Interleukin-1 receptor type 1 is essential for control of cerebral but not systemic listeriosis

Listeria monocytogenes may infect the central nervous system and several peripheral organs. To explore the function of IL-1 receptor type 1 (IL-1R1) in cerebral versus systemic listeriosis, IL-1R1(-/-) and wild-type mice were infected either intracerebrally or intraperitoneally with L. monocytogenes. After intracerebral infection with various numbers of attenuated Listeria, IL-1R1(-/-) mice succumbed due to an insufficient control of intracerebral Listeria, whereas all wild-type mice survived, efficiently restricting growth of Listeria. IL-1R1(-/-) mice recruited increased numbers of leukocytes, especially granulocytes, to the brain compared with wild-type mice. In contrast, both IL-1R1(-/-) and wild-type mice survived a primary and secondary intraperitoneal infection with Listeria without differences in the hepatic bacterial load. In addition, both strains developed similar frequencies of Listeria-specific CD4 and CD8 T cells after primary and secondary intraperitoneal infection. However, an intraperitoneal immunization before intracerebral challenge infection neither protected IL-1R1(-/-) mice from death nor reduced the intracerebral bacterial load, although numbers of intracerebral Listeria-specific CD4 and CD8 T cells and levels of inducible nitric oxide synthase, tumor necrosis factor, and interferon-gamma mRNA were identical in IL-1R1(-/-) and wild-type mice. Collectively, these findings illustrate a crucial role of IL-1R1 in cerebral but not systemic listeriosis.     

CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by Recombinant Listeria monocytogenes during Secondary Infection

Understanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuated actA-deficient Listeria monocytogenes, on the CD8(+)-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinant L. monocytogenes. Challenges of immune mice with actA-deficient and with wild-type recombinant L. monocytogenes generated similar numbers of CD8(+) T cells specific for the NP118-126 epitope. High-dose immunization with actA-deficient L. monocytogenes resulted in substantial numbers of CD8(+) T cells specific for the L. monocytogenes LLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinant L. monocytogenes resulted in rapid control of the infection and decreased CD8(+)-T-cell responses against both the secreted and nonsecreted form of the recombinant antigen compared to the response of na?ve mice. In contrast, mice immunized with a low dose of actA-deficient L. monocytogenes had approximately 10-fold fewer effector and memory T cells specific for LLO91-99 and a substantially higher CD8(+)-T-cell response against the recombinant antigen after challenge with recombinant L. monocytogenes. Although mice immunized with low-dose actA-deficient L. monocytogenes had a substantial recall response to LLO91-99, which reached the same levels by 5 to 7 days postchallenge as that in high-dose-immunized mice, they exhibited decreased ability to control L. monocytogenes replication. Thus, the level of antivector immunity impacts the control of infection and efficiency of priming responses against new antigens introduced with the same vector.     

Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88

Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens. Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear. Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses. We demonstrate that following L. monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses. These T cell responses are sufficient to control secondary infection with a high dose of L. monocytogenes even in the absence of TLR signaling via MyD88. Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals.     

Requirement of secondary lymphoid tissues for the induction of primary and secondary T cell responses against Listeria monocytogenes

Activation of naive T cells is tightly controlled and depends on cognate interactions with professional antigen-presenting cells. We analyzed dependency on secondary lymphoid tissues for the activation of naive and memory CD4(+) and CD8(+) T cells following primary and secondary Listeria monocytogenes infection, respectively. In splenectomized lymphotoxin-beta receptor-deficient mice, lacking all secondary lymphoid tissues, oral infection with L. monocytogenes failed to induce bacteria-specific CD4(+) and CD8(+) T cell responses. Treatment of splenectomized wild-type mice with FTY720, a drug that prevents egress of T cells from lymph nodes, also reduced T cell responses after oral L. monocytogenes infection and blocked T cell responses after intravenous infection. FTY720-treated wild-type and lymphotoxin-beta receptor-deficient mice show only slightly impaired recall responses. However, T cell responses were profoundly inhibited when mice were splenectomized subsequently to recovery from primary infection. T cell transfer experiments demonstrated that the impaired secondary T cell response was not simply due to removal of a large fraction of memory T cells by splenectomy. Overall, these results indicate that not only primary T cell responses, but also secondary T cell responses, highly depend on the lymphoid environment for effective activation.     

