Listeria Monocytogenes-infected Mice Research Articles

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Immunopeptidomics for next-generation bacterial vaccine development.

Antimicrobial resistance is an increasing global threat and alternative treatments substituting failing antibiotics are urgently needed. Vaccines are recognized as highly effective tools to mitigate antimicrobial resistance; however, the selection of bacterial antigens as vaccine candidates remains challenging. In recent years, advances in mass spectrometry-based proteomics have led to the development of so-called immunopeptidomics approaches that allow the untargeted discovery of bacterial epitopes that are presented on the surface of infected cells. Especially for intracellular bacterial pathogens, immunopeptidomics holds great promise to uncover antigens that can be encoded in viral vector- or nucleic acid-based vaccines. This review provides an overview of immunopeptidomics studies on intracellular bacterial pathogens and considers future directions and challenges in advancing towards next-generation vaccines.

Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness.

The type II IFN (IFNγ) enhances antimicrobial activity yet also drives expression of genes that amplify inflammatory responses. Hence, excessive IFNγ stimulation can be pathogenic. Here, we describe a previously unappreciated mechanism whereby IFNγ itself dampens myeloid cell activation. Staining of monocytes from Listeria monocytogenes-infected mice provided evidence of type I IFN-independent reductions in IFNGR1. IFNγ was subsequently found to reduce surface IFNGR1 on cultured murine myeloid cells and human CD14+ peripheral blood mononuclear cells. IFNγ-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFNγ reduced macrophage Ifngr1 transcription by altering chromatin structure at putative Ifngr1 enhancer sites. This is a distinct mechanism from that used by type I IFNs. Ligand-induced reductions in IFNGR1 altered myeloid cell sensitivity to IFNγ, blunting activation of STAT1 and 3. Our data, thus, reveal a mechanism by which IFNGR1 abundance and myeloid cell sensitivity to IFNγ can be modulated in the absence of type I IFNs. Multiple mechanisms, thus, exist to calibrate macrophage IFNGR1 abundance, likely permitting the fine tuning of macrophage activation and inflammation.

Essential role of IκBNS for in vivo CD4+ T-cell activation, proliferation, and Th1-cell differentiation during Listeria monocytogenes infection in mice.

Acquisition of effector functions in T cells is guided by transcription factors, including NF‐κB, that itself is tightly controlled by inhibitory proteins. The atypical NF‐κB inhibitor, IκBNS, is involved in the development of Th1, Th17, and regulatory T (Treg) cells. However, it remained unclear to which extend IκBNS contributed to the acquisition of effector function in T cells specifically responding to a pathogen during in vivo infection. Tracking of adoptively transferred T cells in Listeria monocytogenes infected mice antigen‐specific activation of CD4+ T cells following in vivo pathogen encounter to strongly rely on IκBNS. While IκBNS was largely dispensable for the acquisition of cytotoxic effector function in CD8+ T cells, IκBNS‐deficient Th1 effector cells exhibited significantly reduced proliferation, marked changes in the pattern of activation marker expression, and reduced production of the Th1‐cell cytokines IFN‐γ, IL‐2, and TNF‐α. Complementary in vitro analyses using cells from novel reporter and inducible knockout mice revealed that IκBNS predominantly affects the early phase of Th1‐cell differentiation while its function in terminally differentiated cells appears to be negligible. Our data suggest IκBNS as a potential target to modulate specifically CD4+ T‐cell responses.

MTORC1 regulates mannose-6-phosphate receptor transport and T-cell vulnerability to regulatory T cells by controlling kinesin KIF13A

