ABSTRACT
There have been no studies on the susceptibility and host immune responses to an intranasal infection with Listeria monocytogenes. In this study, we compared the susceptibilities and cytokine responses between intranasal and intravenous infections with L. monocytogenes in mice. Moreover, we compared efficiency of acquisition of host resistance to L. monocytogenes infection between intranasally and intravenously immunized mice because an intranasal immunization of vaccines is reportedly available for induction of adaptive immunity against various infectious pathogens. The susceptibility to an intranasal infection with L. monocytogenes was markedly lower than that to the intravenous infection. The bacterial growth in the lungs, spleens, and livers was substantially similar between intranasally and intravenously infected mice. Titers of endogenous gamma interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) in the spleens, livers, and lungs were parallel to bacterial numbers in each organ of mice during intranasal infection and intravenous infection. IFN-γ-deficient mice and TNF-α-deficient mice were highly susceptible to intranasal infection as well as intravenous infection. Susceptibilities to intranasal and intravenous L. monocytogenes infection were the same in these cytokine-deficient mice. These results suggest that both IFN-γ and TNF-α play critical roles in host resistance to intranasal L. monocytogenes infection as well as the intravenous infection. Acquisition of host resistance to intravenous and intranasal L. monocytogenes infection was induced in intranasally immunized mice as well as intravenously immunized mice, suggesting that intranasal immunization is effective for prevention of a systemic infection with L. monocytogenes.
Listeria monocytogenes is an intracellular-growing bacterium that is ordinarily nonpathogenic to healthy persons; however, it is important as an opportunistic pathogen. The individuals at highest risk are pregnant women and their fetuses, newborn infants, debilitated elderly persons, and immunocompromised hosts. Epidemiological investigations provided evidence that L. monocytogenes may be transmitted as an enteric pathogen by contaminated foods, e.g., vegetables, milk, and dairy products, suggesting that natural L. monocytogenes infection occurs by the oral route (8, 16, 30). Alternatively, the bacterium can be reportedly detected in soil as a saprophytic organism in a plant-soil environment (7, 18, 33, 38).
Host resistance to L. monocytogenes infection is controlled by cell-mediated immunity that is regulated by cytokines. Studies including in vivo administration of anticytokine antibodies and gene knockout technology demonstrated that endogenous cytokines such as gamma interferon (IFN-γ), tumor necrosis factor alpha(TNF-α), interleukin-6 (IL-6), and IL-12 play important roles in host resistance against L. monocytogenes infection (3, 11, 12, 15, 17, 19, 26, 28). By contrast, IL-4 and IL-10 reportedly show the detrimental role in the resistance (4, 10, 36, 37).
Intranasal immunization with various vaccines for acquisition of adaptive immunity against infectious pathogens has reportedly become available (2, 13, 35). Therefore, we were interested in the susceptibility to an intranasal infection with L. monocytogenes, the host's cytokine responses, and their roles in the intranasal infection. Moreover, whether intranasal vaccination may be available for acquiring immunity to secondary challenge with L. monocytogenes is of interest. In this study, we demonstrate that immunodeficient mice such as IFN-γ−/− mice and TNF-α−/− mice were extremely susceptible to an intranasal infection with L. monocytogenes as well as the intravenous infection. Moreover, we show that an intranasal vaccination is effective for acquiring potent host resistance to both intranasal and intravenous L. monocytogenes infections.
MATERIALS AND METHODS
Animals.Male C57BL/6 mice, IFN-γ-deficient (IFN-γ−/−) on a C57BL/6 × Sv129 (32), the corresponding IFN-γ+/+ mice, TNF-α-deficient (TNF-α−/−) mice on a C57BL/6 × Sv129 background (34), and the corresponding TNF-α+/+ mice were used in this study. C57BL/6 mice were purchased from CLEA Japan, Inc., Tokyo, Japan. Mice were used when they were 6 to 8 weeks old. Animals were cared for under specific-pathogen-free conditions in the Institute for Animal Experiment, Hirosaki University School of Medicine. All animal experiments in this paper followed the Guidelines for Animal Experimentation of Hirosaki University.
