Specific depletion reveals a novel role for neutrophil-mediated protection in the liver during Listeria monocytogenes infection

Previous studies have suggested that neutrophils are required for resistance during infection with multiple pathogenic microorganisms. However, the depleting antibody used in those studies binds to both Ly6G and Ly6C (anti-Gr-1; clone RB6-8C5). This antibody has been shown to deplete not only neutrophils but also monocytes and a subset of CD8(+) T cells. Recently, an antibody against Ly6G, which specifically depletes neutrophils, was characterized. In the present study, neutrophils are depleted using the antibody against Ly6G during infection with the intracellular bacterium Listeria monocytogenes (LM). Our data show that neutrophil-depleted mice are much less susceptible to infection than mice depleted with anti-Gr-1. Although neutrophils are required for clearance of LM, their importance is more pronounced in the liver and during a high-dose bacterial challenge. Furthermore, we demonstrate that the protection mediated by neutrophils is due to the production of TNF-α, but not IFN-γ. Additionally, neutrophils are not required for the recruitment of monocytes or the generation of adaptive T-cell responses during LM infection. This study highlights the importance of neutrophils during LM infection, and indicate that depletion of neutrophils is less detrimental to the host than depletion of all Gr-1-expressing cell populations.     

Neural route of cerebral Listeria monocytogenes murine infection: role of immune response mechanisms in controlling bacterial neuroinvasion

The pathologic features of cerebral Listeria monocytogenes infection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG-1)-deficient mice, L. monocytogenes spreads to the brain via a neural route. The neural route of invasion is suggested by (i) the immunostaining of L. monocytogenes in the trigeminal ganglia (TG) and brain stem but not in other areas of the brain; (ii) the kinetics of bacterial loads in snout, TG, and brain; and (iii) the increased resistance of mice infected with a plcB bacterial mutant (unable to spread from cell to cell). Gamma interferon (IFN-gamma) plays a protective role in neuroinvasion; inducible nitric oxide synthase (iNOS) accounts only partially for the protection, as shown by a comparison of the susceptibilities of IFN-gamma receptor (IFN-gamma R)-deficient, iNOS-deficient, and wild-type mice to snout infection with L. monocytogenes. The dramatically enhanced susceptibility of RAG-1-deficient, IFN-gamma R gene-deficient mice indicated the overall importance of innate immune cells in the release of protective levels of IFN-gamma. The source of IFN-gamma appeared to be NK cells, as shown by use of RAG-1-deficient, gamma-chain receptor gene-deficient mice; NK cells played a relevant protective role in neuroinvasion through a perforin-independent mechanism. In vitro evidence indicated that IFN-gamma can directly induce bacteriostatic mechanisms in neural tissue.     

Effects of administration of murine recombinant IL-4 on the resistance of mice to Listeria monocytogenes infection.

Mice given recombinant murine interleukin-4 as a single i.v. bolus concomitant with a Listeria monocytogenes challenge did not display increased anti-listeria resistance. Under certain conditions, IL-4 administration slightly increased the bacterial burdens in the spleens and livers of infected mice. This finding is consistent with previous reports that endogenous IL-4, or transfer of IL-4 producing TH2 cells, can be detrimental to host defense against microbial infection.     

Multiplication of Listeria monocytogenes in a murine hepatocyte cell line.

Listeria monocytogenes was shown to invade and multiply in a murine hepatocyte cell line (ATCC TIB73). Hemolytic and nonhemolytic L. monocytogenes strains exhibited similar abilities to invade hepatocytes, but only hemolytic L. monocytogenes multiplied within this cell line. Microscopic evaluation of monolayers stained with Wright stain demonstrated focal necrosis (plaques) in the hepatocyte monolayers, with large numbers of intracellular listeriae visible within the hepatocytes that lined the margins of these plaques. Murine recombinant interleukin-1 alpha, human recombinant tumor necrosis factor alpha, and murine recombinant gamma interferon did not affect the multiplication of L. monocytogenes in the hepatocytes. These data confirm in vivo observations of the intracellular multiplication of L. monocytogenes in hepatic lesions in infected mice.     

