Pablo A. González

Salmonella Escape from Antigen Presentation Can Be Overcome by Targeting Bacteria to Fcγ Receptors on Dendritic Cells

Dendritic cells (DCs) are professional APCs with the unique ability to activate naive T cells, which is required for initiation of the adaptive immune response against pathogens. Therefore, interfering with DC function would be advantageous for pathogen survival and dissemination. In this study we provide evidence suggesting that Salmonella enterica serovar typhimurium, the causative agent of typhoid disease in the mouse, interferes with DC function. Our results indicate that by avoiding lysosomal degradation, S. typhimurium impairs the ability of DCs to present bacterial Ags on MHC class I and II molecules to T cells. This process could correspond to a novel mechanism developed by this pathogen to evade adaptive immunity. In contrast, when S. typhimurium is targeted to FcγRs on DCs by coating bacteria with Salmonella-specific IgG, bacterial Ags are efficiently processed and presented on MHC class I and class II molecules. This enhanced Ag presentation leads to a robust activation of bacteria-specific T cells. Laser confocal microscopy experiments show that virulent S. typhimurium is rerouted to the lysosomal degradation pathway of DCs when internalized through FcγR. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows degradation signs only when coated with IgG and captured by FcγRs on DCs. Therefore, our data support a potential role for bacteria-specific IgG on the augmentation of Ag processing and presentation by DCs to T cells during the immune response against intracellular bacteria.

Respiratory syncytial virus impairs T cell activation by preventing synapse assembly with dendritic cells

Respiratory syncytial virus (RSV) infection is one of the leading causes of infant hospitalization and a major health and economic burden worldwide. Infection with this virus induces an exacerbated innate proinflammatory immune response characterized by abundant immune cell infiltration into the airways and lung tissue damage. RSV also impairs the induction of an adequate adaptive T cell immune response, which favors virus pathogenesis. Unfortunately, to date there are no efficient vaccines against this virus. Recent in vitro and in vivo studies suggest that RSV infection can prevent T cell activation, a phenomenon attributed in part to cytokines and chemokines secreted by RSV-infected cells. Efficient immunity against viruses is promoted by dendritic cells (DCs), professional antigen-presenting cells, that prime antigen-specific helper and cytotoxic T cells. Therefore, it would be to the advantage of RSV to impair DC function and prevent the induction of T cell immunity. Here, we show that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells. Inhibition of T cell activation by RSV was observed independently of the type of TCR ligand on the DC surface and applied to cognate-, allo-, and superantigen stimulation. As a result of exposure to RSV-infected DCs, T cells became unresponsive to subsequent TCR engagement. RSV-mediated impairment in T cell activation required DC-T cell contact and involved inhibition of immunological synapse assembly among these cells. Our data suggest that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance.

Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells

ABSTRACT Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime naïve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.

Host immunity during RSV pathogenesis

Infection by respiratory syncytial virus (RSV) is the leading cause of childhood hospitalization as well as a major health and economic burden worldwide. Unfortunately, RSV infection provides only limited immune protection to reinfection, mostly due to inadequate immunological memory, which leads to an exacerbated inflammatory response in the respiratory tract promoting airway damage during virus clearance. This exacerbated and inefficient immune-inflammatory response triggered by RSV, has often been attributed to the induction of a Th2-biased immunity specific for some of the RSV antigens. These features of RSV infection suggest that the virus might possess molecular mechanisms to enhance allergic-type immunity in the host in order to prevent clearance by cytotoxic T cells and ensure survival and dissemination to other hosts. In this review, we discuss recent findings that contribute to explain the components of the innate and adaptive immune response that are involved in RSV-mediated disease exacerbation. Further, the virulence mechanisms used by RSV to avoid activation of protective immune responses are described.

Maternal Hypothyroxinemia Impairs Spatial Learning and Synaptic Nature and Function in the Offspring

Neurological deficits in the offspring caused by human maternal hypothyroxinemia are thought to be irreversible. To understand the mechanism responsible for these neurological alterations, we induced maternal hypothyroxinemia in pregnant rats. Behavior and synapse function were evaluated in the offspring of thyroid hormone-deficient rats. Our data indicate that, when compared with controls, hypothyroxinemic mothers bear litters that, in adulthood, show prolonged latencies during the learning process in the water maze test. Impaired learning capacity caused by hypothyroxinemia was consistent with cellular and molecular alterations, including: 1) lack of increase of phosphorylated c-fos on the second day of the water maze test; 2) impaired induction of long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area 1 of the hippocampus Ammons horn stratum radiatum, despite normal responses for input/output experiments; 3) increase of postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartic acid receptor subunit 1, and tyrosine receptor kinase B levels in brain extracts; and 4) significant increase of PSD-95 at the PSDs and failure of this molecule to colocalize with N-methyl-D-aspartic acid receptor subunit 1, as it was shown by control rats. Our findings suggest that maternal hypothyroxinemia is a harmful condition for the offspring that can affect key molecular components for synaptic function and spatial learning.

