institut de Chimie Moléculaire et des Matériaux d'Orsay; FranciaFil: Ehrt, Sabine. Cornell University; Estados UnidosFil: Sygusch, Jurgen. University of Montreal; CanadáFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados Unido

Dynamics of interferon-gamma release assay and cytokine profiles in blood and respiratory tract specimens from mice with tuberculosis and the effect of therapy

There are limitations on diagnostic methods to differentiate between active and latent tuberculosis (TB), and the prediction of latent progression to TB disease is yet complex. Traditionally, tuberculosis-specific host immune response was visualized using the tuberculin skin test. Nowadays, IFN-γ release assays (IGRA) provide a more specific and sensitive tool, by which exposure to Mtb could be determined. However, the merit of IGRA aids in diagnosing active TB is yet unclear. We adapted IGRA for use in mice, and quantifying bead-based flow cytometry techniques were used to assess cytokine profiles during the course of untreated infection and to investigate the value of IGRA and cytokines as biomarkers for therapy response. High variability of IGRA results during progression of active TB infection related to various phases of infection was obtained. However, a significant decrease in IGRA results and in levels of IFN-γ, IL-17, IP-10 or MIG was observed and appeared to be associated with successful therapy. This outcome does not support the value of IGRA to accurately diagnose active TB or to monitor infection progression. However, IGRA proved to be a useful biomarker to monitor therapy success. In addition, different cytokines might serve as biomarkers

Paracoccidioides brasilinsis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in the Lung-Draining Lymph Nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response

Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression rofiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

<p>Abstract</p> <p>Background</p> <p>Immunization of BALB/c mice with a recombinant adenovirus expressing <it>Mycobacterium tuberculosis </it>(<it>M. tuberculosis</it>) antigen 85A (Ad85A) protects against aerosol challenge with <it>M. tuberculosis </it>only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of <it>M. tuberculosis </it>growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.</p> <p>Method</p> <p>Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.</p> <p>Results</p> <p>CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene <it>Cxcr6</it>, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.</p> <p>Conclusions</p> <p>Our microarray analysis represents the first <it>ex vivo </it>study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of <it>M. tuberculosis </it>growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.</p

Resident Cardiac Immune Cells and Expression of the Ectonucleotidase Enzymes CD39 and CD73 after Ischemic Injury

BACKGROUND: The ectoenzymes CD39 and CD73 are expressed by a broad range of immune cells and promote the extracellular degradation of nucleotides to anti-inflammatory adenosine. This study explored the abundance of CD73 and CD39 on circulating and resident cardiac leukocytes and coronary endothelial cells under control conditions and in response to inflammation following myocardial ischemia and reperfusion (I/R). METHODS AND RESULTS: A method was elaborated to permit FACS analysis of non-myocardial cells (resident leukocytes, coronary endothelium and CD31(-) CD45(-) cells) of the unstressed heart. Under control conditions the murine heart contained 2.3 × 10(3) resident leukocytes/mg tissue, the most prominent fraction being antigen-presenting mononuclear cells (CD11b(+) CD11c(+) F4/80(+) MHCII(+)) followed by B-cells, monocytes and T-cells. CD73 was highly expressed on circulating and resident cardiac lymphoid cells with little expression on myeloid cells, while the opposite was true for CD39. Cardiomyocytes and erythrocytes do not measurably express CD39/CD73 and CD39 dominates on coronary endothelium. Three days after I/R, CD73 was significantly upregulated on invading granulocytes (2.8-fold) and T-cells (1.5-fold). Compared with coronary endothelial cells, CD73 associated with leukocytes comprised 2/3 of the total cardiac CD73. CONCLUSION: Our study suggests that extracellular ATP formed during I/R is preferentially degraded by CD39 present on myeloid cells, while the formation of immunosuppressive adenosine is mainly catalysed by CD73 present on granulocytes and lymphoid cells. Upregulated CD73 on granulocytes and T-cells infiltrating the injured heart is consistent with the existence of an autocrine adenosinergic loop which may promote the healing process

CD8+ DC, but Not CD8−DC, Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

Tuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8alpha+ DC isolated from infected mice (iCD8+ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8+ DC recipients than sham-treated control mice. The adoptive transfer of the CD8alpha(-)DC from the infected mice (iCD8(-) DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8(-) DC produced significantly higher levels of IL-10 than iCD8+ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.The data indicate that in vivo BCG-primed CD8+ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases

The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNα, TNFα and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60

The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNα, TNFα, IL12p40, TGFβ and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNα, TNFα and IL12p40 expression was found in infection with NHV, in which expression of TGFβ was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNα and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNα, TNFα and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection

Interrelationship between Dendritic Cell Trafficking and Francisella tularensis Dissemination following Airway Infection

Francisella tularensis, the etiological agent of the inhalation tularemia, multiplies in a variety of cultured mammalian cells. Nevertheless, evidence for its in vivo intracellular residence is less conclusive. Dendritic cells (DC) that are adapted for engulfing bacteria and migration towards lymphatic organs could serve as potential targets for bacterial residence and trafficking. Here, we focus on the in vivo interactions of F. tularensis with DC following airway infection of mice. Lethal airway infection of mice with the live vaccine strain (LVS) results in trafficking of a CD11bhigh/CD11cmed/autofluorescencelow DC subset from the respiratory tract to the draining mediastinal lymph node (MdLN). Simultaneously, a rapid, massive bacterial colonization of the MdLN occurs, characterized by large bacterial foci formation. Analysis of bacteria in the MdLN revealed a major population of extracellular bacteria, which co-exists with a substantial fraction of intracellular bacteria. The intracellular bacteria are viable and reside in cells sorted for DC marker expression. Moreover, in vivo vital staining experiments indicate that most of these intracellular bacteria (∼75%) reside in cells that have migrated from the airways to the MdLN after infection. The correlation between DC and bacteria accumulation in the MdLN was further demonstrated by manipulating DC migration to the MdLN through two independent pathways. Impairment of DC migration to the MdLN, either by a sphingosine-1-phosphate receptor agonist (FTY720) or by the D prostanoid receptor 1 agonist (BW245C), resulted in reduced bacterial colonization of MdLN. Moreover, BW245C treatment delayed the onset of morbidity and the time to death of the infected mice. Taken together, these results suggest that DC can serve as an inhabitation niche for F. tularensis in the early stages of infection, and that DC trafficking plays a role in pathogen dissemination. This underscores the therapeutic potential of DC migration impairing drugs in tularemia treatment