Toll-like receptor 2 is required for optimal control of Listeria monocytogenes infection

The control of Listeria monocytogenes infection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb to Listeria infection. In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice. Here we show that TLR2-deficient mice are more susceptible to systemic infection by Listeria than are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils. The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated by Listeria and were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection. Therefore, the present data demonstrate a role for TLR2 in the control of Listeria infection, but other MyD88-dependent signals may contribute to host resistance.     

Listeriolysin as a virulence factor in Listeria monocytogenes infection of neonatal mice and murine decidual tissue.

Listeriolysin is a 60-kDa protein which allows the growth of Listeria monocytogenes in macrophages and other cells and has been shown to be a virulence factor in Listeria infections of adult mice. However, the neonate and fetoplacental unit are major populations susceptible to listeriosis. Recent data indicate that macrophage and T-cell functions are markedly inhibited in these young mice, and the virulence of listeriolysin-negative (HLY-) and listeriolysin-positive (HLY+) Listeria cells in the setting of such inhibited macrophage and T-cell functions has not previously been examined. We now compare CNL 85/162, a transposon-induced, HLY- Listeria strain, and CNL 85/163, a spontaneous HLY+ revertant. We found that all 18 neonates injected with CNL 85/163 (HLY+) died within 12 days after an injection of 10(4) Listeria cells per mouse. In contrast, all 16 neonates injected with 1,000 times more CNL 85/162 (HLY-) cells survived more than 14 days. Three days after injection, growth of CNL 85/163 (HLY+) in the internal organs was more than 5 log greater than that of CNL 85/162 (HLY-). We also found that CNL 85/162 (HLY-) did not proliferate well in decidual tissue, which is a major component of the placental region. Our studies indicate that HLY- bacteria are not virulent in the neonate and the fetoplacental unit despite the inhibited immune functions at these sites.     

The role of macrophage migration inhibitory factor in lethal Listeria monocytogenes infection in mice

Macrophage migration inhibitory factor (MIF) has been characterized as a proinflammatory cytokine. Previous studies have indicated that MIF may play a beneficial role or a detrimental role in microbial infections, depending on pathogens. In this study, we investigated the role of MIF in Listeria monocytogenes infection. The MIF titers increased 6h after lethal L. monocytogenes infection but not in the sublethal infection. The elimination of bacteria from the spleens and livers was not affected by anti-MIF antibody (Ab) injection in the sublethal infection, whereas anti-MIF Ab treatment rescued mice from the lethal infection, suggesting that MIF plays a deteriorating role in lethal L. monocytogenes infection. Anti-MIF Ab treatment significantly augmented interleukin (IL)-10 production in the spleens and livers 24h after infection, suggesting that MIF might down-regulate IL-10 production. Although the administration of anti-IL-10 monoclonal Ab showed no significant effect on the bacterial growth in the organs, the bacterial infection was deteriorated by the combined administration of Abs against MIF and IL-10. On the other hand, anti-MIF Ab treatment also increased in the serum cortisol titer 6h after infection compared with the control immunoglobulin G-injected group. Depletion of endogenous IL-10 decreased serum cortisol titers. These results suggested that IL-10 and cortisol might be involved in the deteriorating effect of MIF on lethal L. monocytogenes infection.     

Critical role of neutrophils in eliminating Listeria monocytogenes from the central nervous system during experimental murine listeriosis

Neutrophils are the main inflammatory cell present in lesions involving the central nervous system (CNS) during human and murine listeriosis. In this study, administration of the neutrophil-depleting monoclonal antibody RB6-8C5 during experimental murine listeriosis facilitated the multiplication of Listeria monocytogenes in the CNS. These data suggest that neutrophils play a key role in eliminating bacteria that gain access to the CNS compartment. In addition, we provide evidence that their migration into the CNS may be necessary for the subsequent recruitment of macrophages and activated lymphocytes.     

Restricted replication of Listeria monocytogenes in a gamma interferon-activated murine hepatocyte line.

