Adrianus W. M. van der Velden

Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow–resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell–mediated recall responses in the bone marrow.

Salmonella Rapidly Kill Dendritic Cells via a Caspase-1- Dependent Mechanism

Dendritic cells provide a critical link between innate and acquired immunity. In this study, we demonstrate that the bacterial pathogen Salmonella enterica serovar Typhimurium can efficiently kill these professional phagocytes via a mechanism that is dependent on sipB and the Salmonella pathogenicity island 1-encoded type III protein secretion system. Rapid phosphatidylserine redistribution, caspase activation, and loss of plasma membrane integrity were characteristic of dendritic cells infected with wild-type Salmonella, but not sipB mutant bacteria. Caspase-1 was particularly important in this process because Salmonella-induced dendritic cell death was dramatically reduced in the presence of a caspase-1-specific inhibitor. Furthermore, dendritic cells obtained from caspase-1-deficient mice, but not heterozygous littermate control mice, were resistant to Salmonella-induced cytotoxicity. We hypothesize that Salmonella have evolved the ability to selectively kill professional APCs to combat, exploit, or evade immune defense mechanisms.

Down-Modulation of TCR Expression by Salmonella enterica serovar Typhimurium

T cell-mediated adaptive immunity is required to help clear infection with the facultative intracellular bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), yet development of T cell-mediated adaptive immunity to S. Typhimurium has been described as slow and inefficient. A key step in inducing T cell-mediated adaptive immunity is T cell priming; the activation, proliferation, and differentiation of naive T cells following initial encounter with Ag. We previously demonstrated that S. Typhimurium had a direct inhibitory effect on naive T cells from mouse, blocking their proliferation. In this study, we show that S. Typhimurium down-modulates expression of the TCR β-chain, a molecule that is essential for Ag recognition and T cell function. Specifically, we demonstrate that reduced amounts of surface and intracellular TCR-β protein and decreased levels of tcrβ transcript are expressed by T cells cultured in the presence of S. Typhimurium. We further show that the down-modulation of TCR-β expression requires contact between S. Typhimurium and the T cells and that once contact occurs, a factor capable of reducing TCR-β expression is secreted. These results provide new insight into the mechanism by which S. Typhimurium may inhibit T cell priming and avoid clearance by the adaptive immune system.

Recruitment of Rab27a to Phagosomes Controls Microbial Antigen Cross-Presentation by Dendritic Cells

ABSTRACT Polyreactive immunoglobulins (Ig) and complement components are present in tissues and blood of healthy individuals. They facilitate pathogen uptake and inactivation in lysosomes of phagocytes and thereby provide rapid protection against infection. Dendritic cells (DCs) are phagocytes that can acquire peptides from phagocytosed antigen to elicit cytotoxic immune responses by CD8+ T lymphocytes. The mechanisms that select peptides for cross-presentation are not fully resolved. Here we investigated the role of polyreactive Ig and complement in directing phagosomal antigen processing for cross-presentation. Phagocytosis facilitated by serum opsonization required the presence of Ig for effective antigen cross-presentation of microbe-derived antigen. The presence of complement C3 in serum promoted phagocytosis, yet phagosomes were defective in antigen degradation. The small GTPase Rab27a was recently implicated in antigen cross-presentation and was rapidly recruited to phagosomes only when Ig was present. Our data suggest that prebinding of antigen by polyreactive Ig potentiates the efficiency of antigen cross-presentation to CD8+ T cells through recruitment of Rab27a.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l‐asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative l‐asparaginase exhibits l‐asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that l‐asparagine is a nutrient important for T cell activation and that l‐asparagine deprivation, such as that mediated by the Salmonella Typhimurium l‐asparaginase, causes suppression of activation‐induced mammalian target of rapamycin signaling, autophagy, Myc expression, and l‐lactate secretion. We also show that l‐asparagine deprivation mediated by the Salmonella Typhimurium l‐asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l‐asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation‐induced metabolic reprogramming.