Bacteria. L. monocytogenes 10403S strain was provided by T. Yamamoto, Division of Microbiology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan, with D. A. Portnoy's approval (Department of Molecular and Cellular Biology, Division of Infectious Diseases, School of Public Health, University of California, Berkley). Bacteria grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) were dispensed and stored at −80°C until use (20). The concentration of cell suspension was adjusted spectrophotometrically at 550 nm. Mice were infected intranasally with 0.01 ml or intravenously with 0.2 ml of a solution containing various doses of L. monocytogenes cells in 0.01 M phosphate-buffered saline (pH 7.4). For intragastric feeding, we slipped an animal-feeding needle (0.9 by 70 mm; Natsume Factory Inc., Tokyo, Japan) past the pharynx into the stomach, where the inoculum was injected. In some experiments, mice were reinfected intranasally or intravenously at 4 weeks after primary infections.
Determination of numbers of viable L. monocytogenes cells in the organs.The livers, spleens, and lungs of infected animals were homogenized in phosphate-buffered saline or 1% (wt/vol) 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfate (CHAPS) (Wako Pure Chemical Co., Osaka, Japan) on tryptic soy agar (Difco Laboratories). Colonies were routinely counted 24 h later.
Preparation of organ extracts for cytokine assays.The liver, spleen, and lung homogenates for IFN-γ and TNF-α assays were prepared as follows. The organs were aseptically removed from the mice and suspended in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 1% CHAPS, and 10% (wt/vol) homogenates were prepared with a Dounce grinder. They were clarified by centrifuging at 2,000 × g, e.g., for 20 min. The organ extracts were stored at −80°C until the cytokine assays were performed (20).
Cytokine assays.TNF-α and IFN-γ were determined by double sandwich enzyme linked immunosorbent assays (ELISAs) as described previously (22). Purified hamster anti-mouse TNF-α monoclonal antibody (Genzyme Co., Boston, Mass.) and rabbit anti-recombinant mouse TNF-α globulin (Genzyme) were used for TNF-α ELISA. All TNF-α ELISAs were run with recombinant mouse TNF-α (Genzyme). Purified rat anti-mouse IFN-γ monoclonal antibody produced by hybridoma R4-6A2 (31) and rabbit anti-recombinant mouse IFN-γ serum were used for IFN-γ ELISA. All IFN-γ ELISAs were run with recombinant mouse IFN-γ produced and purified by Genentech, Inc., San Francisco, Calif.
Statistical evaluation of the data.Data were expressed as means ± standard deviation, and the Wilcoxon rank sum test was used to determine the significance of the differences of bacterial counts in the organs between the control and experimental groups. The generalized Wilcoxon test was used to determine the significance of differences in the survival rates. The 50% lethal dose (LD50) was calculated by the method of Reed and Muench (27). Each experiment was repeated at least three times and accepted as valid only when the trials showed similar results.
RESULTS
Susceptibility of mice to intranasal infection with L. monocytogenes.C57BL/6 mice were infected intranasally with different doses of viable L. monocytogenes, and survival rates were observed for 10 days (Fig. 1). Survival rates were decreased, depending on the increase in the infectious dose. The LD50 of L. monocytogenes for an intranasal infection was 2 × 108 CFU, while that for an intravenous infection was 5 × 105 CFU (data not shown). When mice were infected intragastrically with even 2 × 109 CFU of L. monocytogenes, the mice all survived (data not shown).
Susceptibility to an intranasal infection with L. monocytogenes in mice. Ten mice per group were infected intranasally with different doses of L. monocytogenes. Survival rates did not change between day 10 and day 20 after infection.
Kinetics of the growth of L. monocytogenes in the organs of mice infected intranasally or intravenously.Next, the fates of L. monocytogenes in the livers, spleens, and lungs from intranasally and intravenously infected mice were compared. C57BL/6 mice were infected intranasally with 2 × 107 CFU or intravenously with 5 × 104 CFU of L. monocytogenes, and the number of bacteria in the livers, spleens, and lungs was determined on days 1, 2, 3, 5, and 8 after intranasal infection (Fig. 2A) and after intravenous infection (Fig. 2B). Bacterial growth in these organs began after day 1 of intranasal infection. The peak of bacterial numbers in the livers and lungs coincided for both infections, while the peak in the spleens was delayed in intranasally infected mice. L. monocytogenes was detected in the lungs on days 2 to 5 after intranasal infection, whereas bacteria were transiently detected on day 3 after intravenous infection.