The intracellular pathogen Listeria monocytogenes replicates mainly in resting macrophages and hepatocytes residing in infected tissues. Both innate and acquired resistance strongly depend on activation of listericidal capacities of macrophages by gamma interferon (IFN-gamma) produced by natural killer cells and T lymphocytes. In contrast to macrophages, hepatocytes have been considered to serve purely as a cellular habitat, prolonging survival of the pathogen in the host. By using an immortalized murine hepatocyte line, the relationship between L. monocytogenes and this cell type has been analyzed in more detail. Our data reveal that hepatocytes are able to eradicate listeriolysin-deficient (avirulent) L. monocytogenes but fail to control growth of listeriolysin-expressing (virulent) L. monocytogenes organisms. Following stimulation with IFN-gamma, hepatocytes gained the capacity to restrict growth of virulent L. monocytogenes, although less efficiently than the highly listericidal IFN-gamma-activated macrophages. Hepatocytes costimulated with a combination of IFN-gamma, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) expressed the highest antilisterial activities. Although IFN-gamma-stimulated hepatocytes produced demonstrable levels of reactive nitrogen intermediates (RNI), the results of inhibition studies do not support a major role for these molecules in antilisterial hepatocyte activities. In contrast, inhibition of RNI produced by macrophages neutralized their antilisterial effects. IFN-gamma-stimulated, L. monocytogenes-infected hepatocytes expressed TNF-alpha mRNA, suggesting that they are a source of this cytokine during listeriosis. These studies suggest a novel function for hepatocytes in listeriosis: first, IFN-gamma-stimulated hepatocytes could contribute to listerial growth restriction in the liver, and second, through secretion of proinflammatory cytokines, they could promote phagocyte influx to the site of listerial growth.     

Evidence for a significant role of CD4+ T cells in adoptive immunity to Listeria monocytogenes in the liver.

Although the ability of CD8+ T cells to adoptively immunize mice against Listeria monocytogenes in the spleen is well established, the role of different T-cell subsets in anti-bacterial protection in the liver, a major target of Listeria infection, remains unclear. Therefore, the ability of sorted CD4+ and CD8+ T cells to adoptively immunize mice against a L. monocytogenes infection in the liver was studied. The results show that positively sorted CD4+ T cells from day 7 Listeria-immune mice were as effective as sorted CD8+ cells in transferring significant anti-Listeria protection in the liver. Similar findings were obtained when CD4+ and CD8+ T cells, negatively selected by antibody-induced complement-mediated depletion in vitro, were used for adoptive transfer. CD8+ T cells, however, were more efficient than CD4+ T cells in transferring protection in the spleen. Taken together, the results show that CD4+ T cells are at least as protective as CD8+ T cells against a L. monocytogenes infection in the liver, thereby arguing against the view that CD4+ T cells are of limited importance in adoptive immunity against listeriosis.     

Mouse peptidoglycan recognition protein PGLYRP-1 plays a role in the host innate immune response against Listeria monocytogenes infection

The role of mouse peptidoglycan recognition protein PGLYRP-1 in innate immunity against Listeria monocytogenes infection was studied. The recombinant mouse PGLYRP-1 and a polyclonal antibody specific to PGLYRP-1 were prepared. The mouse PGLYRP-1 showed antibacterial activities against L. monocytogenes and other Gram-positive bacteria. PGLYRP-1 mRNA expression was induced in the spleens and livers of mice infected with L. monocytogenes. The viable bacterial number increased, and the production of cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was reduced in mice when mice had been injected with anti-PGLYRP-1 antibody before infection. The levels of IFN-γ and TNF-α titers in the organs were higher and the viable bacterial number was reduced in mice injected with recombinant mouse PGLYRP-1 (rmPGLYRP-1) before infection. PGLYRP-1 could directly induce these cytokines in spleen cell cultures. The elimination of intracellular bacteria was upregulated in NMuLi hepatocyte cells overexpressing PGLYRP-1. The enhancement of the elimination of L. monocytogenes from the organs was observed in IFN-γ(-/-) mice by rmPGLYRP-1 administration but not in TNF-α(-/-) mice. These results suggest that PGLYRP-1 plays a role in innate immunity against L. monocytogenes infection by inducing TNF-α.     