Phenotypic, Morphological, and Functional Heterogeneity of Splenic Immature Myeloid Cells in the Host Response to Tularemia

ABSTRACT Recent studies have linked accumulation of the Gr-1+ CD11b+ cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1+ CD11b+ cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1+ CD11b+ cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1+ CD11b+ cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11b+ Ly6Chi Ly6G− cells but not granulocytic, CD11b+ Ly6Cint Ly6G+ cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11b+ Ly6Chi Ly6G− cells were able to suppress the proliferation of T cells, the large presence of Gr-1+ CD11b+ cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia.

CD11b+ Ly6Chi Ly6G− Immature Myeloid Cells Recruited in Response to Salmonella enterica Serovar Typhimurium Infection Exhibit Protective and Immunosuppressive Properties

ABSTRACT Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b+ Ly6Chi Ly6G− and CD11b+ Ly6Cint Ly6G+ cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Recruitment of CD11b+ Ly6Chi Ly6G− but not CD11b+ Ly6Cint Ly6G+ cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b+ Ly6Chi Ly6G− cells exhibited a mononuclear morphology, whereas the CD11b+ Ly6Cint Ly6G+ cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b+ Ly6Chi Ly6G− cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro. However, significant proliferation of T cells was observed only when the ability of the CD11b+ Ly6Chi Ly6G− cells to produce nitric oxide was blocked. CD11b+ Ly6Chi Ly6G− cells recruited in response to S. Typhimurium infection could also present antigen to T cells in vivo, but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b+ Ly6Chi Ly6G− immature myeloid cells recruited in response to S. Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.

Visible Fluorescence Detection of Type III Protein Secretion from Bacterial Pathogens

Type III protein secretion is essential for many gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b+ F4/80+, CD11b+ Ly6Cint Ly6G+ and CD11b+ Ly6Chi Ly6G− cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.

Murine Gammaherpesvirus 68 Pathogenesis Is Independent of Caspase-1 and Caspase-11 in Mice and Impairs Interleukin-1β Production upon Extrinsic Stimulation in Culture

ABSTRACT Gammaherpesviruses establish lifelong infections that are associated with the development of cancer. These viruses subvert many aspects of the innate and adaptive immune response of the host. The inflammasome, a macromolecular protein complex that controls inflammatory responses to intracellular danger signals generated by pathogens, is both activated and subverted during human gammaherpesvirus infection in culture. The impact of the inflammasome response on gammaherpesvirus replication and latency in vivo is not known. Caspase-1 is the inflammasome effector protease that cleaves the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. We infected caspase-1-deficient mice with murine gammaherpesvirus 68 (MHV68) and observed no impact on acute replication in the lung or latency and reactivation from latency in the spleen. This led us to examine the effect of viral infection on inflammasome responses in bone marrow-derived macrophages. We determined that infection of macrophages with MHV68 led to a robust interferon response but failed to activate caspase-1 or induce the secretion of IL-1β. In addition, MHV68 infection led to a reduction in IL-1β production after extrinsic lipopolysaccharide stimulation or upon coinfection with Salmonella enterica serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1β transcript level and was independent of the RTA, the viral lytic replication and transcription activator. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1β production during the initial stages of infection. IMPORTANCE Gammaherpesviruses persist for the lifetime of the host. To accomplish this, they must evade recognition and clearance by the immune system. The inflammasome consists of proteins that detect foreign molecules in the cell and respond by secreting proinflammatory signaling proteins that recruit immune cells to clear the infection. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1, a critical inflammasome component. This led us to investigate whether the virus actively impairs the inflammasome response. We found that the inflammasome was not activated upon macrophage cell infection with murine gammaherpesvirus 68. Infection also prevented the host cell inflammasome response to other pathogen-associated molecular patterns, indicated by reduced production of the proinflammatory cytokine IL-1β upon bacterial coinfection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1β in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal.