Listeria Monocytogenes In Mice Research Articles

Virulence of Listeria Monocytogenes in Mice is Enhanced by Deletion of Pathogenicity Island 4

Virulence of Listeria Monocytogenes in Mice is Enhanced by Deletion of Pathogenicity Island 4

Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses.

Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection ( Jakic et al., 2021 ). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection. Graphic abstract: Figure 1.Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing Listeria monocytogenes) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.

Topical Simvastatin as Host-Directed Therapy against Severity of Cutaneous Leishmaniasis in Mice.

We recently demonstrated that statins mediate protection against intracellular pathogens, Mycobacterium tuberculosis and Listeria monocytogenes in mice. Here, we investigated the immunomodulatory potential of simvastatin as a topical or systemic host-directed drug therapy in controlling inflammatory responses in an experimental mouse model of cutaneous leishmaniasis caused by Leishmania major (LV39). In an ear infection model, topical application of simvastatin directly on established lesions significantly reduced severity of the disease reflected by ear lesion size and ulceration. The host protective effect was further accompanied by decreased parasite burden in the ear and draining lymph nodes in both BALB/c and C57BL/6 mice. Pre-treatment of these mice on a low-fat cholesterol diet and systemic simvastatin also reduced footpad swelling, as well as parasite burdens and ulceration/necrosis in the more robust footpad infection model, demonstrating the prophylactic potential of simvastatin for cutaneous leishmaniasis. Mechanistically, following L. major infection, simvastatin-treated primary macrophages responded with significantly reduced cholesterol levels and increased production of hydrogen peroxide. Furthermore, simvastatin-treated macrophages displayed enhanced phagosome maturation, as revealed by increased LAMP-3 expression in fluorescent microscopy and Western blot analysis. These findings demonstrate that simvastatin treatment enhances host protection against L. major by increasing macrophage phagosome maturation and killing effector functions.

Study of cellular immune responses to the Internalin B protein extracted from Listeria monocytogenes in Mice

This study was carried out to investigate the immunological activity of internalin B (InLB) protein which extracted from the cell wall of Listeria monocytogenes. Immunological activity was determined in vitro by the phagocytic activity assay and in vivo by bacterial clearance test in ninety six male white Swiss BALB/C mice were divided into five equal groups, G1 intrapertonailly injected with 0.5ml of InLB 50µg/ml;G2 intrapertonailly injected with 0.5ml of InLB 50µg/ml and inoculated with 1.2×103CFU/ml of L. monocytogenes.; G3 intrapertonailly injected with 0.5ml of InLB 50µg/ml followed by L. monocytogenes 24 hours later; G4 intrapertonailly injected with1.2×103CFU/ml L. monocytogenes a positive control; G5 intrapertonailly injected with 0.5ml of PBS a negative control, The results of the in vitro study showed the InLB at 50µg/ml induce the phagocytic activity by increase the Nitric oxide (NO) production as compared with Phytohemagglutinin-p (PHA); the results of the in vivo study was carried for 2weeks; at 4,8and 12days bacterial clearance assay; tissue of the internal organs (liver) of the G3 showed decrease in number of the bacteria at 12days (6×10-2 CFU) as compared to G2 and G4 (2×10-3CFU and 1.8×10-4CFU) respectively.The result presented in this study contribute for the first time in Iraq; that internalin B given intrapertonailly injection in mice for 12 days improves the immune responses by decreasing the bacterial number in liver tissue.

127 : Role of TGF-beta signaling in circulating and tissue resident memory CD8+ T cells

Tissue resident memory (Trm) CD8 + T cells are a newly described memory T cell populations that are distinct from circulating memory T cells. The Trm CD8 + T cell responses are critical for protection against pathogens that invade the host via skin and mucosal tissues. Transforming growth factor- β eta (TGF- β eta) is thought to be important for controlling the numbers of KLRG + effector T cells and promoting the residence of the Trm CD8 + T cells in several nonlymhpoid tissues including intestine. We will present data on how TGF- β eta signaling differentially regulates circulating and tissue resident memory CD8 + T cells after systemic infection with Listeria monocytogenes in mice.