Protective T cell immunity against respiratory syncytial virus is efficiently induced by recombinant BCG

Respiratory syncytial virus (RSV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, because of an inefficient immunological memory, RSV infection provides limited immune protection against reinfection. Furthermore, RSV can induce an inadequate Th2-type immune response that causes severe respiratory tract inflammation and obstruction. It is thought that effective RSV clearance requires the induction of balanced Th1-type immunity, involving the activation of IFN-γ-secreting cytotoxic T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette–Guérin (BCG), which has been used in newborns for decades in several countries as a tuberculosis vaccine. Here, we show that immunization with recombinant BCG strains expressing RSV antigens promotes protective Th1-type immunity against RSV in mice. Activation of RSV-specific T cells producing IFN-γ and IL-2 was efficiently obtained after immunization with recombinant BCG. This type of T cell immunity was protective against RSV challenge and caused a significant reduction of inflammatory cell infiltration in the airways. Furthermore, mice immunized with recombinant BCG showed no weight loss and reduced lung viral loads. These data strongly support recombinant BCG as an efficient vaccine against RSV because of its capacity to promote protective Th1 immunity.

Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis

ABSTRACT Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. IMPORTANCE This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame. This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.

Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease

Subunit vaccines comprised of glycoprotein D (gD-2) failed to prevent HSV-2 highlighting need for novel strategies. To test the hypothesis that deletion of gD-2 unmasks protective antigens, we evaluated the efficacy and safety of an HSV-2 virus deleted in gD-2 and complemented allowing a single round of replication on cells expressing HSV-1 gD (ΔgD−/+gD−1). Subcutaneous immunization of C57BL/6 or BALB/c mice with ΔgD−/+gD1 provided 100% protection against lethal intravaginal or skin challenges and prevented latency. ΔgD−/+gD1 elicited no disease in SCID mice, whereas 1000-fold lower doses of wild-type virus were lethal. HSV-specific antibodies were detected in serum (titer 1:800,000) following immunization and in vaginal washes after intravaginal challenge. The antibodies elicited cell-mediated cytotoxicity, but little neutralizing activity. Passive transfer of immune serum completely protected wild-type, but not Fcγ-receptor or neonatal Fc-receptor knock-out mice. These studies demonstrate that non-neutralizing Fc-mediated humoral responses confer protection and support advancement of this attenuated vaccine. DOI: http://dx.doi.org/10.7554/eLife.06054.001

Modulation of nuclear factor‐κB activity can influence the susceptibility to systemic lupus erythematosus

Autoimmune diseases, such as systemic lupus erythematosus (SLE), result from deficiencies in self‐antigen tolerance processes, which require regulated dendritic cell (DC) function. In this study we evaluated the phenotype of DCs during the onset of SLE in a mouse model, in which deletion of the inhibitory receptor FcγRIIb leads to the production of anti‐nuclear antibodies and glomerulonephritis. Splenic DCs from FcγRIIb‐deficient mice suffering from SLE showed increased expression of co‐stimulatory molecules. Furthermore, diseased mice showed an altered function of the nuclear factor‐κB (NF‐κB) transcription factor, which is involved in DC maturation. Compared with healthy animals, expression of the inhibitory molecule IκB‐α was significantly decreased in mice suffering from SLE. Consistently, pharmacological inhibition of NF‐κB activity in FcγRIIb‐deficient mice led to reduced susceptibility to SLE and prevented symptoms, such as anti‐nuclear antibodies and kidney damage. Our data suggest that the occurrence of SLE is significantly influenced by alterations of NF‐κB function, which can be considered as a new therapeutic target for this disease.

HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression

HSV triggers intracellular calcium release to promote viral entry. We hypothesized that Akt signaling induces the calcium responses and contributes to HSV entry. Exposure of human cervical and primary genital tract epithelial, neuronal, or keratinocyte cells to HSV serotype 2 resulted in rapid phosphorylation of Akt. Silencing of Akt with small interfering RNA prevented the calcium responses, blocked viral entry, and inhibited plaque formation by 90% compared to control siRNA. Susceptibility to infection was partially restored if Akt was reintroduced into silenced cells with an Akt‐expressing plasmid. HSV‐2 variants deleted in glycoproteins B or D failed to induce Akt phosphorylation, and coimmunoprecipitation studies indicated that Akt interacts with glycoprotein B. Cell‐surface expression of Akt was rapidly induced in response to HSV exposure. Miltefosine (50 μM), a licensed drug that blocks Akt phosphorylation, inhibited HSV‐induced calcium release, viral entry, and plaque formation following infection with acyclovir‐sensitive and resistant clinical isolates. Miltefosine blocked amplification of HSV from explanted ganglia to epithelial cells; viral yields were significantly less in miltefosine compared to control‐treated cocultures (P<0.01). Together, these findings identify a novel role for Akt in viral entry, link Akt and calcium signaling, and suggest a new target for HSV treatment and suppression.— Cheshenko, N., Trepanier, J. B., Stefanidou, M., Buckley, N., Gonzalez, P., Jacobs, W., and Herold, B. C. HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression. FASEB J. 27, 2584–2599 (2013). www.fasebj.org