Mannose-6-phosphate receptor (M6PR) that facilitates cellular uptake of M6P-bearing proteins, including serine-protease granzyme-B (Gzm-B) has an important role in T-cell activation, migration and contraction. However, molecular mechanisms controlling M6PR expression in T cells remain poorly understood. Here, we show that M6PR expression on T cells is distinctively controlled by two common γ-chain cytokines interleukin-2 (IL-2) and IL-7, and the differential M6PR expression is not caused by an altered synthesis of M6PR protein, but is a result of distinct regulation of kinesin-3 motor-protein KIF13A that transport M6PR onto cell surfaces. Using signaling pathway-specific inhibitors, we determine that IL-2 and IL-7 distinctly regulate KIF13A and β1-adaptin and cell-surface M6PR by controlling a kinase mammalian target of rapamycin complex-1 (mTORC1). Inflammatory cytokine IL-2 and prosurvival cytokine IL-7 induce strong and weak activation of mTORC1, leading to up- and downregulation of motor-protein KIF13A and KIF13A-motorized M6PR on T cells, and formation of IL-2 and IL-7 effectors with M6PRhigh and M6PRlow cell-surface expression, respectively. Inhibition of mTORC1 by rapamycin reduces T-cell expression of KIF13A and cell-surface M6PR, and increases T-cell survival in Listeria monocytogenes-infected mice. Using regulatory T (Treg)-cell-enriched mouse tumor model, we determine that M6PRhigh IL-2 effectors but not M6PRlow IL-7 effectors adoptively transferred into tumors are vulnerable to Treg Gzm-B-mediated cell apoptosis. Inhibition of mTORC1 or small interfering RNA-mediated knockdown of KIF13A or M6PR renders IL-2 effectors refractory to Treg Gzm-B lethal hit. Overall, our data offer novel mechanistic insights into T-cell M6PR regulation, and Treg-resistant/Treg-susceptible phenomenon. Furthermore, regulation of T-cell fate vis-à-vis Treg suppression via the mTORC1-KIF13A-M6PR axis provides a proof of concept for therapeutic strategies to target cancer, infectious and autoimmune diseases.

CD8(+) Tcells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) Tcells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) Tcells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) Tcells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) Tcells. Such mechanism may reduce the chances for pathogen immune evasion.

CpG oligodeoxynucleotide induces bone marrow precursor cells into myeloid-derived suppressor cells

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) perform a number of functions in different immunological settings. In standard in vitro experiments, DCs are produced from mouse bone marrow (BM) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Our previous study demonstrated that BM precursor cells could differentiate into MDSCs when co-cultured with poly (I:C). In the present study, BM precursor cells cultured in GM-CSF and IL-4 were treated with CpG oligodeoxynucleotide (CpG ODN). We observed that Gr1+CD11b+ cells exhibiting MDSC functions accumulated in the co-culture system. A similar phenomenon was also observed in Listeria monocytogenes-infected mice. In conclusion, we demonstrated that prolonged CpG ODN stimulation could inhibit the development of DCs and induce the differentiation of BM precursor cells into MDSCs.

Angelica sinensisModulates Immunohematopoietic Response and Increases Survival of Mice Infected withListeria monocytogenes

The effects of a dry extract of the roots of Angelica sinensis (Oliv.) Diels (ASE) on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM) in normal and Listeria monocytogenes-infected mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in infected mice. Prophylactic administration of ASE (10, 25, and 50 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation to control values. The dose of 50 mg/kg ASE was the optimal biologically active dose in infected mice, and this dose schedule significantly increased survival of mice infected with a lethal dose of L. monocytogenes, with survival rate up to 30%. Investigation of the production of colony-stimulating factors revealed a dose-dependent increased colony-stimulating activity in the serum of infected mice, with higher response produced by the 50 mg/kg dose. Notably, no effects were observed with the 100 mg/kg dose, compared with infected nontreated controls. Further studies to investigate the production of factors such as inteferon-γ and tumor necrosis factor-α demonstrated increased levels of both cytokines in mice infected with L. monocytogenes and treated with 50 mg/kg ASE. We propose that ASE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (colony-stimulating factors, interferon-γ, and tumor necrosis factor-α).

Exacerbating Role of γδ T Cells in Chronic Colitis of T-Cell Receptor α Mutant Mice

T-cell receptor (TCR) gammadelta T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in gammadelta T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of gammadelta T cells in chronic colitis has not been fully identified. TCRalpha-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in gammadelta T-cell population, represent an excellent model to investigate the role of gammadelta T cells in UC-like colitis. To identify the role of gammadelta T cells in this colitis, we herein have generated TCRgamma-deficient mice through deletion of all TCR Cgamma genes (Cgamma1, Cgamma2, Cgamma3, and Cgamma4) using the Cre/loxP site-specific recombination system and subsequently crossing these mice with TCRalpha-deficient mice. An increase in colonic gammadelta T cells was associated with the development of human UC as well as UC-like disease seen in TCRalpha-deficient mice. Interestingly, the newly established TCRalpha(-/-) x TCRgamma(-/-) double mutant mice developed significantly less severe colitis as compared with TCRalpha-deficient mice. The suppression of colitis in TCRalpha(-/-) x TCRgamma(-/-) double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration. gammadelta T cells are involved in the exacerbation of UC-like chronic disease. Therefore, gammadelta T cells may represent a promising therapeutic target for the treatment of human UC.

