Modeling Infectious Disease in Mice: Co-Adaptation and the Role of Host-Specific IFNγ Responses

Molecular and Cellular Biolog

H2-M3–restricted CD8+ T cells are not required for MHC class Ib–restricted immunity against Listeria monocytogenes

Studies using major histocompatibility complex (MHC)-Ia–deficient mice have shown that MHC-Ib–restricted CD8+ T cells can clear infections caused by intracellular pathogens such as Listeria monocytogenes. M3-restricted CD8+ T cells, which recognize short hydrophobic N-formylated peptides, appear to comprise a substantial portion of the MHC-Ib–restricted T cell response in the mouse model of L. monocytogenes infection. In this study, we isolated formyltransferase (fmt) mutant strains of L. monocytogenes that lacked the ability to add formyl groups to nascent polypeptides. These fmt mutant Listeria strains did not produce antigens that could be recognized by M3-restricted T cells. We showed that immunization of MHC-Ia–deficient mice with fmt mutant Listeria resulted in stimulation of a protective memory response that cleared subsequent challenge with wild-type L. monocytogenes, despite the fact that M3-restricted CD8+ T cells did not proliferate in these mice. These data suggest that M3-restricted T cells are not required for protection against L. monocytogenes and underscore the importance of searching for new antigen-presenting molecules among the large MHC-Ib family of proteins

IFNγ Inhibits the Cytosolic Replication of Shigella flexneri via the Cytoplasmic RNA Sensor RIG-I

The activation of host cells by interferon gamma (IFNγ) is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs) and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1), an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR) retanoic acid-inducible gene I (RIG-I), a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS)—but not cytosolic Nod-like receptors (NLRs)—are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5′-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing this bacterium. Additionally, these findings expand our understanding of how IFNγ recognizes, and ultimately restricts, bacterial pathogens within host cells

H2-M3-Restricted CD8\u3csup\u3e+\u3c/sup\u3e T Cells are not Required for MHC Class Ib-Restricted Immunity Against \u3cem\u3eListeria monocytogenes\u3c/em\u3e

Studies using major histocompatibility complex (MHC)-Ia–deficient mice have shown that MHC-Ib–restricted CD8+ T cells can clear infections caused by intracellular pathogens such as Listeria monocytogenes. M3-restricted CD8+ T cells, which recognize short hydrophobic N-formylated peptides, appear to comprise a substantial portion of the MHC-Ib–restricted T cell response in the mouse model of L. monocytogenes infection. In this study, we isolated formyltransferase (fmt) mutant strains of L. monocytogenes that lacked the ability to add formyl groups to nascent polypeptides. These fmt mutant Listeria strains did not produce antigens that could be recognized by M3-restricted T cells. We showed that immunization of MHC-Ia–deficient mice with fmt mutant Listeria resulted in stimulation of a protective memory response that cleared subsequent challenge with wild-type L. monocytogenes, despite the fact that M3-restricted CD8+ T cells did not proliferate in these mice. These data suggest that M3-restricted T cells are not required for protection against L. monocytogenes and underscore the importance of searching for new antigen-presenting molecules among the large MHC-Ib family of proteins

The Intestinal Flora Is Required to Support Antibody Responses to Systemic Immunization in Infant and Germ Free Mice

The presence of a complex and diverse intestinal flora is functionally important for regulating intestinal mucosal immune responses. However, the extent to which a balanced intestinal flora regulates systemic immune responses is still being defined. In order to specifically examine whether the acquisition of a less complex flora influences responses to immunization in the pre-weaning stages of life, we utilize a model in which infant mice acquire an intestinal flora from their mothers that has been altered by broad-spectrum antibiotics. In this model, pregnant dams are treated with a cocktail of antibiotics that alters both the density and microbial diversity of the intestinal flora. After challenge with a subcutaneous immunization, the antibiotic altered flora infant mice have lower antigen specific antibody titers compared to control age-matched mice. In a second model, we examined germ free (GF) mice to analyze how the complete lack of flora influences the ability to mount normal antibody responses following subcutaneous immunization. GF mice do not respond well to immunization and introduction of a normal flora into GF mice restores the capacity of these mice to respond. These results indicate that a gastrointestinal flora reduced in density and complexity at critical time points during development adversely impacts immune responses to systemic antigens