Kinetics of the growth of L. monocytogenes in the organs of mice infected intranasally or intravenously. Mice were infected with 2 × 107 CFU of L. monocytogenes intranasally (A) or with 5 × 104 CFU of L. monocytogenes intravenously (B). Bacterial numbers were determined at the indicated times. Each point represents the mean ± standard deviation (error bar) for a group of four mice.
Kinetics of endogenous IFN-γ and TNF-α production in the organs of mice infected with L. monocytogenes intranasally or intravenously.Endogenous productions of IFN-γ and TNF-α in the livers, spleens, and lungs were compared between mice intranasally and intravenously infected with L. monocytogenes. C57BL/6 mice were infected intranasally with 2 × 107 CFU or intravenously with 5 × 104 CFU of L. monocytogenes, and the titers of IFN-γ and TNF-α in the organs were determined on days 1, 2, 3, 5, and 8 after infection (Fig. 3 and Fig. 4). In intranasal infection, IFN-γ was detected only on day 1 in the livers, while the production peaked on day 3, being parallel with the growth of bacteria in the spleens and lungs (Fig. 3A). IFN-γ production in intranasally infected mice was lower in the livers and spleens, compared with that in intravenously infected mice (Fig. 3B). In the lungs, on the contrary, IFN-γ production in intranasally infected mice was higher than that in intravenously infected mice. TNF-α production in the livers and spleens of intranasally infected mice was also lower than that in intravenously infected mice, whereas the titers in the lungs showed the higher level in intranasally infected mice (Fig. 4).
Kinetics of endogenous IFN-γ production in the organs of mice infected intranasally or intravenously with L. monocytogenes. Mice were infected with 2 × 107 CFU of L. monocytogenes intranasally (A) or with 5 × 104 CFU of L. monocytogenes intravenously (B). IFN-γ titers were determined at the indicated times. Each point represents the mean + standard deviation (error bar) for a group of four mice.
Kinetics of endogenous TNF-α production in the organs of mice infected intranasally or intravenously with L. monocytogenes. Mice were infected with 2 × 107 CFU of L. monocytogenes intranasally (A) or with 5 × 104 CFU of L. monocytogenes intravenously (B). TNF-α titers were determined at the indicated times. Each point represents the mean + standard deviation (error bar) for a group of four mice.
Susceptibilities of cytokine gene knockout mice to intranasal or intravenous infection with L. monocytogenes.We compared susceptibilities of IFN-γ−/− mice and TNF-α−/− mice to intranasal and intravenous infections with L. monocytogenes. Cytokine gene knockout mice were infected with 102 CFU to 104 CFU of L. monocytogenes, and survival rates were observed for 20 days (Fig. 5). IFN-γ+/+ mice and TNF-α+/+ mice all survived intranasal and intravenous infections with these infectious doses (data not shown), while both IFN-γ−/− mice and TNF-α−/− mice were highly susceptible to intranasal and intravenous infections. Approximate half of both gene knockout mice succumbed when they were infected intranasally with 103 CFU of L. monocytogenes (Fig. 5A and C). By contrast, TNF-α−/− mice were more susceptible than IFN-γ−/− mice when they were infected intravenously with L. monocytogenes (Fig. 5B and D). For instance, all IFN-γ−/− mice survived, but half of the TNF-α−/− mice succumbed when they were infected with 102 CFU of L. monocytogenes. On the other hand, half of the IFN-γ−/− mice and half of the TNF-α−/− mice succumbed to an intranasal infection with 103 CFU of L. monocytogenes, and most of the IFN-γ−/− mice and all of the TNF-α−/− mice died when they were infected with 104 CFU of L. monocytogenes (Fig. 5A and C). The LD50 of intranasal and intravenous infections in each group was calculated (Table 1). In intravenous infections, LD50s for IFN-γ−/− mice and TNF-α−/− mice were 700- and 8,000-fold higher than those for wild-type mice, respectively. In intranasal infections, by contrast, the LD50s for IFN-γ−/− mice and TNF-α−/− mice were 140,000- and 280,000-fold higher than those for wild-type mice, respectively. Moreover, the susceptibility of TNF-α−/− mice was 11.6-fold higher than that of IFN-γ−/− mice that were intravenously infected, while the susceptibility of TNF-α−/− mice was only 2-fold higher than that of IFN-γ−/− mice that were intranasally infected.
