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Infection and Immunity, September 2003, p. 4891-4900, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.4891-4900.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Increased Mortality and Dysregulated Cytokine Production in Tumor Necrosis Factor Receptor 1-Deficient Mice following Systemic Klebsiella pneumoniae Infection

Thomas A. Moore,^* Michelle L. Perry, Andrew G. Getsoian, Christine L. Monteleon, Anna L. Cogen, and Theodore J. Standiford

Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, Michigan

Received 24 February 2003/ Returned for modification 17 April 2003/ Accepted 27 May 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
A significant clinical complication of pulmonary infections with Klebsiella pneumoniae is peripheral blood dissemination, resulting in a systemic infection concurrent with the localized pulmonary infection. In this context, little is known about the role of tumor necrosis factor receptor 1 (TNFR1)-mediated innate immune responses during systemic Klebsiella infections. Mice lacking TNFR1 were significantly more susceptible to Klebsiella-induced mortality following intravenous inoculation. Bacterial clearance was impaired in TNFR1-deficient mice at early times following infection. Unexpectedly, bacterial burdens at the onset of mortality (days 2 to 3 postinfection) were not higher in mice lacking TNFR1. However, elevated production of liver-associated proinflammatory cytokines (interleukin-12, tumor necrosis factor alpha [TNF-[, and gamma interferon [IFN-]) and chemokines (MIP-1, MIP-2, and MCP-1) was observed within the first 24 h of infection. Additionally, excessive plasma-associated IFN- was also observed late in the course of infection (day 3). Spleen cells from day-3 infected TNFR1-deficient mice secreted markedly enhanced levels of IFN- when cultured in vitro. Additionally, there was a marked increase in the total number of activated lymphocyte subsets as indicated by CD69 upregulation. A notable exception was the sharp decrease in the frequency of splenic NK T cells in infected TNFR1 knockout (KO) mice. Anti-TNF- therapy in TNFR1 KO mice significantly reduced chemokine production and liver injury. Combined, these data indicate a dysregulated antibacterial host response following intravenous Klebsiella infection in the absence of TNFR1 signaling, resulting in heightened cytokine production and hyperactivation of specific splenic lymphocyte subsets.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Klebsiella pneumoniae is a leading cause of nosocomial and community-acquired gram-negative bacterial pneumonia, resulting in a severe pyogenic infection with high mortality rates in the absence of therapeutic intervention (3, 34, 37). A significant clinical complication of K. pneumoniae infection is peripheral blood dissemination, resulting in bacteremia concurrent with the localized pulmonary infection (19, 49). Inability to clear blood-borne bacteria can lead to a state of overwhelming bacteremia, which can culminate in multiple organ dysfunction syndrome and increased mortality.

Tumor necrosis factor alpha (TNF-) is a proinflammatory cytokine that is rapidly produced following pathogenic insult, resulting in the initiation of a proinflammatory cytokine cascade which can have both beneficial and detrimental effects. Secretion of TNF- has been shown to be critical for the resolution of a variety of bacterial, fungal, and parasitic infections. The absence of TNF- bioactivity results in significant increases in mortality which correlate with an inability to clear the infectious agent (10, 17, 22, 26, 43, 45, 48). Conversely, excessive TNF- production in the context of systemic bacterial infection, abdominal sepsis, or endotoxemia results in increased mortality and organ injury (20, 21, 36, 46). The biological activities of TNF- are mediated by two distinct but structurally related receptors belonging to the tumor necrosis factor receptor (TNFR) supergene family (39). TNFR1 (p55) contains an intracytoplasmic death domain and has been shown to mediate the majority of the biological effects of TNF-, including apoptosis, toxic shock, germinal center formation, and adhesion molecule upregulation. TNFR2 (p75) lacks a death domain, and its role in mediating TNF- activity is less well established. Recently, however, it has been reported that membrane-bound TNF- is more efficient at activating TNFR2 than the soluble form of TNF-. Additionally, TNFR2 ligation amplifies TNFR1 triggering, resulting in enhanced TNFR1-mediated activity. The absence of TNFR1 has been shown to be detrimental to resistance directed against a wide variety of microbial pathogens (1, 6, 30-32, 35, 42). These data indicate the importance of TNFR1 ligation and signal transduction for successful host antibacterial responses. In contrast, TNFR2-deficient mice have largely displayed a minimal phenotype when challenged with a variety of pathogens (6, 30, 31, 42).