Bacteremia is required for invasion of the murine central nervous system by Listeria monocytogenes

The ability of the facultative intracellular pathogenListeria monocytogenesto penetrate the central nervous system (CNS) was studied by following the kinetics of brain invasion and histological lesions during an acute intravenous (i.v.) infection in the mouse. CNS invasion occurred during the early phase of infection and produced severe meningoencephalitis characterized by multiple granulomatous foci predominantly located in the brainstem and associated with diffuse meningitis and an intense inflammatory reaction involving the choroid plexuses. Bacterial counts in the brain could reach 104.5–105.8by day 5 of infection with 1–2×106bacteria i.v., depending upon the bacterial strain used. It was found that CNS invasion was highly dependent upon the level and the duration of bacteremia, thus indicating that persistent bacteremia is essential to induce meningoencephalitis toL. monocytogenes.     

Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model

Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.     

Characterization of T-cell receptor gamma delta T cells appearing at the early phase of murine Listeria monocytogenes infection.

It has been shown that T-cell receptor (TcR) gamma delta + CD4- CD8- T cells increase in number and have an important role in early protection in murine Listeria monocytogenes infection. In this report, to characterize further the phenotype of the gamma delta T cells in listeriosis, we analysed V region gene usage and in vitro antigen recognition of the TcR gamma delta T cells in the peritoneal cavity of mice at the early phase after i.p. infection with a sublethal dose of L. monocytogenes. The gamma delta T cells predominantly expressed V delta 6 which has been reported to be expressed by TcR gamma delta-bearing foetal thymocyte hybridomas specific to mycobacterial and self heat-shock protein (hsp) 60. These early appearing CD3+ CD4- CD8- T cells in Listeria-infected mice, which were reported to be TcR gamma delta T cells, increased in proportion and in size by in vitro stimulation with recombinant hsp 60 from Mycobacterium bovis and purified protein derivative from M. tuberculosis but not by stimulation with heat-killed L. monocytogenes. A 65,000 MW molecule was detected in the lysate of viable L. monocytogenes but not in the lysate of heat-killed L. monocytogenes by a monoclonal antibody (mAb) raised against mycobacterial hsp 60. These results suggest that the V delta 6-bearing peripheral gamma delta T cells are activated by recognizing listerial hsp 60 expressed by viable L. monocytogenes. The hsp 60-reactive V delta 6-bearing T cells may have an important role in protection against L. monocytogenes and other parasites that express hsp 60 at high level.     

The cellular niche of Listeria monocytogenes infection changes rapidly in the spleen

The spleen is an important organ for the host response to systemic bacterial infections. Many cell types and cell surface receptors have been shown to play role in the capture and control of bacteria, yet these are often studied individually and a coherent picture has yet to emerge of how various phagocytes collaborate to control bacterial infection. We analyzed the cellular distribution of Listeria monocytogenes (LM) in situ during the early phase of infection. Using an immunohistochemistry approach, five distinct phagocyte populations contained LM after i.v. challenge and accounted for roughly all bacterial signal in tissue sections. Our analysis showed that LM was initially captured by a wide range of phagocytes in the marginal zone, where the growth of LM appeared to be controlled. The cellular distribution of LM within phagocyte populations changed rapidly during the first few hours, decreasing in marginal zone macrophages and transiently increasing in CD11c⁺ DC. After 4–6 h LM was transported to the periarteriolar lymphoid sheath where the infective foci developed and LM grew exponentially.     

4-1BB (CD137) is required for rapid clearance of Listeria monocytogenes infection

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is a T-cell-costimulatory receptor that is expressed on activated T cells, dendritic cells, and NK cells. Little has been reported about its role in early host defense against bacterial infection. In this study, we report that 4-1BB-deficient (4-1BB(-/-)) mice are much more susceptible to Listeria monocytogenes (intracellular bacteria) infections than wild-type mice. Upon L. monocytogenes infection, 4-1BB(-/-) mice showed a lower survival rate, a higher bacterial burden in organs, and larger hepatic microabscesses than 4-1BB(+/+) mice. 4-1BB(-/-) mice also had impairment in clearance of bacteria from the bloodstream. Neutrophils from 4-1BB(+/+) mice constitutively expressed 4-1BB, which could be activated to induce intracellular Ca(2+) influx by ligation with anti-4-1BB antibody. On the other hand, neutrophils from 4-1BB(-/-) mice were defective in reactive oxygen species generation, phagocytic activities, and intracellular Ca(2+) mobilization. In addition, mice pretreated with anti-4-1BB monoclonal antibody were much more resistant to L. monocytogenes infection than control antibody-treated mice. Our results support the notion that 4-1BB may play a major role in host defense against intracellular pathogens through neutrophil activation.     