Increased susceptibility to primary infection with Listeria monocytogenes in germfree mice may be due to lack of accumulation of L-selectin+ CD44+ T cells in sites of inflammation.

The host defense of germfree (GF) mice against primary infection with Listeria monocytogenes was compared with that of specific-pathogen-free (SPF) mice. In SPF mice, the numbers of bacteria in the peritoneal cavity, liver, and spleen decreased gradually to undetectable levels by day 8 after intraperitoneal infection with a sublethal dose (2 X 10(3) CFU) of L. monocytogenes. On the other hand, the elimination of bacteria in these organs of GF mice was significantly impaired at this stage after inoculation. We have reported previously that T cells coexpressing L-selectin and CD44 play an important role in protection against L. monocytogenes through trafficking to sites of inflammation. Consistent with our previous findings, the number of unique L-selectin+ CD44+ T cells in the peritoneal cavity was remarkably increased on day 8 after infection in SPF mice, whereas such an increase was not evident in GF mice at this stage. Listeria-specific T-cell proliferation was normally detected in the lymph node cells of GF mice inoculated with L. monocytogenes, whereas the T-cell-proliferative response of the peritoneal exudate cells of GF mice was significantly impaired compared with that of SPF mice. These results suggest that the priming of T cells against listerial antigens normally occurs in the peripheral lymphoid organs of GF mice but the trafficking of the activated T cells to the inflamed sites may be severely impaired in GF mice, resulting in increased susceptibility to infection with L. monocytogenes.     

The characterization of testicular cell (TC)-specific T-cell clones induced by intratesticular Listeria monocytogenes infection: TC-specific T cells with atypical cytokine profile transfer orchitis.

A unilateral infection of Listeria monocytogenes into the testis of mice induces not only Listeria-specific T cells but also autoreactive T cells that can transfer experimental autoimmune orchitis (EAO) into naive mice. To investigate the characteristics of the autoreactive T cells, we established six testicular cell (TC)-specific T-cell clones from the spleen of the intratesticularly infected mice. All the clones expressed CD4 and T-cell receptor (TCR) alpha beta, and four of the six clones expressed V beta 8. They showed proliferative response to TC in the presence of syngeneic spleen antigen-presenting cells, but did not cross-react to Listeria antigen (Ag). They produced interferon-gamma (IFN-gamma) when stimulated with TC, but interleukin-2 (IL-2), IL-4 and IL-10 were undetectable. IL-2 production was not detected even when they were restimulated with TC after a 10-day resting culture without Ag and IL-2, although they proliferated in the restimulation culture. Even in the presence of anti-IL-2 mAb, the TC-specific T-cell clones showed proliferative response against TC. The observations indicate that the TC-specific IFN-gamma-producing T cells proliferate in the absence of autocrine. Both intravenous and intratesticular injection of these clones transferred EAO in syngeneic naive mice. These results suggest that L. monocytogenes infection in the testis induces autoreactive orchitogenic CD4+ T cells without cross-reactivity to bacterial Ag. Furthermore, these data demonstrate that CD4+ T cells with an atypical cytokine profile can efficiently cause EAO.     

Effective induction of acquired resistance to Listeria monocytogenes by immunizing mice with in vivo-infected dendritic cells

Splenic dendritic cells (DCs) obtained from mice at 48 h after Listeria monocytogenes infection exhibited up-regulation of CD80 and produced higher titers of gamma interferon (IFN-gamma) and interleukin-12 (IL-12) than did DCs obtained from uninfected mice. Mice immunized with DCs obtained from mice that had been infected with L. monocytogenes 48 h before acquired host resistance to lethal infection with L. monocytogenes at 4 and 8 weeks. Immunization with DCs from heat-killed L. monocytogenes failed to induce resistance. Acquired antilisterial resistance is specific, since the immunized mice could not be protected from Salmonella enterica serovar Typhimurium infection. Infected DCs stimulated proliferation of naive CD4(+) and CD8(+) cells in vitro, suggesting that in vivo-infected DCs activate CD8(+) T cells, which are critical in acquired antilisterial resistance, as well as CD4(+) T cells. When wild-type mice were immunized with DCs from IFN-gamma-deficient mice, they were protected against a lethal L. monocytogenes challenge. In contrast, when mice were immunized with DCs from anti-IL-12 p40 monoclonal antibody-injected mice, they failed to gain acquired antilisterial resistance. These results suggest that DC-derived IL-12, but not IFN-gamma, may play a critical role in induction of acquired antilisterial resistance. Our present results suggest that splenic DCs obtained from mice infected with L. monocytogenes in vivo may be an effective immunogen with which to induce antigen-specific immunity.     