It has previously been shown that TNF- neutralization during localized K. pneumoniae pulmonary infection resulted in greatly increased mortality and bacterial burdens, indicating that the interaction of TNF- with one of its receptors (most likely TNFR1) is critical for the resolution of an organ-specific K. pneumoniae infection (22). Recently, however, the differential requirement for gamma interferon (IFN-) during pulmonary and systemic infection with K. pneumoniae was reported (28). Clearance of a localized lung infection was critically dependent on IFN- production, while resolution of a blood-borne infection was IFN- independent. These results raised the possibility that the requirement of TNF--mediated signaling during Klebsiella infection may differ depending on the type and site of infection. Here we report on the relative importance of TNFR1- and TNFR2-mediated signaling during systemic, blood-borne gram-negative bacterial infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. B6.129-Tnfrsf1a^tm1Mak (TNFR1-deficient), B6.129-Tnfrsf1b^tm1Mwm (TNFR2-deficient), and C57BL/6J wild-type mice were purchased from the Jackson Laboratory and housed under specific-pathogen-free conditions within the animal care facility at the University of Michigan until the day of sacrifice. All experimental animal procedures were approved by the University Committee on Use and Care of Animals at the University of Michigan.

K. pneumoniae inoculation. K. pneumoniae strain 43816 (serotype 2) (American Type Culture Collection, Manassas, Va.) was grown in trypic soy broth (Difco, Detroit, Mich.) overnight at 37°C. Bacterial concentrations were determined by measuring the absorbance at 600 nm and were compared to a predetermined standard curve. Bacteria were then diluted to the desired concentrations for inoculation. For intravenous infection, mice were warmed under a heat lamp for an appropriate time to allow vasodilation of the tail vein. Bacteria [(5 to 8) x 10⁴], diluted in pyrogen-free saline, were injected into the tail vein in a 0.5-ml volume with a 27-gauge needle. For all experiments, an aliquot of the inoculated K. pneumoniae suspension was serially diluted onto blood agar plates to confirm the actual dose of injected bacteria.

Whole-liver and -spleen homogenization for CFU and cytokine analyses. At designated times, mice were euthanized by inhalation of CO₂. Livers or spleens were perfused with 2 to 3 ml of phosphate-buffered saline-5 mM EDTA and were removed for analyses as previously described (16, 27). Briefly, tissues were homogenized by using a tissue homogenizer (Biospec Products, Bartlesville, Okla.) in 1 ml of phosphate-buffered saline-Complete protease inhibitor cocktail (Boehringer Mannheim Biochemicals, Chicago, Ill.). For determination of CFU in organs, a small aliquot of tissue homogenate was serially diluted, plated onto blood agar plates, and incubated at 37°C, and colonies were counted.

For total-liver cytokine analyses by enzyme-linked immunosorbent assays (ELISA), tissue homogenates were sonicated briefly to ensure complete cellular disruption and then centrifuged at 1,500 x g for 10 min. The supernatants were collected and assessed for cytokine levels by ELISA. Murine interleukin-12 (IL-12), TNF-, IL-10, IFN-, macrophage inflammatory protein 1 (MIP-1), monocyte chemoattractant protein 1 (MCP-1), and MIP-2 were quantitated by using a modification of a sandwich ELISA method (11). Assays were shown to be specific for the indicated murine cytokine and not to cross-react with any other murine cytokines tested.

Peripheral blood CFU and cytokine analyses. For determination of bacterial numbers in peripheral blood, mice were euthanized, and heparinized blood was collected by cardiac puncture at the indicated times. Serial dilutions were plated onto blood agar plates and incubated at 37°C, and colonies were counted.

For IFN-, TNF-, and IL-12 analyses, blood samples were spun at 10,000 x g for 15 min, and plasma samples were then collected and frozen at -70°C until cytokine analysis. Cytokines were analyzed by using commercially available OptEIA ELISA kits according to the manufacturer's procedures (BD PharMingen, San Diego, Calif.).

Plasma AST analyses. Levels of aspartate aminotransferase (AST) in plasma, as an indication of hepatic cellular injury, were determined in peripheral blood samples collected 1 day after K. pneumoniae inoculation. Heparinized blood samples were spun at 10,000 x g for 15 min; then plasma samples were collected and frozen at -70°C until AST activities were analyzed. AST activity was quantitated by the Clinical Chemistry Laboratory at the University of Michigan Medical Center by use of an automated spectrophotometric assay.

In vivo TNF- neutralization. Mice were injected intraperitoneally with 500 µg of a neutralizing anti-TNF- monoclonal antibody (clone MP6-XT3) 2 h prior to intravenous inoculation with K. pneumoniae. For survival studies, mice received a second injection of 250 µg on day 2 postinfection.

