Mary Jo Wick

Bacterial strategies for overcoming host innate and adaptive immune responses

In higher organisms a variety of host defense mechanisms control the resident microflora and, in most cases, effectively prevent invasive microbial disease. However, it appears that microbial organisms have coevolved with their hosts to overcome protective host barriers and, in selected cases, actually take advantage of innate host responses. Many microbial pathogens avoid host recognition or dampen the subsequent immune activation through sophisticated interactions with host responses, but some pathogens benefit from the stimulation of inflammatory reactions. This review will describe the spectrum of strategies used by microbes to avoid or provoke activation of the hosts immune response as well as our current understanding of the role this immunomodulatory interference plays during microbial pathogenesis.

The innate immune response differs in primary and secondary Salmonella infection

This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mφ) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-γ were, unexpectedly, neutrophils and Mφ, the former having intracellular stores of IFN-γ that were released during infection. IFN-γ-producing phagocytes greatly outnumbered IFN-γ-producing NK cells, NKT cells, and T cells during the primary response. TNF-α production was also dominated by neutrophils and Mφ, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-α early during primary infection. The TNF-α response was reduced in a secondary response, but remained dominated by neutrophils and Mφ. Moreover, no significant IFN-γ production by Mφ was associated with the secondary response. Indeed, only NK1.1+ cells and T cells produced IFN-γ in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-γ, and demonstrate that immune status influences the nature of the innate response.

Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter

Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8α+, CD8α−CD4−, and CD8α−CD4+ DC, from these organs in response to Salmonella. Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8α+ and CD8α− splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-α was produced by a large percentage of CD8α− DC, while only a minor proportion of CD8α+ DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8α+ DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion.

The phoP locus influences processing and presentation of Salmonella typhimurium antigens by activated macrophages

The destruction and processing of bacteria by activated macrophages facilitates the presentation of antigens to T cells and thereby promotes the induction of specific immunity. The PhoP‐PhoQ regulatory system that controls the synthesis of many Salmonella proteins required for virulence and survival within macrophages is one mechanism that this particular intracellular pathogen has evolved to resist destruction. To address whether the phoP locus also influences antigen processing during the interaction of Salmonella typhimurium with macrophages, we tested the effect of phoP mutations on the processing and presentation of model antigens expressed by the bacteria. Activated macrophages processed phoP− bacteria with greater efficiency than wild‐type bacteria, as measured by the response of antigen‐specific T‐hybridoma cells; Salmonella constitutively expressing PhoP were processed even less efficiently than wild‐type Salmonella. After heat‐inactivation, however, both wild‐type and phoP− bacteria were efficiently processed. The altered processing and presentation efficiency was not due to differences in the level of antigen expressed by the bacteria or differences in the level of bacterial uptake by the macrophages. In addition, phoP‐regulated gene expression was shown to influence processing of antigen phagocytosed independently of the bacteria. Thus, phoP‐regulated gene products decrease the processing and presentation of S. typhimurium antigens, demonstrating a rote 1or this virulence locus in the inhibition of the induction of specific immunity.

Salmonella enterica Serovar Typhimurium-Induced Maturation of Bone Marrow-Derived Dendritic Cells

ABSTRACT Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating withEscherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. colifor OVA(257-264)/Kb and OVA(265-277)/I-Abpresentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/Kb complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-Ab complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQvirulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-Ab presentation but not for OVA(257-264)/Kb presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar TyphimuriumphoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show thatSalmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-Ab presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.

Differential Involvement of Dendritic Cell Subsets During Acute Salmonella Infection

Within murine CD11c+ dendritic cells (DC), CD8α+, CD8α−CD4+, and CD8α−CD4− subsets are defined. This study characterized the localization, number, and function of these subsets during acute Salmonella typhimurium infection. Immunohistochemical and flow cytometric analyses of spleens from mice orally infected with virulent S. typhimurium revealed that in situ redistribution and alteration in the absolute number and function of DC occurred in a subset-specific manner during infection. CD8α−CD4+ DC present at B cell follicle borders in the spleen of naive mice were absent 5 days post-Salmonella infection, despite no overall change in the absolute number of CD8α−CD4+ splenic DC. CD8α+ and CD8α−CD4− DC were prominently associated with the red pulp, and the frequency of these cells increased strikingly 5 days post-Salmonella infection. Significant quantitative increases in both CD8α+ and CD8α−CD4− subsets were associated with the in situ redistribution. Examination of Salmonella-infected TAP1−/−/β2-microglobulin−/− mice, which lack CD8α+ T cells, confirmed the differential subset-specific modulations in the DC populations both in situ and quantitatively. Ex vivo intracellular cytokine analysis showed significantly increased frequencies of CD8α+ DC producing TNF-α at days 2 and 5 postinfection. In contrast, CD4+ DC producing TNF-α were transiently increased followed by a significant reduction. No significant increase in IL-12p40 or IL-10 production by splenic DC was detected during the first 5 days post-S. typhimurium infection. Together these data reveal differential modulation of splenic DC subsets with regard to organization, number, and cytokine production during the course of acute Salmonella infection.