Susceptibility to an intranasal infection with L. monocytogenes in cytokine gene knockout mice. IFN-γ−/− mice were infected intranasally (A) or intravenously (B) with different doses of L. monocytogenes cells. TNF-α−/− mice were infected intranasally (C) or intravenously (D) with different doses of L. monocytogenes. The survival of each group was observed until day 20 after infection. Twelve mice were used in each group.
Comparison of LD50 between intranasal and intravenous infections in wild-type mice and cytokine deficient mice
Acquired host resistance to an intravenous challenge in mice immunized intranasally or intravenously with L. monocytogenes.We compared development of acquired immunity to an intravenous challenge of L. monocytogenes in mice between intranasal and intravenous immunizations with the same dose of the pathogen. C57BL/6 mice were immunized intranasally or intravenously with 5 × 104 CFU of L. monocytogenes, and they were challenged intravenously with 5 × 104 CFU of L. monocytogenes 28 days later. The bacterial numbers in the livers, spleens, and lungs were determined at 48 h after the secondary challenge (Table 2). As a negative control, nonimmunized mice were challenged with the same dose of bacteria. The growth of bacteria in the organs of both intranasally immunized mice and intravenously immunized mice was significantly inhibited, compared with that of the nonimmunized controls (P < 0.01).
Antilisterial resistance to an intravenous challenge with L. monocytogenes in intranasally immunized mice
Acquired host resistance to an intranasal challenge in mice immunized intranasally or intravenously with L. monocytogenes.We also compared development of acquired immunity to an intranasal challenge with L. monocytogenes in mice that were intranasally and intravenously immunized with the same dose of the pathogen. C57BL/6 mice were immunized intranasally or intravenously with 5 × 104 CFU of L. monocytogenes, and they were challenged intranasally with 106 CFU of L. monocytogenes 28 days later. The bacterial numbers in the livers, spleens, and lungs were determined at 72 h after the secondary challenge (Table 3). The growth of bacteria in the organs of intravenously immunized mice was significantly inhibited, compared with that of the nonimmunized controls. However, bacterial numbers of intranasally immunized mice were significantly reduced in the spleens (P < 0.01), but not in the livers and lungs (P > 0.05), compared with those of the nonimmunized controls. We also examined the ability of acquired resistance in mice that had been immunized intranasally with the higher dose of L. monocytogenes. The growth of bacteria in the organs of immunized mice was significantly inhibited, compared with that of the nonimmunized controls, when mice were immunized intranasally with 106 CFU of L. monocytogenes (Table 3).
Antilisterial resistance to an intranasal challenge with L. monocytogenes in intranasally immunized mice
DISCUSSION
In this study, we showed that the susceptibility to intranasal L. monocytogenes infection was markedly lower than that to the intravenous infection in immunocompetent mice. However, we demonstrated that immunodeficient mice such as IFN-γ−/− mice and TNF-α−/− mice were extremely susceptible to the intranasal infection as well as the intravenous infection. Moreover, we demonstrated that intranasal immunization is effective method to develop acquired immunity to systemic L. monocytogenes infection.
Lethal L. monocytogenes infection is reportedly responsible for hepatitis (29), meningoencephalitis (9), and pancreatitis (6). A recent report (5) documented that listeriolysin O, a member of the family of thiol-activated preforming toxins, is involved in L. monocytogenes-induced death. In this study, the slight infiltration of polymorphonuclear cells was observed in the lung tissues obtained from intranasally infected mice, but there were no significant differences of magnitude of inflammation in the lung tissues of mice between those subjected to intranasal infection and intravenous infection (data not shown), suggesting that pneumonia may not be the cause of death in intranasal infection.
L. monocytogenes was persistently detected in the lungs on days 2 to 5 after intranasal infection (Fig. 2A), whereas bacteria were transiently detected on day 3 after intravenous infection (Fig. 2B), indicating that occurrence of L. monocytogenes infection in lungs is more efficient in the intranasal route than in the intravenous route. The bacterial growth in the livers and spleens was substantially similar between intranasal infection and intravenous infection, although commencement of bacterial growth in the spleens was delayed in intranasal infection (Fig. 2). It should be noted that significant bacterial growth in the livers and spleens was not observed on day 1 after intranasal infection, different from the case of intravenous infection (Fig. 2). Similarly, our previous study showed that bacterial growth in livers and spleens began on day 2 when L. monocytogenes infection was induced by an oral route (23, 25). These results suggest that L. monocytogenes can finally result in systemic infection irrespective of routes of invasion.
Our previous study demonstrated that various endogenous cytokines could be detected in the circulation and infectious foci in parallel with bacterial growth during L. monocytogenes infection (20-22, 24, 25). In this study, kinetics of endogenous IFN-γ and TNF-α in the spleens, livers, and lungs were compared between intranasal infection and intravenous infection (Fig. 3 and Fig. 4). The characteristic of intranasal infection was observed in endogenous IFN-γ production. Namely, the titers were higher in the lungs but the production was lower in the spleens and livers of intranasally infected mice, compared with those in the intravenously infected group. It has been recognized that both IFN-γ and TNF-α play central roles in host resistance against L. monocytogenes infection (3, 11, 12, 19, 26, 28). In this study, the critical roles of IFN-γ and TNF-α in L. monocytogenes infection were confirmed in cytokine gene-deficient mice because both IFN-γ−/− mice and TNF-α−/− mice were extremely susceptible to intranasal infection (Fig. 5). Immunocompetent mice were 400-fold less sensitive to intranasal infection than to intravenous infection (Table 1). By contrast, the differences of susceptibilities between intranasal infection and intravenous infection were only 2-fold in IFN-γ−/− mice and 11.6-fold in TNF-α−/− mice (Table 1), suggesting that the implication of both cytokines in host resistance to the intranasal infection might be stronger than that to the intravenous infection. Moreover, the results suggest that IFN-γ dependency might be higher in host resistance to an intranasal infection with L. monocytogenes than that to the intravenous infection.
Recently, intranasal immunization with various vaccines for acquisition of adaptive immunity against infectious diseases has reportedly become available (2, 13, 35). Therefore, the mouse model of an intranasal infection with L. monocytogenes used herein must be a useful model to analyze the mechanism of immune responses induced by intranasal immunization. In the present study, acquisition of host resistance to intravenous L. monocytogenes infection was upregulated in the spleens, livers, and lungs of intranasally immunized mice as well as intravenously immunized mice (Table 2), suggesting that intranasal immunization with L. monocytogenes is effective for prevention of the systemic infection. By contrast, when mice were immunized with the same dose of L. monocytogenes intranasally or intravenously, the elimination of bacteria was significantly enhanced in the spleens but not in the livers and lungs of intranasally immunized mice (Table 3). The higher dose of L. monocytogenes was required to acquire the effective elimination of bacteria from the livers and lungs in intranasally infected mice (Table 3). Recent studies demonstrated that dendritic cells from lungs and liver preferentially induce differentiation of T-helper 2 cells (14) and that lung-derived dendritic cells induce the differentiation of T-regulatory cells (1). Cytokines derived from T-helper 2 cells and T-regulatory cells, such as IL-4 and IL-10, are known to inhibit antilisterial resistance (4, 10, 36, 37). Hence, there is a possibility that intranasal infection with L. monocytogenes may induce T-helper 2 cells and T-regulatory cells in these organs.
In conclusion, an intranasal vaccination is effective for acquiring potent host resistance to systemic infection with L. monocytogenes infections. We are now attempting to determine the best condition, including the form of immunogens and adjuvants, under which to induce acquired immunity to L. monocytogenes infection by intranasal immunization and to determine the immune responses to intranasal L. monocytogenes infection in lungs.
ACKNOWLEDGMENTS
This work was supported in part by grants-in-aid for General Scientific Research (10670247 and 13670261) provided by the Japanese Ministry of Education, Science, Sports, and Culture.
FOOTNOTES
- Received 18 March 2002.
- Returned for modification 23 April 2002.
- Accepted 23 May 2002.
- Copyright © 2002 American Society for Microbiology