In vitro splenocyte culture and IFN- secretion analyses. Spleens from uninfected and day-3 infected C57BL/6 and TNFR1-deficient mice were removed for analyses. Single-cell suspensions were prepared, red blood cells were lysed, and cells were plated at a concentration of 6 x 10⁵ per well in 48-well tissue culture plates in a final volume of 600 µl. Cells were cultured for 24 h in complete tissue culture medium (RPMI plus 10% fetal calf serum, 100 µM nonessential amino acids, 1 mM sodium pyruvate, 55 µM 2-mercaptoethanol, and penicillin-streptomycin; Gibco Life Technologies, Rockville, Md.) with or without 50 ng of phorbol myristate acetate (PMA)/ml and 100 ng of ionomycin/ml. Cell culture supernatants were harvested and stored at -70°C until analysis for IFN- levels. IFN- production was detected using commercially available capture and detection antibodies (R&D Systems, Minneapolis, Minn.). The ELISA detection limit was routinely <32 pg/ml.

Multiparameter flow cytometric analyses of splenic lymphocytes. Single-cell splenocyte suspensions from uninfected and infected C57BL/6 or TNFR1-deficient mice were isolated as described above. All staining reagents were purchased from PharMingen (San Diego, Calif.). Nonspecific antibody binding was blocked by incubation with an anti-CD16/32 antibody (clone 2.4G2) prior to specific antigen staining. The activation statuses of lymphocyte subsets were determined by incubation with an anti-CD69-biotin antibody (clone H1.2F3) detected with streptavidin-allophycocyanin. Specific lymphocyte subsets were detected by using the following directly conjugated (with fluorescein isothiocyanate, phycoerythrin, or PerCP-Cy5.5) antibodies: anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD161 (anti-NK1.1; clone PK136), anti-Tcrß (clone H57-597), and anti-Tcr (clone GL3). Cells were analyzed on a dual-laser FACScalibur cytometer (Becton Dickinson, San Jose, Calif.) with excitation wavelengths of 488 and 635 nm. Data analyses were performed using the Cellquest (Becton Dickinson) software package.

Statistical analyses. Statistical significance was determined by the unpaired, two-tailed Student t test; analysis of variance for multiple group comparisons by use of the Student-Newman-Keuls posttest; and Fisher's exact test. Calculations were performed using InStat 3 for Macintosh (GraphPad Software, San Diego, Calif.). Statistical analyses of survival curves were performed by the log rank test using the Prism 3 for Macintosh software program (GraphPad Software).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased mortality in TNFR1- but not TNFR2-deficient mice following intravenous K. pneumoniae infection. To determine the importance of TNFR1 or TNFR2 signaling during systemic blood-borne bacterial infections, TNFR1- or TNFR2-deficient mice and C57BL/6 wild-type mice were intravenously inoculated with K. pneumoniae and monitored for survival. In the first series of experiments, 100% of the TNFR1-deficient mice succumbed to an inoculation dose of 7 x 10⁴ CFU by day 5, with mortality beginning as early as day 1 postinfection (Fig. 1A). In contrast, 40% of infected C57BL/6 mice survived the infection. In the second series of experiments, TNFR2-deficient mice were no more susceptible than their wild-type counterparts when inoculated with 5 x 10⁴ CFU (Fig. 1B).

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FIG. 1. Increased mortality in TNFR1- but not TNFR2-deficient mice following intravenous K. pneumoniae infection. (A) TNFR1-deficient mice and their C57BL/6 wild-type controls were intravenously inoculated with 6.8 x 104 K. pneumoniae bacteria, and survival was monitored over 8 days. TNFR1-deficient mice displayed increased mortality as early as day 1 postinfection, and by day 5 all were dead (P < 0.0002 for comparison with control mice). Survival curves were generated from two independent experiments with a total of 18 mice per group. (B) TNFR2-deficient mice and their wild-type controls were intravenously inoculated with 5 x 104 bacteria and monitored for survival over 8 days. Mortality rates were identical for the two groups of animals. Survival curves were generated from three independent experiments with a total of 25 mice per group.

Impaired early but not late bacterial clearance in TNFR1-deficient mice following intravenous infection. TNFR1-deficient mice were significantly more susceptible to blood-borne Klebsiella infection than either TNFR2-deficient or wild-type mice. To determine whether impaired bacterial clearance was responsible for this enhanced mortality, the kinetics of bacterial clearance in the blood and liver were examined for TNFR1-deficient mice (Table 1, experiment A). TNFR1-deficient mice displayed an early impairment in bacterial clearance at 6 h postinfection. Impaired clearance of bacteria from blood continued through the first 24 h of infection, after which clearance kinetics were similar to those of wild-type animals. Interestingly, the clearance of liver-associated bacteria in TNFR1-deficient mice, while impaired at 6 h, was not impaired at later times (>24 h) compared to that in wild-type mice. In fact, the trend was for improved bacterial clearance relative to that of infected wild-type mice, reaching levels of significance at 48 h postinfection. These data indicate an impaired ability to clear bacteria early during the course of infection in mice lacking TNFR1. However, bacterial burdens at the onset of mortality (days 2 to 3) were not greater than those in infected wild-type animals.