CLASSICAL MHC CLASS I PEPTIDE PRESENTATION OF A BACTERIAL FUSION PROTEIN BY BONE MARROW-DERIVED DENDRITIC CELLS

Dendritic cells (DC) expanded in the presence of GM‐CSF from the bone marrow of C57BL/6 mice process Gram‐negative bacteria expressing the model antigen Crl‐OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257u2009–u2009264) processed by DC co‐incubated with E. coli expressing Crl‐OVA, which contains the Kb‐binding OVA(257u2009–u2009264) epitope, occurs by a cytosolic MHC‐I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1−/− mice co‐incubated with E. coli expressing Crl‐OVA did not result in Kb presentation of OVA(257u2009–u2009264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257u2009–u2009264) on Kb when C57BL/6 DC phagocytosed and processed E. coli expressing Crl‐OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257u2009–u2009264) processed from bacteria expressing Crl‐OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257u2009–u2009264) on neighboring bystander Kb‐expressing antigen‐presenting cells after BALB/c (H‐2d) DC phagocytosed E. coli expressing Crl‐OVA could not be detected. Together, these data support a cytosolic MHC‐I presentation pathway for OVA(257u2009–u2009264) processed from E. coli expressing Crl‐OVA by bone marrow‐derived DC.

Processing of bacterial antigens for peptide presentation on MHC class I molecules

Summary: Professional antigen‐presenting cells (pAPC) can process and present exogenous antigens on major histocompatibility complex class 1 (MHC‐I) molecules. This unusual pathway for antigen presentation may represent a physiologically important step in the course of priming and tolerance induction of CD8+ T cells. In addition, it may play an important role in immunological surveillance for pathogens that survive in vacuolar compartments in APC. The goal of the present review is to discuss recent studies on the processing of bacterial‐derived antigens for presentation on MHC‐1 molecules. The antigen presentation emphasized will include bacteria that remain confined in vacuolar compartments. This is in contrast to antigens derived from bacteria that have intrinsic properties allowing translocation across membranes and access into the classical MHC‐I presentation pathway In particular, presentation of bacterial antigens by dendritic cells (DC) will be emphasized, and MHC‐I presentation of antigens derived from apoptotic cells, particularly cells induced to undergo apoptosis by microbial infection, will be presented. Finally, some special aspects of the interaction between bacteria and DC will be discussed as ii relates to DC maturation, antigen presentation and T‐cell stimulation.

Liver Dendritic Cells Present Bacterial Antigens and Produce Cytokines upon Salmonella Encounter

The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c+ cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8α−CD4− double negative cells constituted the majority of liver CD11c+ (∼85%) with few cells expressing CD8α or CD4. Flow cytometry analysis of freshly isolated CD11c+ cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c+ MHC class IIhigh cells had a greater capacity to internalize Salmonella relative to CD11c+ MHC class IIlow cells. Moreover, both CD8α− and CD8α+ liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c+ cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c+ cells infected with Salmonella expressing GFP showed that both CD8α− and CD8α+ DC produced IL-12p40 and TNF-α. The majority of cytokine-positive cells did not contain bacteria (GFP−) whereas only a minor fraction of cytokine-positive cells were GFP+. Furthermore, only ∼30–50% of liver DC containing bacteria (GFP+) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4+ and CD8+ T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.

Interaction of bacteria with antigen presenting cells: influences on antigen presentation and antibacterial immunity.

Abstract Macrophages and dendritic cells are phagocytic antigen-presenting cells involved in the immune response to bacteria, linking the innate and adaptive responses during bacterial infection. Antigens from bacteria are processed for presentation on MHC class II and MHC class I to stimulate CD4+ and CD8+ T cells, respectively. Many bacterial pathogens have developed strategies to interfere with the host’s capacity to mount an immune response by interfering with antigen processing pathways or otherwise modulating the adaptive immune response. Understanding the mechanisms bacteria use to avoid immune recognition may facilitate the development of new strategies to eradicate pathogens from infected hosts.