Prophylactic administration of Withania somnifera extract increases host resistance in Listeria monocytogenes infected mice

In this study, we demonstrated that Withania somnifera L. extract (WSE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 100, 250 and 500 mg/kg for 10 days, with survival rates up to 30%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte–macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors (CSFs) revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with WSE. Further studies to investigate the levels of interferon-gamma (INF-γ) and lymphocyte cell proliferation were undertaken. We observed dose-dependent increases in cell proliferation and in the levels of INF-γ in mice infected with L. monocytogenes and treated with WSE. All together, our results suggest that WSE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow, proliferation of lymphocytes and increased INF-γ levels.

Fatal infection in mice after injection of immunosuppressive serum fractions from surgical patients.

Following surgical or accidental trauma many patients show suppression of cellular immunity. In this investigation sera from severely burned patients and patients undergoing aortic aneurysm repair were studied. Sera shown to suppress phytohaemagglutinin-induced blastogenesis of normal human lymphocytes were fractionated using ion exchange and G25 Sephadex chromatography. Suppressive activity was largely confined to a low molecular weight (LMW) fraction and was dose dependent. LMW fractions of normal sera had no significant suppressive activity. The purpose of this study was to test the causal relationship between immunosuppressive serum and decreased resistance to bacterial infection. Listeria monocytogenes infected mice were used as an in vivo model to test suppression of cellular immunity. Injection of LMW fractions of suppressive sera significantly increased mortality in these mice, but had no effect on non-infected mice. There was good correlation between the in vitro and in vivo effects of the suppressive fractions. These results suggest that a circulating factor in the serum of injured patients suppresses cellular immunity and may be responsible for impaired resistance to infection.

The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice.

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or DeltaactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset. In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes. Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C(high) monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.

TNF/iNOS-Producing Dendritic Cells Mediate Innate Immune Defense against Bacterial Infection

Dendritic cells (DCs) present microbial antigens to T cells and provide inflammatory signals that modulate T cell differentiation. While the role of DCs in adaptive immunity is well established, their involvement in innate immune defenses is less well defined. We have identified a TNF/iNOS-producing (Tip)-DC subset in spleens of Listeria monocytogenes-infected mice that is absent from CCR2-deficient mice. The absence of Tip-DCs results in profound TNF and iNOS deficiencies and an inability to clear primary bacterial infection. CD8 and CD4 T cell responses to L. monocytogenes antigens are preserved in CCR2-deficient mice, indicating that Tip-DCs are not essential for T cell priming. Tip-DCs, as the predominant source of TNF and iNOS during L. monocytogenes infection, orchestrate and mediate innate immune defense against this intracellular bacterial pathogen.

Protective effects of Chlorella vulgaris in lead-exposed mice infected with Listeria monocytogenes

Chlorella vulgaris extract (CVE) was examined for its chelating effects on the myelosuppression induced by lead in Listeria monocytogenes-infected mice. The reduction in the number of bone marrow granulocyte-macrophage progenitors (CFU-GM) observed after the infection was more severe in the groups previously exposed to lead. Extramedullar hematopoiesis, which was drastically increased after the infection, was not altered by the presence of lead. Treatment with CVE, given simultaneously or following lead exposure, restored to control values the myelosuppression observed in infected/lead-exposed mice and produced a significant increase in serum colony-stimulating activity. The benefits of the CVE treatment were also evident in the recovery of thymus weight, since the reduction produced by the infection was further potentiated by lead exposure. The efficacy of CVE was evident when infected and infected/lead-exposed mice were challenged with a lethal dose of L. monocytogenes after a 10-day treatment with 50 mg/kg CVE/day, given simultaneously to the exposure to 1300 ppm lead acetate in drinking water. Survival rates of 30% for the infected group and of 20% for the infected/lead-exposed groups were observed. Evidence that these protective effects of CVE are partly due to its chelating effect was given by the changes observed in blood lead levels. We have observed in the group receiving the CVE/lead simultaneous exposure a dramatic reduction of 66.03% in blood lead levels, when compared to lead-exposed nontreated control. On the other hand, CVE treatment following lead exposure produced a much less effective chelating effect. CVE treatments for 3 or 10 days, starting 24 h following lead exposure, produced a reduction in blood lead levels of 13.5% and 17%, respectively, compared to lead-exposed nontreated controls. The significantly better response observed with the simultaneous CVE/lead administration indicates that the immunomodulation effect of CVE plays an important role in the ability of this algae to reduce blood lead levels. In this regard, additional experiments with gene knockout C57BL/6 mice lacking a functional IFN-γ gene demonstrated that this cytokine is of paramount importance in the protection afforded by CVE. The antibacterial evaluation measured by the rate of survival demonstrated that, in face of a 100% survival in the control group composed of normal C57BL/6 mice, which are resistant to L. monocytogenes, we observed no protection whatsoever in the IFN-γ knockout C57BL/6 mice treated with CVE and inoculated with L. monocytogenes.

EFFECTS OF CHLORELLA VULGARIS EXTRACT ON CYTOKINES PRODUCTION IN LISTERIA MONOCYTOGENES INFECTED MICE

ABSTRACTIn this study, we have investigated the effects of the unicellular-green-algae Chlorella vulgaris on the production of INF-γ, IL-2, IL-4 and IL-10 in normal and Listeria monocytogenes infected mice. Our results demonstrated that in normal/non infected mice, CVE administration produced no effects in the levels of all cytokines studied. However, Listeria monocytogenes infection enhanced the production of INF-γ and IL-2 at 48 and 72 h after the bacteria inoculation. Interestingly, the treatment with five consecutive doses of 50 mg/Kg/day of Chlorella vulgaris given previously to infection, led to further increases in INF-γ and IL-2 levels at 48 and 72 h in relation to the presence of infection alone. No changes in IL-4 and IL-10 production were observed in Listeria monocytogenes and CVE treated/infected mice. These results are in accordance with the literature, which shows that CVE is a biological response modifier that enhances resistance to Listeria monocytogenes through augmentation of IL-2 and IFN-γ.

Addition of Maitake D-fraction Reduces the Effective Dosage of Vancomycin for the Treatment of Listeria-Infected Mice

Maitake D-fraction, β1,6-glucan having β1,3-branches, has been reported to activate the immune system of the host. To elucidate whether the D-fraction can reduce the clinical effective dosage of antibiotics in the treatment of opportunistic bacterial infection, we examined the effects of D-fraction on the treatment of Listeria monocytogenes-infected mice in combination with vancomycine (VCM), the only antibiotic used for methicillin-resistant Staphylococcus aureus (MRSA). Listeria-infection was introduced by its inoculation into the abdominal cavity of mice. Without treatment, all inoculated mice died within 3 days after the inoculation. In contrast, in the mice treated with combined therapy of D-faction (10 mg /kg per day) and VCM (10 mg /kg per day), the survival rate was maintained at 60% on the 10th day after the inoculation, which was superior to that of mice treated with VCM alone (10 mg /kg per day). To investigate the mechanism underlying the reinforcement of VCM treatment by the D-fraction, the activities of macrophages and splenic T cells of Listeria-infected mice were evaluated. In mice administered with both D-frac-tion and VCM, macrophages produced 2.7 times as much interleukin-1 as that of non-treated control mice. The bactericidal activity of splenic T cells was also enhanced by 2.6 times of that of non-treated control mice. These results indicate that D-fraction activates immuno-competent cells, induced cytokine production, and consequently enhanced the bactericidal activities of the splenic T cells against Listeria monocytogenes, suggesting the clinical benefit of D-fraction in the case of anti-bacterial treatment for patients with high risks.

Cytokine Profile and Natural Killer Cell Activity in Listeria Monocytogenes Infected Mice Treated Orally with Petiveria Alliacea Extract

In this work, we investigated the effects of Petiveria alliacea extract on the production of Th1-type and Th2-type cytokines and on NK cells activity in normal and Listeria monocytogenes infected mice. Our results demonstrated that in normal/non-infected mice P. alliacea administration led to increased levels of Interleukin-2 (IL-2). The infection alone enhanced INF-γ levels and NK cell activity at 48 and 72 hours of infection. The treatment with five consecutive doses of 1000mg/kg/day of P. alliacea extract, given previously to infection, led to further increases in IL-2 levels, in relation to normal/non-infected/P. alliacea treated controls, and in INF-γ levels at 72h of infection, compared to infected mice. On the other hand, the production of IL-4 and IL-10 were not altered either by the infection or by the treatment with P. alliacea extract. NK cells activity increased at 48h and 72h following the inoculation of the bacteria. When mice were treated with P. alliacea previously to infection, NK activity was higher than that observed at 48h, 72h and 120h of infection in the infected animal. Based on these findings we suggest that P. alliacea up-regulates anti-bacterial immune response by enhancing both Th1 function and the activity of NK cells.