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TABLE 1. Bacterial burdens in TNFR1- and TNFR2-deficient mice following intravenous K. pneumoniae infectiona

The kinetics of bacterial infection in TNFR2-deficient mice were similar to those in infected wild-type control mice, with only a transitory increase in levels of blood-borne bacteria noted at 24 h postinfection (Table 1, experiment B).

Liver injury in TNFR1-deficient mice and wild-type mice following intravenous infection. The unimpaired clearance of bacteria from the livers of TNFR1-deficient mice at the onset of mortality was unexpected in light of the increased mortality observed in these animals. To determine if excessive liver injury may have contributed to increased mortality, release of the hepatocyte-associated enzyme AST into peripheral blood was examined. At 24 h postinfection, TNFR1-deficient, TNFR2-deficient, and wild-type control mice all had significantly elevated, but similar, plasma AST levels compared to those of uninfected animals (Fig. 2). By 48 h postinfection, AST levels had dropped to near baseline in all three groups of infected animals (data not shown). These data suggest that excessive liver injury is not likely to explain the increased mortality seen in TNFR1-deficient mice.

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FIG. 2. Liver injury in TNFR1- or TNFR2-deficient mice and wild-type mice following intravenous infection. Plasma AST levels, as an indication of liver cellular injury, were determined 24 h following bacterial infection. All three groups of infected mice displayed a 10-fold increase in AST activity 24 h postinfection. Data are presented as mean AST activities from three independent experiments with a total of 18 to 21 mice per group. Error bars, standard errors of the means.

Increased production of liver-associated proinflammatory cytokines and chemokines in TNFR1-deficient mice. The data obtained thus far indicated that the increased mortality seen in TNFR1-deficient mice was not due to impaired bacterial clearance or excessive liver injury. To determine if dysregulated cytokine production may in part contribute to increased mortality, hepatic cytokine levels were measured at various times following intravenous infection. Rapid upregulation of liver-associated IL-12 and TNF- levels was noted within 6 h postinfection in wild-type animals, with levels decreasing over the next 48 h (Fig. 3). Interestingly, IL-12 and TNF- levels were significantly higher in TNFR1-deficient mice than in wild-type mice at 6 h following infection and remained elevated through 48 h. IFN- production was also modestly increased in TNFR1-deficient mice, but only at 6 h postinfection (Fig. 3). Unlike that of these proinflammatory cytokines, hepatic IL-10 production was not markedly upregulated in either wild-type or TNFR1-deficient mice at any time point examined (data not shown).

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FIG. 3. Increased production of liver-associated proinflammatory cytokines and chemokines in TNFR1-deficient mice following intravenous Klebsiella infection. Livers from infected TNFR1-deficient mice or infected C57BL/6 mice were removed at 6, 24, 48, and 72 h postinfection, and cytokine or chemokine production was assessed by ELISA as described in Materials and Methods. Induction of cytokines or chemokines is displayed as mean fold induction above that in uninfected controls. Error bars, standard errors of the means. Data were generated from two to four independent experiments with a total of 12 to 24 mice per group. *, P < 0.01; , P < 0.05.

Rapid chemokine production has been shown to be critical in several models of infectious disease. Secretion of hepatic MIP-1, MCP-1, and MIP-2 was rapidly upregulated following Klebsiella infection in both wild-type and TNFR1-deficient mice (Fig. 3). However, levels were significantly elevated in TNFR1-deficient mice at both 6 and 24 h postinfection.

Increased plasma-associated proinflammatory cytokine levels in TNFR1-deficient mice. To determine if excessive systemic proinflammatory cytokine production may contribute to the increased mortality seen in TNFR1-deficient mice, blood plasma cytokine levels were measured at early (6 h) and late (72 h) times following infection. Coincident with elevated liver-associated TNF- production 6 h postinfection, plasma TNF- levels were threefold higher in TNFR1-deficient mice than in infected control mice (Table 2). Additionally, TNFR1-deficient mice lacked appreciable levels of IL-10 in plasma at 6 h, unlike infected wild-type mice. Unexpectedly, at 72 h following infection, plasma IFN- levels in TNFR1-deficient mice were 10-fold higher than those in wild-type mice. Of significance, liver-associated IFN- levels were not elevated at 72 h postinfection in TNFR1-deficient mice (data not shown), suggesting that this increased plasma IFN- was not of hepatic origin. No differences in plasma TNF- and IL-10 levels were observed between TNFR1-deficient mice and wild-type mice at 72 h postinfection.

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TABLE 2. Plasma cytokine levels in TNFR1 KO mice following infectiona

Enhanced IFN- production by TNFR1-deficient splenocytes following infection. Increased plasma IFN- levels in the absence of increased liver-associated IFN- levels 3 days following infection suggested a nonhepatic source of IFN-. Because the spleen is continuously exposed to blood-borne bacterial infections, we examined IFN- production by isolated splenocytes on day 3 postinfection. Spleen cells were isolated from uninfected and infected wild-type mice and TNFR1-deficient animals and were then cultured in vitro for 24 h with low-dose PMA and ionomycin stimulation. As can be seen in Fig. 4, suboptimal stimulation failed to induce IFN- production from either uninfected animal group. Spleen cells from day-3 infected wild-type mice secreted minimal levels of IFN- following in vitro culture. In noticeable contrast, spleen cells isolated from infected TNFR1-deficient mice displayed significant IFN- secretion. When higher concentrations of PMA and ionomycin (capable of stimulating IFN- production from naïve animals) were tested, the same pattern was observed, namely, significantly more IFN- production by spleen cells from TNFR1-deficient animals than by those from infected control animals (data not shown).

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FIG. 4. Enhanced IFN- production by TNFR1-deficient splenocytes following infection. Spleen cells were isolated from uninfected and day-3 infected TNFR1-deficient and C57BL/6 wild-type control animals as described in Materials and Methods. Cells were cultured in vitro with exogenous PMA (50 ng/ml) and ionomycin (100 ng/ml). After 24 h of culture, supernatants were analyzed for IFN- secretion. Note that these doses failed to induce detectable IFN- secretion from uninfected wild-type or TNFR1-deficient splenocytes. Wild-type infected spleen cells produced IFN- at levels slightly above the threshold of detection. In contrast, TNFR1-deficient spleen cells secreted significant levels of IFN- following in vitro culture. Data are expressed as mean IFN- production from two independent experiments with a total of seven animals, with each individual spleen cultured in duplicate. Error bars, standard errors of the means.

Increased activation and total number of splenic lymphocyte subsets in TNFR1-deficient mice following infection. Elevated IFN- secretion by splenocytes isolated from day-3 infected TNFR1-deficient mice suggested increased activation of IFN--secreting spleen lymphocytes. To determine the activation statuses of specific lymphocyte populations, splenocytes were isolated from day-3 infected wild-type and TNFR1-deficient mice and analyzed for upregulation of CD69 expression as an indication of cellular activation. Significantly higher CD69 expression was observed on CD4⁺ T cells, CD8⁺ T cells, CD4^- CD8^- (DN) T cells, T cells, and NK cells from day-3 infected TNFR1-deficient splenocytes than on those from infected C57BL/6 animals (Fig. 5). In addition to increased frequencies of activated lymphocytes, the total numbers of CD69⁺ DN T cells and T cells and, to a lesser extent, those of CD4⁺ T cells and CD8⁺ T cells were markedly increased in the spleens of infected TNFR1-deficient mice relative to the total numbers of activated lymphocyte subsets in infected wild-type mice (Fig. 6). Lymphocyte numbers were the same in wild-type and TNFR1-deficient mice prior to infection, indicating that the increased numbers observed after infection were not due to differences in basal numbers of cells.

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FIG. 5. Increased activation of splenic lymphocyte subsets in TNFR1-deficient mice following infection. Wild-type and TNFR1-deficient mice were intravenously infected, and splenocytes were isolated 3 days postinfection. Cells were stained for CD69 expression on lymphocyte subsets as described in Materials and Methods. Data in dot plots are from events pregated as indicated on the left. The percentage of the cell population of interest (horizontal axis) expressing CD69 is given in the upper right quadrant. Data are representative of two independent experiments with seven mice per group.

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FIG. 6. Increased total numbers of activated splenic lymphocyte subsets following infection in TNFR1-deficient mice. Wild-type and TNFR1-deficient mice were intravenously infected, and splenocytes were isolated 3 days postinfection. The percentage of cells expressing CD69 was determined (Fig. 5), and the total number of activated cells was calculated by multiplying the frequency of expression by the total number of splenocytes of that subset. Infected TNFR1-deficient spleens contained significantly more activated DN-ß T cells and T cells and, to a lesser extent, more CD4+ T cells and CD8+ T cells than infected wild-type spleens (*, P < 0.01). Dotted line indicates baseline number of cells prior to infection. Data are representative of two independent experiments with seven mice per group.

Decreased numbers of splenic NK T cells in TNFR1-deficient mice following infection. When the activation statuses of NK T cells (defined as NK1.1⁺ ß-Tcr⁺ cells) in infected wild-type and TNFR1-deficient animals were compared, only modest increases in CD69 expression were observed (data not shown). Unexpectedly, however, the frequency of NK T cells was dramatically reduced in day-3 infected TNFR1-deficient spleens (Fig. 7). Additionally, the total number of NK T cells was reduced twofold in TNFR1-deficient spleens, correlating with the decreased frequency of the cells. Combined, these data (Fig. 5 to 7) indicate dysregulated, excessive activation of the majority of potential IFN--producing cell populations within the spleen, with the notable exception of NK T cells.

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FIG. 7. Decreased numbers of splenic NK T cells in TNFR1-deficient mice following infection. The frequency of splenic NK T cells (defined as NK1.1+ ßTcr+ Tcr-) was determined as described in Materials and Methods. The frequencies of splenic NK T cells in infected wild-type and TNFR1-deficient mice are given within the appropriate dot plots. Data are representative of two independent experiments with seven mice per group.

Reduced liver injury in TNFR1-deficient mice following TNF- neutralization. Elevated liver- and blood-associated TNF- levels were observed in TNFR1-deficient mice during the first 24 h of infection (Fig. 3; Table 2). Because excessive TNF- production has been shown to be detrimental during systemic infections, we administered a neutralizing anti-TNF- antibody to TNFR1-deficient mice prior to infection. TNF- neutralization resulted in a twofold reduction in plasma AST levels in TNFR1-deficient mice at 24 h postinfection, indicating significantly reduced liver injury (Fig. 8).

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FIG. 8. Reduced liver injury in TNFR1-deficient mice following TNF- neutralization. An anti-TNF- monoclonal antibody was intraperitoneally administered 2 h prior to infection. TNFR1 knockout (KO) mice were then intravenously inoculated with K. pneumoniae and analyzed 24 h postinfection. Plasma AST levels, as an indication of liver cellular injury, were determined 24 h following bacterial infection. Anti-TNF- treatment reduced AST levels by 50% in TNFR1-deficient mice following infection (*, P < 0.01).

Decreased liver-associated chemokine production in TNFR1-deficient mice following TNF- neutralization. Intravenous Klebsiella infection induced significantly greater secretion of liver-associated chemokines 24 h postinfection in TNFR1-deficient mice than in control infected animals (Fig. 3). TNF- neutralization dramatically reduced secretion of MIP-2, MCP-1, and, to a lesser degree, MIP-1 in TNFR1-deficient animals (Fig. 9). Additionally, IL-12, TNF-, and IFN- production were also decreased following anti-TNF- treatment of TNFR1-deficient mice (data not shown).

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FIG. 9. Decreased chemokine production in TNFR1-deficient mice following TNF- neutralization. TNFR1-deficient mice were pretreated with anti-TNF- 2 h prior to intravenous K. pneumoniae infection. Livers were excised 24 h postinfection, and induction of the chemokines MIP-1, MIP-2, and MCP-1 was determined by ELISA as described in Materials and Methods. Anti-TNF- treatment significantly reduced the excessive induction of MIP-2 (*, P < 0.001) and MCP-1 (, P < 0.05), along with a consistent trend for reduced MIP-1 levels (P = 0.052), in TNFR1-deficient mice. Data are presented as mean fold induction (over levels in uninfected animals) from three independent experiments with a total of 15 mice per group. Error bars, standard errors of the means.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice lacking TNFR1 are significantly more susceptible to Klebsiella-induced mortality following intravenous inoculation. In contrast, TNFR2-deficient mice are no more susceptible than their wild-type counterparts. Unexpectedly, the onset of mortality seen in TNFR1-deficient mice from day 2 postinfection and later did not correlate with increased bacterial burden. However, elevated production of liver-associated proinflammatory cytokines (IL-12, TNF-, and IFN-) and chemokines (MIP-1, MIP-2, and MCP-1) within the first 24 h of infection was observed. Additionally, excessive plasma-associated IFN- levels were noted in TNFR1-deficient mice. Spleen cells from day-3 infected TNFR1-deficient mice secreted markedly enhanced levels of IFN- when cultured in vitro, likely accounting for the elevated plasma IFN- levels noted at this time point following infection. TNFR1-deficient spleens also contained significantly more activated lymphocytes 3 days following infection than spleens from infected C57BL/6 control animals. A notable exception, however, was the sharp decrease in the frequency and total number of splenic NK T cells in infected TNFR1-deficient mice. Combined, these data indicate a dysregulated antibacterial host response following intravenous Klebsiella infection in the absence of TNFR1 signaling, resulting in elevated cytokine or chemokine production and hyperactivation of specific splenic lymphocyte subsets. The excessive liver-associated chemokine production seen in TNFR1-deficient mice was significantly reduced following anti-TNF- therapy. Anti-TNF- pretreatment of TNFR1-deficient mice also resulted in reduced liver injury at 24 h postinfection.

Our observation that TNFR1-deficient mice are more susceptible to blood-borne Klebsiella-induced mortality is in general agreement with previously published data on a variety of pathogenic organisms. TNFR1-deficient mice have been reported to be significantly more susceptible to intravenous Listeria infection, which correlated with impaired bacterial clearance (31, 32, 35). In a systemic streptococcal infection model, TNFR1-deficient mice displayed increased mortality and increased blood bacterial counts (30). In contrast to reports of impaired bacterial clearance, TNFR1-deficient mice have also been reported to display enhanced bacterial clearance in a pulmonary model of Pseudomonas pneumonia (40). In addition to increased susceptibility to bacterial infections, TNFR1-deficient mice have been shown to be more susceptible to fungal and parasitic infections (6, 42).

Elevated plasma IFN- production late during the course of infection in TNFR1-deficient mice was unanticipated. This increase is unlikely to be of hepatic origin, because no increased liver-associated IFN- levels (protein or mRNA) were observed 3 days following infection. Splenic lymphocytes were examined as a possible source of systemically released IFN-. In vitro culture of spleen cells from TNFR1-deficient mice in the presence of suboptimal doses of PMA and ionomycin resulted in significant secretion of IFN-. It should be noted that the dose of in vitro stimulation used failed to induce IFN- secretion from uninfected C57BL/6 or TNFR1-deficient splenocytes. Only minimal amounts of IFN- were detected from cultured wild-type spleen cells from day-3 infected animals. These data suggest that spleen lymphocytes from TNFR1-deficient mice are excessively activated at later times following infection in vivo, rendering them more sensitive to in vitro activation and subsequent IFN- secretion. Interestingly, when spleen cells taken from TNFR1-deficient mice at day 1 postinfection were cultured in a parallel manner, no enhanced IFN- secretion was observed, suggesting that mechanisms inducing increased IFN- production are activated later in the infection. These data are similar to those reported by Zhao et al. for a systemic Yersinia enterocolitica infection model (50). They reported enhanced IFN- production by mitogen-activated splenocytes from TNFR1-deficient animals following infection. Additionally, increased pulmonary IFN- and TNF- levels have been reported following Histoplasma infection (1).

Flow cytometric analyses of spleen lymphocyte subsets from day-3 infected TNFR1-deficient mice substantiated the excessive activation status of these cells. A marked upregulation of CD69 expression was observed on the majority of potential IFN--producing lymphocytes, including CD4⁺ T cells, CD8⁺ T cells, DN T cells, T cells, and NK cells. Of these lymphocyte subsets, all but NK cells also showed higher absolute cell numbers. While we did not directly examine which subset(s) was producing IFN-, these data strongly suggest that the number of potential IFN--secreting activated lymphocytes is significantly increased in the spleens of TNFR1-deficient mice 3 days postinfection. When spleen cells taken from TNFR1-deficient and wild-type mice at day 1 postinfection were examined for CD69 upregulation, no differences in activation status between various lymphocyte subpopulations were noted, in agreement with the lack of detectable secreted IFN- levels following in vitro culture at this time point.

Unexpectedly, splenic NK T cells were reduced in frequency and total numbers in TNFR1-deficient mice 3 days following infection. NK T cells are a unique T-cell subset capable of producing high levels of IL-4 and/or IFN- following activation (9, 13, 38). Moreover, they have been shown to regulate other lymphocytes during the course of an immune response (4, 33, 44). Of relevance to our observations, Nakano and colleagues reported enhanced T-cell-derived IFN- production following NK T-cell depletion, suggesting NK T-cell-mediated suppression of T-cell activity (29). This suggests the possibility that the disappearance of NK T cells is responsible, at least in part, for the increased plasma-associated IFN- levels. We are currently unsure as to the mechanism responsible for the reduction of NK T-cell numbers in TNFR1-deficient mice following infection. One possibility is activation-induced apoptosis. At an earlier time following infection (day 1), we did not observe a decrease in the number of splenic NK T cells in TNFR1-deficient mice, indicating that the disappearance of these cells occurred later during the infection. Another possibility is increased trafficking of NK T cells out of the spleen at later times following infection.

TNFR1-deficient mice displayed excessive production of liver-associated cytokines, in particular the chemokines MIP-2, MCP-1, and MIP-1. Neutrophil recruitment and sequestration in the liver have been shown to induce liver damage (2, 5, 15). Since MIP-2 is a potent neutrophil chemoattractant, it is possible that excessive MIP-2 production could lead to increased tissue damage and/or organ failure. Neutralization of MIP-2 activity has been shown to reduce neutrophil influx, leading to decreased liver injury and mortality in several models (7, 12, 23, 47). Since MIP-2 is only one of several chemokines known to induce neutrophil chemotaxis, it is likely that MIP-2 neutralization reduces, but does not eliminate, neutrophil recruitment. We have initiated studies examining the effects of MIP-2 neutralization in TNFR1-deficient and wild-type animals. Preliminary results suggest a significant reduction in liver injury following MIP-2 neutralization in both TNFR1-deficient and wild-type mice (data not shown).

Mice rendered deficient in MIP-1 have been shown to have less tissue inflammation following infection. This might suggest that excessive production of MIP-1 may result in increased tissue damage and mortality. However, these animals lacking MIP-1 activity were also more susceptible to a variety of pathogenic organisms, again suggesting that under- or overproduction of MIP-1 may be detrimental to the host (8, 14, 24, 41). Similar observations have been made by examining MCP-1 in a variety of infection models (18, 25). Combined, these data suggest that the accumulative effect of excessive MIP-1 and MCP-1 may contribute to the increased mortality in TNFR1-deficient mice.

Elevated hepatic TNF- secretion following infection was seen in TNFR1-deficient mice following intravenous K. pneumoniae infection. This liver-associated production is a likely source of the increased plasma TNF- levels noted at 6 h postinfection. We have not observed increased TNF- production by cultured spleen cells removed from TNFR1-deficient mice at 1 or 3 days postinfection, lending support to the hypothesis that the liver is the source of elevated systemic TNF- levels. Increased TNF- production by TNFR1-deficient mice has been reported in several other pathogenic models. Systemic LPS administration resulted in higher serum TNF- levels in TNFR1-deficient mice than in LPS-treated wild-type control animals (31). Pulmonary challenge with Pseudomonas aeruginosa resulted in increased TNF- levels in bronchoalveolar lavage fluids ofTNFR1-deficient mice (40), while infection with Histoplasma capsulatum stimulated increased lung-associated TNF- production (1). Combined, our data and these indicate excessive TNF- production in the absence of TNFR1-mediated signaling.

The liver injury noted in TNFR1-deficient mice following bacterial infection intrigued us. This observation clearly indicates that not all TNF--mediated liver injury occurred through TNFR1-mediated signaling. This injury suggests two possibilities: (i) it may be due to TNF- signaling through the intact TNFR2 receptor, or 2) it may be non-TNF- mediated. Interestingly, TNF- neutralization of TNFR1-deficient infected mice reduced plasma AST levels by only 50% at 24 h postinfection. These mice still retained functional TNFR2, which preferentially utilizes membrane-bound TNF- rather than soluble TNF-. It is possible that anti-TNF- treatment was unable to efficiently block membrane-bound TNF- signaling through TNFR2. Alternatively, liver injury in TNFR1-deficient mice may occur in a TNF--independent manner.

The dramatic reduction in liver-associated MIP-2, MIP-1, and MCP-1 levels in anti-TNF--treated TNFR1-deficient mice suggests TNF--mediated chemokine induction in TNFR1-deficient mice. Anti-TNF- treatment has been reported to reduce MIP-1 production in an endotoxemia model (41). However, ours is the first report of TNF- directly modulating the production of CXC and CC chemokines in the context of systemic blood-borne bacterial infections in the absence of TNFR1-mediated signaling. This observation may have broader implications for proposed therapeutic TNF- neutralization studies and points to potential unanticipated downstream effects of TNF- neutralization on neutrophil and monocyte/macrophage chemotaxis.

These studies detail the critical importance of TNFR1-mediated signaling during systemic K. pneumoniae infection. TNFR1-deficient mice displayed significantly increased mortality following infection. Surprisingly, this increased mortality occurred in the absence of increased bacterial burdens or increased liver injury. However, excessive proinflammatory cytokine and chemokine production was noted in TNFR1-deficient mice relative to that in infected C57BL/6 animals, suggesting dysregulated production in the absence of TNFR1 signaling. The absence of TNFR1-mediated signaling also resulted in heightened activation of several splenic lymphocyte subsets. Combined, these data indicate the critical importance of TNFR1 expression during systemic gram-negative bacterial infections while suggesting a role for TNFR1-mediated immunoregulation during the latter stages of bacterial infections.

 
This research was supported in part by a research grant from the American Lung Association (T.A.M.) and grants AI49448 (T.A.M.), HL57243, HL58200, and P50HL60289 (T.J.S.) from the National Institutes of Health. T.A.M. is a Parker B. Francis Fellow in Pulmonary Research and an Edward Livingston Trudeau Fellow of the American Lung Association.

 
* Corresponding author. Mailing address: University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, 6301 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0642. Phone: (734) 647-8378. Fax: (734) 764-4556. E-mail: tmoore{at}umich.edu.

Editor: J. N. Weiser

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, September 2003, p. 4891-4900, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.4891-4900.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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