In vivo activity of nisin A and nisin V against Listeria monocytogenes in mice.

BackgroundLantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo.ResultsHere we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 105 cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen.ConclusionThis analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.

Machine Learning Analysis of the Relationship Between Changes in Immunological Parameters and Changes in Resistance to Listeria monocytogenes: A New Approach for Risk Assessment and Systems Immunology

No method has been reported to predict, even approximately, the impact of mild-to-moderate changes in several immunological parameters on resistance to infection. The ability to make such predictions would be useful in risk assessment. In addition, equations that predict host resistance on the basis of changes in components of a complex biological system (the immune system) would fulfill one of the major goals of systems biology. In this study, multiple machine learning classification methods were used to predict the effects of a series of drugs and chemicals on host resistance to Listeria monocytogenes in mice on the basis of changes in several holistic immunological parameters. A data set produced under the sponsorship of the National Toxicology Program (NTP) was used in this study. The NTP data set was found to have a high percentage of missing data and to be noisy (probably due to the intrinsically stochastic nature of immune responses). Data preprocessing steps were used to mitigate these problems. In evaluating the machine learning classifiers, we first randomly partitioned the NTP data set into 10 subsets. Each time, we used nine subsets of the data to train the machine learning classifiers, and the remaining single subset to predict outcomes with regard to host resistance. This process was repeated until all 10 combinations of the 9-1 split of the subsets have been tested. The best of the classifiers predicted host resistance outcome correctly for 94.7% of cases, a result which indicates it is possible to identify mathematical expressions that will be useful for risk assessment and to establish a basis for systems immunology.

Clemastine causes immune suppression through inhibition of extracellular signal-regulated kinase–dependent proinflammatory cytokines

Antihistamines are considered safe and used worldwide against allergy, pruritus, nausea, and cough and as sleeping aids. Nonetheless, a growing number of reports suggest that antihistamines also have immunoregulatory functions. We examined the extent and by what potential mechanisms histamine-1-receptor (H1R) antagonists exert immune suppressive effects. Immune suppression by antihistamines and immunosuppressants was tested in mice infected with Listeria monocytogenes. Potential modes of action were studied invitro by using murine and human cells. We also tested whether injection of clemastine in healthy volunteers affected the activation of peripheral macrophages and monocytes. Finally, therapeutic application of clemastine-mediated immune suppression was tested in a murine model of sepsis. Clemastine and desloratadine strongly reduced innate responses to Listeria monocytogenes in mice as did dexamethasone. The immune suppression was MyD88 independent and characterized by inhibition of the mitogen-activated protein kinase-extracellular signal-regulated kinase signaling pathway, leading to overall impaired innate immunity with reduced TNF-α and IL-6 production. Surprisingly, the observed effects were H1R independent as demonstrated in H1R-deficient mice. Moreover, in a double-blind placebo-controlled clinical trial, 1 intravenous administration of clemastine reduced the TNF-α secretion potential of peripheral blood macrophages and monocytes. This inhibition could be exploited to treat sepsis in mice. The safety profile of antihistamines may need to be revisited. However, antihistamine-mediated immune suppression may also be exploited and find applications in the treatment of inflammatory diseases.

Listeria monocytogenesSerotypes 1/2c and 3c PossessinlH

To examine the serotype specificity of inlH, which encodes the virulence-associated surface protein InlH related to the intracellular survival of Listeria monocytogenes in mice, the presence of inlH in 337 L. monocytogenes strains, representing 11 different serotypes, was studied. A total of 106 strains representing 3 serotypes and 14 pulsed-field gel electrophoresis (PFGE) types were positive for inlH by polymerase chain reaction. inlH was present in all 99 serotype 1/2c and 3 serotype 3c strains. Moreover, 4 out of 129 (3%) serotype 1/2a strains carried inlH. All 106 strains representing serotypes 1/2b, 3a, 3b, 4a, 4b, 4c, 4d, and 7 and 125 out of 129 (97%) serotype 1/2a strains were inlH-negative. The coding sequences of the inlH genes of eight L. monocytogenes strains representing three serotypes and five PFGE types were identical. These results suggest that inlH is specifically present in serotype 1/2c, 3c, and a small fraction of 1/2a L. monocytogenes strains and exists as a single allele.

Fermentation Metabolites fromLactobacillus gasseriandPropionibacterium freudenreichiiExert Bacteriocidal Effects in Mice

Lactobacillus gasseri OLL 2716 promotes the elimination of Helicobacter pylori and is utilized in yogurts that are specifically labeled as health foods. On the other hand, milk whey fermented by Propionibacterium freudenreichii ET-3, which increases the numbers of Bifidobacterium, is effective for intestinal disorders. We previously demonstrated that oral administration of L. gasseri and P. freudenreichii fermentation metabolites (LP-FM) improved calf intestinal microflora and reduced the incidence of diarrhea. However, the detailed immunological mechanisms responsible for these effects remain to be fully understood. In this study, we investigated whether LP-FM stimulates the innate immune response and promotes the elimination of Listeria monocytogenes in mice. The C57BL/6 female mice that were treated with LP-FM or L. gasseri fermentation metabolites alone for 4 weeks had more peripheral white blood cells than the untreated control mice. In particular, LP-FM-treated mice had higher CD4- and CD8-positive T-cell counts. The levels of reactive oxygen and nitrogen species produced by peritoneal macrophages were also higher in LP-FM-treated mice. Furthermore, LP-FM-treated mice that were infected with L. monocytogenes exhibited significant enhancement of the elimination of Listeria from the spleen and the liver in comparison with untreated control mice infected with Listeria. The activation of innate immunity by LP-FM was increased by the combination of fermentation metabolites from P. freudenreichii. These results suggest that LP-FM, which contains metabolites from L. gasseri and P. freudenreichii, stimulates the function of the innate immune system, thereby significantly promoting the elimination of L. monocytogenes in mice.

C3 Promotes Expansion of CD8+ and CD4+ T Cells in a Listeria monocytogenes Infection

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.

Examination of host immune resistance against Listeria monocytogenes infection in cyclophosphamide-treated mice after dietary lipid administration

Despite the beneficial effects in the resolution of inflammatory disorders due to their immunosuppressive properties, n-3 polyunsaturated fatty acids are associated with a reduction of immune resistance to some microorganisms. Here, we examine the influence of different dietary lipids on host immune resistance against Listeria monocytogenes in mice treated with cyclophosphamide (CPA). Balb/c mice were fed one of four diets, which contained either olive oil (OO), fish oil (FO), hydrogenated coconut oil (HCO) or low fat (LF) for 4 weeks. Subsequently, mice were treated with CPA or PBS, prior to L. monocytogenes infection. Splenocyte proliferation, survival analysis, counts of viable bacteria from spleens and livers, and measurement of pro-inflammatory cytokine levels were determined. The FO-rich diet reduced survival, particularly in CPA-treated mice. CPA was responsible for a significant increase of viable bacteria recovery from spleens and livers within each group fed high fat diets, which was aggravated in mice fed an FO diet. In addition, a significant increase of both TNF-alpha and IL-12p70 levels was detected in this group. These results may acquire a crucial relevance in clinical nutrition, particularly when FO diets are administered to immunocompromised patients. The mechanism(s) that impair(s) the elimination of L. monocytogenes could be associated with a low mitogen-stimulated splenocyte proliferation, and with an alteration of pro-inflammatory cytokine production. The application of the neutropenic agent CPA moderately aggravates the immunosuppressive state mainly in FO-fed animals.

Stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections

We investigated the effect of a primary non-lethal infection with lymphocytic choriomeningitis virus (LCMV) on the course and outcome of a secondary infection with the Gram-negative Salmonella enterica serovar Typhimurium or the Gram-positive Listeria monocytogenes in mice. We found that at each stage of the viral infection the susceptibility of mice to bacterial super-infections changes dramatically and depends also on whether the secondary infection is a Gram-positive or Gram-negative one. The study shows that the outcome of the secondary infection is determined by a delicate balance between the overproduction of and the hypersensitivity to inflammatory cytokines (TNF-alpha and IFN-gamma), as well as by the changes in blood leukocytes occurring in mice in the course of viral infection.

Potential antimicrobial effects of human lactoferrin against oral infection with Listeria monocytogenes in mice

Listeria monocytogenes is a food-borne pathogen that causes serious listeriosis in humans. Antimicrobial effects of human lactoferrin (hLF) against L. monocytogenes have been clearly demonstrated in in vitro studies. However, in vivo studies have not been reported yet. This study investigated whether the oral administration of hLF could inhibit oral infection of listeria in BALB/c mice. The MICs for several strains of L. monocytogenes were determined, and the most sensitive strain was used for the animal work. hLF was administered to BALB/c mice for 7 days, commencing 4 days before oral infection. The effect of hLF was determined by bacterial enumeration and histopathological analysis of the liver and spleen, which are well-known as the major targets of oral listeria infection in mice. In bacterial enumeration, hLF decreased the number of L. monocytogenes cells in the liver. Histopathologically, the size and frequency of necrotic foci in the liver samples decreased with hLF administration. However, these changes were not observed in the spleen samples. The mRNA levels of inflammatory cytokines, such as interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, decreased in the liver of mice receiving hLF. This study has shown that hLF decreases the hepatic colonization of L. monocytogenes, hepatic necrosis and expression of inflammatory cytokines. It revealed that perorally given hLF could mediate antimicrobial and anti-inflammatory activities remote from the gut (i.e. in the liver) of mice challenged with L. monocytogenes.

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with ≥90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.

Characteristics of the gastritis induced by Listeria monocytogenes in mice: microbiology, histopathology, and mRNA expression of inflammatory mediators with time course of infection

Listeria monocytogenes induces the suppurative gastritis in some mice strains. In this study, characteristics of the gastritis caused by L. monocytogenes infection in mice were examined with time course of infection. Mice were administered intragastrically with 1.8×10 8 CFU of L. monocytogenes. Each three mice were sacrificed by cervical dislocation at 1, 3, 5, 7, 10, 14, 17, 21, and 28 days postinoculation (pi), respectively. Bacterial colonization in the stomachs reached the peak at 3 days pi, maintained over 4.3 log 10 CFU/g tissue until 14 days pi, and was cleared by 28 days pi. However, in the spleens and livers, the bacteria could not be detected after 7 days pi. The gastric lesions were the most prominent at between 3 and 7 days pi. The lesions consisted of marked neutrophilic infiltration, edema, vacuolar degeneration and necrosis of muscle cells and were more severe in the nonglandular region and fundus than in the pylorus, and were in submucosa, lamina muscularis, and serosa than in mucosa. mRNA expression of several cytokines (INF-γ, IL-1β, IL-5, IL-6, IL-12, and TNF-α) and chemokines (KC, MCP-1) increased in the gastric tissue of infected mice at 1–7 days pi and slightly decreased at 14 days pi. These findings would be useful for studying the pathological mechanism of human febrile gastroenteritis due to L. monocytogenes infection.

Changes in peritoneal myeloid populations and their proinflammatory cytokine expression during infection with Listeria monocytogenes are altered in the absence of gamma/delta T cells.

Evidence that gamma/delta T cells play a broad, immunoregulatory role has been accumulating steadily. We show here that myeloid cells are disregulated after peritoneal infection with Listeria monocytogenes in mice lacking gamma/delta T cells. Inflammatory populations of neutrophils and monocytes recruited to the site of infection remained longer. Intracellular cytokine analysis showed that frequencies of myeloid cells producing interleukin-12 and tumor necrosis factor alpha were higher and remained elevated longer after infection in mice genetically deficient in gamma/delta T cells. In vivo dye-tracking studies indicated that the majority of inflammatory monocytes differentiated into resident tissue macrophages in situ. In vitro experiments confirmed that monocytes harvested from mice lacking gamma/delta T cells were defective in their maturation process. This evidence suggests that gamma/delta T cells promote differentiation in the monocyte/macrophage lineage. These cells are important for bactericidal activity, inflammatory cytokine production, clearance of inflammatory neutrophils, and ultimately, antigen presentation to T cells. Regulation of monocyte/macrophage differentiation may underlie a broad segment of the phenotypic alterations that have been reported in mice lacking gamma/delta T cells.

Sodium pentobarbital anesthesia transiently enhances the severity of infection following intragastric, but not intravenous, inoculation of Listeria monocytogenes in mice

In the present study we observed that mice anesthetized with sodium pentobarbital were far more susceptible to gastrointestinal challenge with Listeria monocytogenes than were unanaesthetized mice. The effect of pentobarbital anesthesia was transient (gone within 2 h) and did not alter the severity of infection following i.v. challenge with L. monocytogenes. Treatment with pharmacological inhibitors of gastric acidity (i.e. cimetidine and omeprazole), or intestinal motility (loperamide), did not duplicate the effect of pentobarbital on gastrointestinal listeriosis. These findings suggest that sodium pentobarbital anesthesia causes a short-lived but striking diminution in resistance to gastrointestinal listeriosis in mice, via an undefined mechanism.

The effect of processed meat and meat starter cultures on gastrointestinal colonization and virulence of Listeria monocytogenes in mice

Listeria monocytogenes is a foodborne pathogen of major concern to the food industry in general and the meat industry in particular. The aim of this study was firstly to identify a strain of Listeria that was virulent in SPF BALB/c mice. Secondly, to investigate if a traditional meat starter culture (FloraCarn) and nontraditional meat starter (NTMS) cultures of dairy product and human origin ( Lactobacillus and bifidobacteria) inhibit this pathogen in vivo. In addition, the inhibition of Listeria was investigated in vitro. In vitro inhibition was investigated using an agar inhibition assay, where soft agar containing the pathogen was laid over colonies of NTMS cultures, and inhibition expressed as the zones of inhibition developing around the colonies. For assessment of virulence, mice were intragastrically challenged with broth cultures of five strains of Listeria. For assessment of anti-listeria effect in vivo, the Listeria strain proven to be most pathogenic (LM3) was given to mice in salami batter containing no other added cultures (control) or batter inoculated with either (1) FloraCarn, (2) a NTMS culture, or (3) a combination of FloraCarn and a NTMS culture. The batter was given to mice after a 3-day fermentation and faecal levels of pathogen and body weight were monitored. Intragastric challenge with LM3, but no other strains, resulted in a significant weight loss ( p<0.05) and up to 10 6 colony forming units (cfu) of LM3 per gram faeces. No weight loss was observed in animals fed with salami batter containing LM3. Consumption of salami batter fermented by a combination of NTMS culture ( Lactobacillus acidophilus LAFTI® L10) and FloraCarn reduced faecal levels of the pathogen by 2.5 log units compared to the control. Consumption of salami batter fermented with FloraCarn and LAFTI® L10 (L10) alone reduced faecal levels by 0.5–1 and 1.5 log units, respectively. Of the NTMS cultures investigated here, L10 displayed the greatest inhibition of LM3 in vitro. These results indicate that the ability of pathogenic Listeria to cause listeriosis is dependent on the nature of the food in which the pathogen is present, and that a traditional meat starter culture (FloraCarn) and some NTMS cultures, particularly L10, inhibit growth of the pathogen during passage through the gastrointestinal tract.

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice.

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.