CD8+ T Cells Restrict Yersinia pseudotuberculosis Infection: Bypass of Anti-Phagocytosis by Targeting Antigen-Presenting Cells

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8+ T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8+ T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated TH1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8+ T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis–associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8+ T cells are critical for hosts to overcome the anti-phagocytic action of Yops.Molecular and Cellular Biolog

Anthrax toxinmediated delivery of a cytotoxic T-cell epitope in

ABSTRACT The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO 91-99 ) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO 91-99 fusion protein to stimulate a CTL response against the epitope in BALB͞c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO 91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications. All viruses and some bacterial and protozoan pathogens have evolved the ability to survive and replicate within mammalian cells. Immune recognition of these cytoplasmic pathogens depends upon the cell surface display of peptide antigens derived from pathogen-associated proteins. These peptides are presented in complexes with host class I molecules encoded by the major histocompatibility complex (MHC-I), and cytotoxic T lymphocytes (CTL) are activated following recognition of the foreign peptide in complex with MHC-I (1). Activated CTL lyse the infected cell, secrete cytokines, and then proliferate and differentiate (2, 3). Each of these steps plays an important role in clearing the host of the pathogen. Lysis of the target cell deprives the organism of its replicative niche and exposes the pathogen to elements of the humoral immune system. Secretion of cytokines has many effects, including enhancement of local immune responses. Proliferation of the CTL clone results in expansion of a set of reactive CTL to effect clearance of the pathogen from other infected cells, whereas differentiation provides a set of long-lived memory cells available to respond more quickly and effectively to subsequent challenge. Vaccines that prime these memory cells provide protection of the host upon reexposure to the pathogen. For a vaccine to mimic infection by cytoplasmic pathogens, it must introduce the target antigen(s) into the cytosol of host cells in vivo. This has been accomplished by expressing heterologous antigens by live viral or bacterial vectors, by using adjuvants, or by delivering DNA expression vectors (DNA vaccines) (4-7). Herein we describe a strategy for introducing CTL epitopes into the cytosol of host cells, in vivo, using delivery components of an intracellularly acting toxin, anthrax toxin. Anthrax toxin is composed of three proteins that act in binary combinations to elicit two toxic effects, lethality and edema (8). Lethal factor (LF) and edema factor (EF) are intracellularly acting proteins, both of which require protective antigen (PA) for their translocation to the cytosol of eukaryotic cells. Initially, LF and EF bind competitively to proteolytically activated PA (PA 63 ) at the cell surface. The protein complex is endocytosed, and LF and EF are translocated to the cytosol following endosomal acidification. There, EF expresses its adenylate cyclase activity, elevating cAMP levels, and LF expresses its as yet undefined activity, inducing lethal overproduction of certain cytokines in its target cells, macrophages (9, 10). The amino-terminal domain of LF (LFn; 255 residues) has been found to contain the information necessary for binding to PA and mediating translocation. The domain alone lacks lethal potential that depends on the putatively enzymatic carboxyl-terminal moiety (11). Recent results have demonstrated that genetically fusing LFn to heterologous polypeptides enables them to be delivered to the cytosol of cultured mammalian cells in the presence of PA To test the potential of the anthrax toxin system to mediate presentation of peptides in complex with MHC-I, we chose to fuse LFn to a CTL epitope from Listeria monocytogenes. L. monocytogenes is a facultative intracellular bacterial pathogen that survives within the cytosol of macrophages. After its entry into the cell by phagocytosis, the bacterium produces listeriolysin O (LLO), which lyses the phagosomal membrane and allows the bacterium to escape to the cytosol. LLO within the cytosol is proteolytically processed, generating short peptides, some of which are presented at the cell surface in complex with MHC-I. Processing of LLO results in the presentation of a nonameric peptide, LLO [91][92][93][94][95][96][97][98][99] MATERIALS AND METHODS Peptide Synthesis. The LLO 91-99 peptide GYKDGNEYI was synthesized on an Applied Biosystems model 432A peptide synthesizer

The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation

A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ producing CD4 T-cells. By contrast, mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T-cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAP) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b+CD103− dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b−CD103+ DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T-cells, but only mucosal vaccination induced effector T-cells that rapidly seeded uterine mucosa with resident memory T-cells (TRM). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T-cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T-cell subsets with distinct migratory properties