The protective role of T-cell receptor Vgamma1+ T cells in primary infection with Listeria monocytogenes

We have previously reported that the T-cell receptor (TCR) gamma delta+ T cells increase in mice infected with an intracellular bacteria Listeria monocytogenes, and the cells predominantly express Vdelta6 and Vgamma1 genes. In this study, we used a monoclonal antibody (mAb) specific to TCR Vgamma1 to estimate the frequency of Vgamma1+ T cells and we discuss their significance in protection against L. monocytogenes. The spleen, liver and peritoneal exudate cells from mice intraperitoneally infected with L. monocytogenes were analysed by flow cytometry. In all the organs investigated, Vgamma1+ cells increased predominantly among TCR gamma delta+ T cells at an early phase (day 5-7) of the infection. To elucidate the significance of the Vgamma1+ T cells in the protection against L. monocytogenes, mice were depleted of TCR Vgamma1+ gamma delta T cells or all TCR gamma delta+ T cells by intraperitoneal inoculation of anti-Vgamma1 mAb or anti-pan TCR gamma delta mAb, respectively, before infection with L. monocytogenes. The bacterial growth in the spleen and the liver examined on day 5 after the infection increased significantly by the depletion of TCR Vgamma1+ T cells. The numbers of L. monocytogenes in TCR Vgamma1+ T-cell-depleted mice were nearly the same as in mice depleted of all TCR gamma delta+ T cells. These results demonstrated that Vgamma1+ T cells are the predominant population of gamma delta T cells in protection against L. monocytogenes at the early phase of the primary infection.     

Analysis of murine genetic predisposition to pneumococcal infection reveals a critical role of alveolar macrophages in maintaining the sterility of the lower respiratory tract

The study of pathogenic mechanisms of disease can be greatly facilitated by studying genetic differences in susceptibility to infection. In the present study, we compared the severity of pneumococcal infection in C57BL/6 (B6) and 129Sv mice. The results showed that 129Sv mice were remarkably more susceptible to pneumococcal infection than B6 mice. Bacterial clearance, proinflammatory mediators, leukocyte recruitment, and phagocyte activities were measured to examine potential immune factors associated with differences in susceptibility to pneumococcal infection. The greater susceptibility of 129Sv mice was associated only with inadequate alveolar macrophage bacterial killing, as indicated by significantly decreased initial bacterial clearance from the respiratory tract. Effective pneumococcal clearance was not dependent upon Toll-like receptor 2 (TLR2) expression, oxidative stress, or matrix metallopeptidase 12 (MMP-12) expression. Furthermore, phagocytosis analysis suggested that the deficiency found in 129Sv alveolar macrophages was not due to a lack of bacterial recognition but, rather, to reduced bacterial uptake. In conclusion, our findings indicate a crucial role of alveolar macrophage phagocytosis during innate defense against pneumococcal infection, which may explain the association of host genetic risk factors with predisposition to pneumococcal infection.     

Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria.

The capacity of the intracellular pathogen Listeria monocytogenes to activate the alternative pathway of human complement was examined. Incubation of L. monocytogenes with human serum in optimal conditions (20% Mg2+EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-chelated serum) consumed (31.3 +/- 3.9)% of C3 hemolytic activity and led to similar amounts of C3 deposition among the 27 strains tested, except for a rough mutant and the penicillin-induced L forms of strain EGD, which bound reduced amounts of C3. The same results were obtained with strains belonging to related species (L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii). Direct evidence is provided that L. monocytogenes induces the deposition of C3b and its cleavage products iC3b and C3d through ester and amide linkages, as demonstrated by the analysis of the released products of radiolabelled purified C3 after treatment with hydroxylamine. Our results clearly demonstrate that L. monocytogenes activates the alternative pathway of human complement, suggesting that bacteria in the blood or in tissues of infected patients are opsonized and targeted to C3 receptor-bearing cells such as macrophages.