Memory phenotype CD8+ T cells with innate function selectively develop in the absence of active Itk

T cells with a memory-like phenotype and possessing innate immune function have been previously identified as CD8(+)CD44(hi) cells. These cells rapidly secrete IFN-gamma upon stimulation with IL-12/IL-18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8(+) T cells in Itk null mice have a phenotype of CD44(hi) similar to memory-like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFN-gamma upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to lipopolysaccharides by secretion of IFN-gamma. Transfer of these cells rescues the ability of IFN-gamma null mice to reduce bacterial burden following L. monocytogenes infection, indicating that these cells are functional CD8(+)CD44(hi) T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory-like CD8(+) T cells with innate function.     

Substantial in vivo proliferation of CD4(+) and CD8(+) T lymphocytes during secondary Listeria monocytogenes infection

In mice Listeria monocytogenes infection induces a strong T cell response. In an attempt to quantitatively analyze the magnitude and kinetics of the CD4(+) and CD8(+) T cell response during L. monocytogenes infection in vivo we used a T cell transfer system that is independent of in vitro cell culture techniques and information about the identity of immunogenic T cell epitopes. Our results demonstrate substantial expansion of the in vivo primed and transferred T cell populations in response to L. monocytogenes. At the peak of response, transferred T cells represented more than one third of the total CD4(+) and CD8(+) T cell populations in blood and spleen of recipient mice. After stimulation in vitro, 40 % of these CD4(+) T cells responded to heat-killed listeriae with the production of IFN-gamma. Thus, our results reveal that in addition to the large CD8(+) T cell population an almost equally large population of Listeria-reactive CD4(+) T cells is generated in response to L. monocytogenes infection.     

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.     

Administration of superantigens protects mice from lethal Listeria monocytogenes infection by enhancing cytotoxic T cells

Superantigens stimulate T-cell-receptor Vbeta-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated with Staphylococcus aureus enterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection with Listeria monocytogenes but not from lethal infection with Streptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore, L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8(+) T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4(+) T cells or CD8(+) T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination of L. monocytogenes-specific CD8(+) cytotoxic T lymphocytes (CTL). It was found that CD4(+) T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killed L. monocytogenes-immunized mice enhanced the protection from challenge of L. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection with L. monocytogenes, which was dependent on the enhanced L. monocytogenes-specific CTL activity in the presence of CD4(+) T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.     

A protective role of gamma delta T cells in primary infection with Listeria monocytogenes in autoimmune non-obese diabetic mice.

We investigated the host defense mechanism in primary infection with Listeria monocytogenes in non-obese diabetic (NOD) mice at pre-diabetic stage showing an impaired responsiveness of the alpha beta T cells to T-cell receptor (TCR) triggering. The NOD mice showed a deteriorated resistance at the late stage after an intraperitoneal infection with L. monocytogenes compared with BALB/c and C57BL/6 mice as assessed by bacterial growth in organs. Consistent with our previous findings, a prominent increase in number of gamma delta T cells was evident at the early stage after infection, while generation of Listeria-specific alpha beta T cells was impaired in these mice. In vivo administration of anti-TCR gamma delta monoclonal antibody (mAb) allowed L. monocytogenes to grow exaggeratedly in the NOD mice. These results imply that gamma delta T cells may be mainly involved in protection against primary infection with L. monocytogenes in NOD mice.     

Th0-like CD4+ T cells protect mice with murine retrovirus-induced immunodeficiency syndrome (MAIDS) against co-infection with Listeria monocytogenes.

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP-BM5 murine leukaemia virus (MuLv) infection. Although LP-BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell-mediated immunity, the mice with established MAIDS infected with LP-BM5 8 weeks previously, showed resistance to an intraperitoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed-type hypersensitivity response to heat-killed Listeria (HKL.) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+ alpha beta T cells but neither by gamma delta T cells nor natural killer (NK) cells. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) were produced by CD4+ T cells from Listeria-infected MAIDS mice in response to the in vitro stimulation with HKL, whereas IFN-gamma but not IL-10 were produced by those from Listeria-infected control mice. These results suggest that T-helper 0 (Th0)-like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice.     

