Listeria Monocytogenes Infection Model Research Articles

Protein Kinase CK2 Controls CD8+ T Cell Effector and Memory Function during Infection.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2β). CK2 promotes cancer progression by activating the NF-κB, PI3K/AKT/mTOR, and JAK/STAT pathways, and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2α deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore, CK2α is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly, using a mouse Listeria monocytogenes infection model, we demonstrate that CK2α is required for CD8+ T cell expansion, maintenance, and effector function in both primary and memory immune responses. Collectively, our study implicates CK2α as an important regulator of mouse CD8+ T cell activation, metabolic reprogramming, and differentiation both in vitro and in vivo.

Sirtuin 5 is Dispensable for CD8+ T Cell Effector and Memory Differentiation.

CD8+ T cell effector and memory differentiation is tightly controlled at multiple levels including transcriptional, metabolic, and epigenetic regulation. Sirtuin 5 (SIRT5) is a protein deacetylase mainly located at mitochondria, but it remains unclear whether SIRT5 plays key roles in regulating CD8+ T cell effector or memory formation. Herein, with adoptive transfer of Sirt5+/+ or Sirt5−/− OT-1 cells and acute Listeria monocytogenes infection model, we demonstrate that SIRT5 deficiency does not affect CD8+ T cell effector function and that SIRT5 is not required for CD8+ T cell memory formation. Moreover, the recall response of SIRT5 deficient memory CD8+ T cells is comparable with Sirt5+/+ memory CD8+ T cells. Together, these observations suggest that SIRT5 is dispensable for the effector function and memory differentiation of CD8+ T cells.

Response: Commentary: An In Silico-In Vitro Pipeline Identifying an HLA-A*02:01+ KRAS G12V+ Spliced Epitope Candidate for a Broad Tumor-Immune Response in Cancer Patients.

Proteasomes are the central cytosolic and nuclear proteases in eukaryotic cells. They degrade a broad range of proteins, mainly cytoplasmic, though they are also active in other cell compartments (1). The peptides produced during protein degradation can be further processed by cytosolic aminopeptidases to preserve amino acid availability for new protein synthesis. Not all proteasomal products are destinated for full degradation. For example, p105 is cleaved by proteasomes, thereby generating a component of the transcription factor NF-κB. Similarly, osteopontin seems to be processed by these proteases, which produce peptides that promote cell migration (2–4). However, the best studied peptide products produced by proteasomes are peptides destined for binding HLA-I complexes (i.e. the HLA-I immunopeptidome) for presentation at the cell surface to CD8+ T cells (5). HLA-I immunopeptidomes have widely been investigated in the context of cancer, radiotherapy, infection, autoimmunity and other conditions. These peptides can be used for detection of specific T cell immune responses and for vaccination purposes against infections and cancer. It is long established that proteases such as trypsin can both cleave peptide bonds but also ligate (repair) these peptide bonds (6, 7). The proteasome is no exception and can produce so-called spliced peptides through proteasome-catalyzed peptide splicing (PCPS). This latter process can occur by combining non-contiguous peptide fragments of either the same molecule – i.e. cis-PCPS – or of two distinct proteins - i.e. trans-PCPS ( Figure 1A ). This results in peptide sequences that are not defined by the genetic code. Cis-spliced peptide identification is challenging: depending on the method of identification used, their estimated frequency in HLA-I immunopeptidomes ranged from 1% to 34% (8). PCPS is not a random process and seems to be ruled by sequence motif preferences and driving forces, which may differ from peptide hydrolysis, although the exact rules are largely unknown (7, 9–11). Cis-spliced peptides seem to be routinely produced and presented by various cells, depicting peptide splicing as a standard activity of proteasomes (7, 9, 12–17). On average, they are produced and presented by HLA-I complexes in smaller amount than non-spliced peptides although exceptions have been described (10, 12, 15, 17). Cis-spliced peptides can target in vivo CD8+ T cell response against bacterial antigens otherwise neglected in a mouse model of Listeria monocytogenes infection (18). They can activate ex vivo CD8+ T cells specific to Listeria monocytogenes or HIV through cross-recognition (19, 20). Cis-spliced epitopes derived from melanoma-associated antigens are recognized by CD8+ T cells in peripheral blood of melanoma patients (13, 17). A melanoma patient with metastasis was cured through adoptive T cell therapy using an autologous tumor-infiltrating lymphocyte (TIL) clone, which was proved, in a later study, to be specific to a cis-spliced epitope rather than any non-spliced peptides derived from the melanoma-associated antigen tyrosinase (21, 22). Despite their theoretically vast sequence variability, cis-spliced peptides might not play a destabilizing role in central and peripheral tolerance and in pruning T cell repertoire (23), although they may be involved in autoreactive CD8+ T cell response in autoimmune diseases such as Type 1 Diabetes (T1D) (24, 25). Open in a separate window Figure 1 Proteasome-catalyzed peptide splicing and the investigated KRAS G12V cis-spliced and non-spliced peptides. (A) Proteasome-generated cis-spliced peptides can be formed by cis-PCPS, when the two splice-reactants, i.e. the non-contiguous peptide fragments ligated by proteasomes, derive from the same polypeptide molecule; the ligation can occur in normal order, i.e. following the orientation from N- to C-terminus of the parental protein (normal cis-PCPS), or in the reverse order (reverse cis-PCPS). (B) The KRAS5-6/8-14 G12V [KL][VVGAVGV] cis-spliced peptide and the KRAS5-13 G12V [KLVVVGAVG] non-spliced peptide, which are investigated in this study and derived from the KRAS2-35 G12V synthetic substrate polypeptide. The G12V mutation is marked in bold.

The TRPM2 ion channel regulates the inflammatory response of neutrophils during Listeria monocytogenes infection

Abstract Calcium signaling is required for most antimicrobial functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating calcium mobilization, cell migration and oxidative stress in neutrophils. However, the precise mechanism by which TRPM2 activation may exert a pro- or anti-inflammatory function remains unclear. In this study, we used a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils’ function. We found that infected Trpm2−/− mice were associated with high bacterial burden in spleen and liver indicating increased susceptibility to Listeria infection. Pronounced migration rates of neutrophils and monocytes to the liver and spleen characterized the inflammatory response to Listeria during the first 18h. In the Trpm2−/− mice, the acute phase infection resulted in septic shock, defined by increased serum levels of TNF-α, IL-6, and IL-10, but no decreased levels of IFN-γ or IL-12. Furthermore, depletion of neutrophils demonstrated the critical role of these cells in regulating acute inflammation by conferring resistance of Trpm2−/− mice to Listeria infection. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2−/− neutrophils. Finally, the increased inflammatory properties of Trpm2−/− neutrophils correlated with cellular calcium overloading and potentiated membrane depolarization, in response to Listeria infection. In conclusion, our findings suggest that TRPM2 channel plays critical roles in the regulation of the inflammatory properties of neutrophils during Listeria monocytogenes infection.

BCAP Regulates Dendritic Cell Maturation Through the Dual-Regulation of NF-κB and PI3K/AKT Signaling During Infection.

The maturation of dendritic cells (DCs) is essential in adaptive immunity. B cell adapter for phosphoinositide 3-kinase (BCAP) has been shown a divergent activities in cell type dependent manner including B cells, NK cells, macrophages, and plasmacytoid DCs (pDCs), however, its role in conventional DCs (cDCs) remains unknown. Here, we report that BCAP negatively regulates Toll-like receptor-induced cDC maturation and inhibits cDCs from inducing antigen-specific T cell responses, thereby weakening the antibacterial adaptive immune responses of mice in a Listeria monocytogenes-infection model. Furthermore, we demonstrate that BCAP simultaneously modulates the activation of the NF-κB and PI3K/AKT signaling by dynamically interacting with, respectively, MyD88 and the p85α subunit of PI3K. Our study thus reveals non-redundant roles for BCAP in regulating cDC maturation and reveals a bilateral signal transduction mechanism.

The TRPM2 Ion Channel Regulates Inflammatory Functions of Neutrophils During Listeria monocytogenes Infection.

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca2+) signaling and the activation of specific Ca2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2−/− mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2−/− mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2−/− infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2−/− neutrophils. Finally, the increased inflammatory properties of Trpm2−/− neutrophils correlated with the dysregulated cytoplasmic concentration of Ca2+ and potentiated membrane depolarization, in response to L. monocytogenes. In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection.

Tim-3 Promotes Listeria monocytogenes Immune Evasion by Suppressing Major Histocompatibility Complex Class I.

T-cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint inhibitor that has therapeutic implications for many tumors and infectious diseases. However, the mechanisms by which Tim-3 promotes immune evasion remain unclear. In this study, we demonstrated that Tim-3 inhibits the expression of major histocompatibility complex class I (MHC-I) in macrophages at both the messenger ribonucleic acid and protein levels by inhibiting the STAT1-NLRC5 signaling pathway. As a result, MHC-I-restricted antigen presentation by macrophages was inhibited by Tim-3 both in vitro and in a Listeria monocytogenes infection model in vivo. Systemic overexpression of Tim-3 or specific knockout of Tim-3 in macrophages significantly attenuated or enhanced CD8+ T-cell activation and infection damage in L monocytogenes-infected mice, respectively. Thus, we identified a new mechanism by which Tim-3 promotes L monocytogenes immune evasion. Further studies on this pathway might shed new light on the physio-pathological roles of Tim-3 and suggest new approaches for intervention.

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ Tcells in L.monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ Tcells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ Tcell response against L.monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

The absence of TNF permits myeloid Arginase 1 expression in experimental L. monocytogenes infection

During an immune response inflammatory macrophages with their wide variety of effector mechanisms including the expression of inducible nitric oxide synthase play an important part in the defense against invading pathogens. The inflammatory phenotype requires the presence of TNF which suppresses alternative activation. In the bacterial Listeria monocytogenes infection model inflammatory macrophages are crucial for protection. After infection, TNF-deficient hosts have a similar number of splenic macrophages but die rapidly. A more detailed analysis of these cells showed that while inducible nitric oxide synthase is expressed at a comparable level TNF-deficient macrophages show an increased expression of Arginase 1.

CD81 controls immunity to Listeria infection through rac-dependent inhibition of proinflammatory mediator release and activation of cytotoxic T cells.

Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81(-/-) mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81(-/-) spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α(+) DCs. In conclusion, this study demonstrates that CD81-Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases.

Innate-like CD4 T cells selected by thymocytes suppress adaptive immune responses against bacterial infections

We have reported a new innate-like CD4 T cell population that expresses cell surface makers of effector/memory cells and produce Th1 and Th2 cytokines immediately upon activation. Unlike conventional CD4 T cells that are selected by thymic epithelial cells, these CD4 T cells, named T-CD4 T cells, are selected by MHC class II expressing thymocytes. Previously, we showed that the presence of T-CD4 T cells protected mice from airway inflammation suggesting an immune regulatory role of T-CD4 T cells. To further understand the function of T-CD4 T cells, we investigated immune responses mediated by T-CD4 T cells during bacterial infection because the generation of antigen specific CD4 T cells contributes to clearance of infection and for the development of immune memory. The current study shows a suppressive effect of T-CD4 T cells on both CD8 and CD4 T cell-mediated immune responses during Listeria and Helicobacter infections. In the mouse model of Listeria monocytogenes infection, T-CD4 T cells resulted in decreasedfrequency of Listeria-specific CD8 T cells and the killing activity of them. Furthermore, mice with T-CD4 T cells developed poor immune memory, demonstrated by reduced expansion of antigen-specific T cells and high bacterial burden upon re-infection. Similarly, the presence of T-CD4 T cells suppressed the generation of antigen-specific CD4 T cells in Helicobacter pylori infected mice. Thus, our studies reveal a novel function of T-CD4 T cells in suppressing anti-bacterial immunity.

Statins inhibit iNOS‐mediated microbicidal potential of activated monocyte‐derived dendritic cells by an IFN‐β‐dependent mechanism

Statins are prescribed to 25 million people worldwide for treating hypercholesterolemia and reducing the risk of cardiovascular diseases. However, the side effects of statins on immunity, and particularly on DC immunobiology, have not been analyzed in-depth. Here, we have investigated the impact of lovastatin treatment during monocyte differentiation into DCs on the responsiveness of the resulting monocyte-derived DCs (moDCs) to TLR-mediated activation. Lovastatin positively regulated TLR4 signaling in LPS-stimulated moDCs, leading to strong activation of p38 MAP-kinase paralleled by increased proinflammatory cytokine and IFN-β production. In contrast, lovastatin promoted negative regulation of IFN-β-mediated autocrine signaling through the IFN-αβ receptor, paralleled by low expression of the transcription factor IRF-1, leading to the inhibition of the enzymes iNOS and HO-1. Defective activation of iNOS/HO-1 resulted in limited cytoprotective capacity against ROS and reduced microbicidal potential. These data were validated using an in vivo model of Listeria monocytogenes infection, which revealed that iNOS activation by splenic inflammatory moDCs, specialized in NO and TNF-α production, was strongly reduced in lovastatin-treated, Listeria-infected mice. Statin treatment could have severe implications in immunity against pathogens due to defective iNOS/HO-1 metabolism activation in inflammatory moDCs that might lead to immune failure.

DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses

Generation of microbicidal reactive oxygen species (ROS) is a pivotal protective component of the innate immune system in many eukaryotes. NOD (nucleotide oligomerisation domain containing protein)-like receptors (NLRs) have been implicated as phylogenetically ancient sensors of intracellular pathogens or endogenous danger signals. NOD2 recognizes the bacterial cell wall component muramyldipeptide leading to NFkappaB and MAPK activation via induced proximity signalling through the serine-threonine kinase RIP2. In addition to the subsequent induction of cytokines and antimicrobial peptides, NOD2 has been shown also to exert a direct antibacterial effect. Using a fluorescence-based ROS detection assay we demonstrate controlled ROS generation as an integral component of NOD2-induced signalling in epithelial cells. We demonstrate that the NAD(P)H oxidase family member DUOX2 is involved in NOD2-dependent ROS production. Coimmunoprecipitation and fluorescence microscopy were used to show that DUOX2 interacts and colocalizes with NOD2 at the plasma membrane. Moreover, simultaneous overexpression of NOD2 and DUOX2 was found to result in cooperative protection against bacterial cytoinvasion using the Listeria monocytogenes infection model. RNAi-based studies revealed that DUOX2 is required for the direct bactericidal properties of NOD2. Our results demonstrate a new role of ROS as effector molecules of protective cellular signalling in response to a defined danger signal carried out by a mammalian intracellular NLR system.

MyD88-Dependent Activation of B220−CD11b+LY-6C+ Dendritic Cells during Brucella melitensis Infection

IFN-gamma is a key cytokine controlling Brucella infection. One of its major function is the stimulation of Brucella-killing effector mechanisms, such as inducible NO synthase (iNOS)/NOS2 activity, in phagocytic cells. In this study, an attempt to identify the main cellular components of the immune response induced by Brucella melitensis in vivo is made. IFN-gamma and iNOS protein were analyzed intracellularly using flow cytometry in chronically infected mice. Although TCRbeta(+)CD4(+) cells were the predominant source of IFN-gamma in the spleen, we also identified CD11b(+)LY-6C(+)LY-6G(-)MHC-II(+) cells as the main iNOS-producing cells in the spleen and the peritoneal cavity. These cells appear similar to inflammatory dendritic cells recently described in the mouse model of Listeria monocytogenes infection and human psoriasis: the TNF/iNOS-producing dendritic cells. Using genetically deficient mice, we demonstrated that the induction of iNOS and IFN-gamma-producing cells due to Brucella infection required TLR4 and TLR9 stimulation coupled to Myd88-dependent signaling pathways. The unique role of MyD88 was confirmed by the lack of impact of Toll-IL-1R domain-containing adaptor inducing IFN-beta deficiency. The reduction of IFN-gamma(+) and iNOS(+) cell frequency observed in MyD88-, TLR4-, and TLR9-deficient mice correlated with a proportional lack of Brucella growth control. Taken together, our results provide new insight into how immune responses fight Brucella infection.

Host Resistance Model to an Intracellular Pathogen

This unit describes a model for the determination of host resistance in mice using a Gram-positive intracellular bacterium, Listeria monocytogenes (LM), and discusses its broad use in immunotoxicological studies. The provides detailed procedures for LM infection and sample analysis, including preparation and infection of mice with LM, recording of sickness behaviors in infected mice, and determination of viable LM numbers in the tissues from infected animals. The protocol also describes use of serum and tissue homogenates for assessment of cytokines. Additionally, background information on LM and LM infection is provided for better understanding and utilization of this model. The LM infection model is useful for the initial screening of possible modulation of immune responses, particularly innate immunity and type-1 cell-mediated immunity (CMI), by environmental factors, as well as for further investigation of the underlying mechanisms of environmental factor-induced changes in immunity.

Differential requirements for the chemokine receptor CCR7 in T cell activation during Listeria monocytogenes infection

Effective priming of T cell responses depends on cognate interactions between naive T cells and professional antigen-presenting cells (APCs). This contact is the result of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, play a central role. We used the murine Listeria monocytogenes infection model to characterize the role of the CCR7/CCR7 ligand system in the generation of T cell responses during bacterial infection. We demonstrate that efficient priming of naive major histocompatibility complex (MHC) class Ia–restricted CD8+ T cells requires CCR7. In contrast, MHC class Ib–restricted CD8+ T cells and MHC class II–restricted CD4+ T cells seem to be less dependent on CCR7; memory T cell responses are independent of CCR7. Infection experiments with bone marrow chimeras or mice reconstituted with purified T cell populations indicate that CCR7 has to be expressed on CD8+ T cells and professional APCs to promote efficient MHC class Ia–restricted T cell priming. Thus, different T cell subtypes and maturation stages have discrete requirements for CCR7.

Listeria monocytogenes as a probe of immune function

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.

Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs). I. Effects of DEPs on early pulmonary responses.

It has been hypothesized that diesel exhaust particles (DEPs) aggravate pulmonary bacterial infection by both innate and cell-mediated immune mechanisms. To test this hypothesis, we investigated the effects of DEP exposure on the functions of alveolar macrophages (AMs) and lymphocytes from lung-draining lymph nodes using a rat Listeria monocytogenes infection model. In the present study, we focused on the effects of DEP exposure on AM functions, including phagocytic activity and secretion of proinflammatory cytokines. The Listeria infection model was characterized by an increase in neutrophil count, albumin content, and acellular lactate dehydrogenase activity in the bronchoalveolar lavage (BAL) fluid at 3 and 7 days postinfection. Short-term DEP inhalation (50 and 100 mg/m(3), 4 hr) resulted in a dose-dependent suppression of lung clearance of Listeria, with the highest bacteria count occurring at day 3. This aggravated bacterial infection was consistent with the inhibitory effect of DEPs on macrophage functions. DEPs suppressed phagocytosis and Listeria-induced basal secretion of interleukin-1ss (IL-1ss) and IL-12 by AMs in a dose-dependent manner. The amount of IL-1ss and IL-12 in the BAL fluid was also reduced by DEP exposure. In addition, DEPs decreased Listeria-induced lipopolysaccharide-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha), IL-1ss, and IL-12 from AMs. These results suggest that DEPs retard bacterial clearance by inhibiting AM phagocytosis and weaken the innate immunity by inhibiting AM secretion of IL-1ss and TNF-alpha. DEPs may also suppress cell-mediated immunity by inhibiting AM secretion of IL-12, a key cytokine for the initiation of T helper type 1 cell development in Listeria infection.

Adjuvanticity of an IL-12 fusion protein expressed by recombinant ΔG-vesicular stomatitis virus

The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described. Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection. In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-ΔG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F. VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored. The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12. Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity. These studies demonstrated that VSVΔG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVΔG-IL12F as a vector for delivery of IL-12F in vivo.

Evaluation of the safety of recombinant P-selectin glycoprotein ligand-immunoglobulin G fusion protein in experimental models of localized and systemic infection.

P-selectin is a major component in the early interaction between platelets, endothelial cells, and inflammatory cells in the initial phases of the innate immune response. The major ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1) and this ligand is expressed on the surface of monocyte, lymphocyte, and neutrophil membranes. A truncated form of recombinant human P-selectin glycoprotein ligand-1 has been covalently linked to immunoglobulin G (rPSGL-Ig) and this fusion peptide functions as a competitive inhibitor of PSGL-1. As an inhibitor of neutrophil-endothelial cell adherence, rPSGL-Ig is in early clinical development for the treatment of ischemia reperfusion injury. To determine the potential for deleterious effects from inhibition in P-selectin-mediated neutrophil attachment in the presence of bacterial infection, the effects of therapeutic doses of rPSGL-Ig were tested in three standard laboratory sepsis models. The experimental models included: the murine systemic Listeria monocytogenes infection model, the Pseudomonas aeruginosa bacteremia model in neutropenic rats, and the cecal ligation and puncture (CLP)-induced peritonitis model in rats. Recombinant human PSGL-Ig had no adverse effects on mortality or immune clearance in systemic bacterial infection in any of the three infection models. The PSGL-1 inhibitor did significantly decrease local neutrophil infiltration and bacterial clearance in the peritoneum following CLP, but this did not increase the systemic levels of proinflammatory cytokines, the quantitative levels of bacteremia, or the overall mortality rate following CLP. The results indicate that rPSGL-Ig did not exacerbate infection in these experimental sepsis models.