Bone marrow cellular composition in Listeria monocytogenes infected mice detected using ER-MP12 and ER-MP20 antibodies: a flow cytometric alternative to differential counting

Detailed assessment of bone marrow cellular composition is essential in the evaluation of various experimental in vivo systems, such as expression of transgenes, null mutations and stimulation of host defence in infection. Traditional morphological analysis of mouse bone marrow is laborious, requires specific cytological expertise, and is somewhat subjective. As an alternative, we have examined whether double labelling of bone marrow with the anti-precursor monoclonal antibodies ER-MP12 and ER-MP20 could be used for differential analysis by flow cytometry, as these antibodies define six relatively homogeneous cell populations in mouse bone marrow. Following a sublethal infection of mice with Listeria monocytogenes, we monitored changes in cellular composition of the bone marrow at various time points in three ways: differential morphological count; single-color flow cytometric analysis using markers for the myeloid, erythroid and lymphoid lineages; and double labelling with ER-MP12 and ER-MP20. As expected, the bone marrow composition changed dramatically during infection, leading to an increase of myeloid cells which peaked after 1 week of infection. Data determined by ER-MP12/20 flow cytometric analysis appeared to be in close agreement with both morphology and lineage marker analysis. In addition, ER-MP12/20 analysis provided more detailed information with regards to the presence of early myeloid precursors compared to lineage marker analysis. These data show that flow cytometric analysis of bone marrow using ER-MP12 and ER-MP20 monoclonal antibodies provides a relatively simple, rapid and objective assay when evaluating cellular composition in the bone marrow of the mouse.

Transient control of interleukin-4-producing natural killer T cells in the livers of Listeria monocytogenes-infected mice by interleukin-12.

Unconstrained development of gamma interferon (IFN-gamma)-secreting natural killer (NK) cells and T helper (Th) 1 cells is central to protection against Listeria monocytogenes. In contrast, interleukin 4 (IL-4) is considered harmful. IL-12 produced by infected macrophages promotes, and IL-4 interferes with, protective antilisterial immunity. The liver NK T lymphocytes, which are a potent source of IL-4, are downregulated at an intermediate stage of listeriosis. Here we demonstrate that endogenous IL-12 participates in the control of IL-4-producing liver NK T lymphocytes during listeriosis. The effects of L. monocytogenes infection on IL-4-producing liver NK T lymphocytes were reversed by antibody neutralization of IL-12 but not of IFN-gamma or tumor necrosis factor alpha (TNF-alpha). IL-4 production by liver NK T lymphocytes was virtually unaffected by heat-killed L. monocytogenes (HKL). Viable L. monocytogenes markedly increased the numbers of IL-12 producers in livers in parallel with an increase in macrophage numbers, whereas HKL failed to do so with similar efficiency. These results indicate that in the liver endogenous IL-12 improves protective immunity against listeriosis by downregulating IL-4-producing NK T lymphocytes. Moreover, our findings that HKL have a low level of IL-12-inducing activity and fail to control IL-4-producing NK T lymphocytes in the liver are consistent with the lesser protective capacity of HKL compared to that of live listeriae.

Down-Regulation of Listeria Monocytogenes-specific Th1 Cytokine Response by treatment of Mice with Goat Antibody to Mouse IgD

Injection of goat anti-mouse IgD antibody (GαM IgD) to mice has been shown to induce polyclonal IgG 1 and IgE production by B cells and IL-4 production by goat Ig-specific T cells. Surface IgD crosslinking also activates function of B cells as antigen presenting cells. Although the GαM IgD treatment is a well established system for regulation of immune response against antigens that bind to B cell receptor, we found that the GαM IgD treatment also influences immune response against irrelevant bacterial antigen. The T cells from the GαM IgD-treated Listeria monocytogenes-infected mice showed increased IL-4 production and decreased IFN-γ and IL-2 production against listerial antigen compared with those from control L. monocytogenes-infected mice. Interestingly, changes were also found in antigen presenting cells in the GaM IgD-treated mice. MHC class II expression of both B cells and macrophages decreased significantly in the GαM IgD-treated mice, suggesting cytokine induced by GαM IgD-treatment may suppress MHC class II expression and modulate APC function in the GαM IgD-treated mice. In accordance with the assumption, T cells from the GαM IgD-treated mice produced high amount of IL-4 and IL-10 in in vitro culture with goat serum which contain goat Ig. These result suggest that GαIgD treatment may modulate APC function in the GαM IgD-treated mice through Th2 type cytokine(s) produced by goat Ig-specific T cells, which results in changes of Th response against irrelevant antigen.