Characterization of early gamma interferon (IFN-gamma) expression during murine listeriosis: identification of NK1.1+ CD11c+ cells as the primary IFN-gamma-expressing cells

Though it is well established that gamma interferon (IFN-gamma) is crucial to the early innate defense of murine listeriosis, its sources remain controversial. In this study, intracellular cytokine staining of IFN-gamma-expressing splenocytes early after Listeria monocytogenes infection revealed that NK1.1(+), CD11c(+), CD8(+) T, and CD4(+) T cells expressed IFN-gamma 24 h after infection. Contrary to the previous report, most IFN-gamma(+) dendritic cells (DC) were CD8alpha(-) DC. Unexpectedly, almost all CD11c(+) IFN-gamma-expressing cells also expressed NK1.1. These NK1.1(+) CD11c(+) cells represented primary IFN-gamma-expressing cells after infection. In situ studies showed these NK1.1(+) CD11c(+) cells were recruited to the borders of infectious foci and expressed IFN-gamma. A significant NK1.1(+) CD11c(+) population was found in uninfected spleen, lymph node, blood, and bone marrow cells. And its number increased significantly in spleen, lymph node, and bone marrow after L. monocytogenes infection. Using interleukin-12 (IL-12) p40(-/-) mice, IFN-gamma expression was found to be largely IL-12 p40 dependent, and the number of IFN-gamma-expressing cells was only about one-third of that of wild-type mice. Moreover, the IFN-gamma expression was absolutely dependent on live L. monocytogenes infection, as no IFN-gamma was detected after inoculation of heat-killed L. monocytogenes. Our findings not only provide an insight into IFN-gamma expression after in vivo infection but may also change the current perceptions of DC and natural killer cells.     

T lymphocyte dynamics during Listeria monocytogenes infection

Infection of mice with Listeria monocytogenes results in a robust T lymphocyte response that clears the pathogen and provides long term immunity from reinfection. The number of splenic CD4+ and CD8+ T cells and natural killer cells increases during primary and recall infection with L. monocytogenes, however the proportional increase is greatest for CD8+ T cells. The proportion of CD8 T cells expressing low levels of CD62L, a sign of activation, was increased among immune splenocytes, suggesting a substantial expansion of L. monocytogenes specific CTL. Analysis of CTL specific for the immunodominant LLO 91-99 epitope showed that essentially all were CD62Llo during the primary response, but that many upregulated expression of CD62L during the memory phase. Interestingly, the antigen specificity of nearly all additional CD62Llo CTL detected in spleens during recall L. monocytogenes infection can be accounted for with MHC class I tetramers complexed with four different epitopes. These studies demonstrate the complex T lymphocyte dynamics during infection with intracellular pathogens.     

CD8+ T cell contraction is controlled by early inflammation

Pathogen-specific CD8(+) T cells expand in number after infection and then their numbers invariably contract by 90-95%, leaving a stable memory cell pool. The chief features of this response are programmed early after infection; however, the factors regulating contraction are mostly undefined. Here we show that antibiotic treatment before Listeria monocytogenes infection induced numbers of protective memory CD8(+) T cells similar to those in control infected mice, by a pathway without contraction. The absence of contraction correlated with decreased early inflammation and interferon-gamma production and an increased fraction of CD8(+) T cells expressing the interleukin 7 receptor at the peak of the response. Thus, contraction is controlled by early inflammation but is not essential for the generation of protective memory CD8(+) T cells after infection.     

Priming of protective anti-Listeria monocytogenes memory CD8+ T cells requires a functional SecA2 secretion system

The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have demonstrated that this secretion system is also linked to the onset of long-term CD8(+) T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8(+) T cells that are required for protection against secondary challenge with wild-type (wt) L. monocytogenes. Since the SecA2 ATPase is well conserved among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3(+) memory CD8(+) T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8(+) T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.     

The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4-CD8alphaalpha+ T cells expressing T cell receptor (TCR)alphabeta or TCRgammadelta and CD4+ CD8alphaalpha+ T cells expressing TCR alphabeta. We show here that CD4+ CD8alphaalpha+ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/alphaEbeta7 integrin is important for homing and maintenance of i-IEL in intestine. Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion of L. monocytogenes into i-EC activated NFkappa-B and subsequently up-regulated the expression of IL-15 gene, which has a NFkappa-B binding site at the promoter region. i-IEL, especially gammadelta T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection with L. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.