Endotoxin Priming Improves Clearance of Pseudomonas aeruginosa in Wild-Type and Interleukin-10 Knockout Mice
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感染与免疫杂志 2005年第11期
Departments of Anesthesiology Microbiology and Immunology, The University of Texas Medical Branch
Shriners Hospital for Children, Galveston, Texas
ABSTRACT
Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN- and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN- and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.
INTRODUCTION
Many investigators have reported that severe trauma or critical illness can precipitate a state of impaired immune function that predisposes the host to infectious complications (3, 25, 33, 37). This syndrome is often termed immunoparalysis or postinflammatory immunosuppression and is characterized by decreased production of gamma interferon (IFN-) and interleukin-12 (IL-12) as well as increased secretion of the anti-inflammatory cytokine IL-10 in response to an infectious challenge. Some researchers have proposed that the state of endotoxin (lipopolysaccharide [LPS]) tolerance is a model of immunoparalysis because LPS-tolerant animals exhibit a pattern of cytokine production that is nearly identical to that observed during postinflammatory immunosuppression (10, 28, 31). LPS tolerance is induced by exposure to sublethal doses of LPS, which results in a suppressed proinflammatory response and improved survival after challenge with a normally lethal dose of LPS (4, 15). LPS tolerance is characterized by decreased LPS-induced production of proinflammatory cytokines, such as tumor necrosis factor alpha and IL-1, as well as reduced secretion of IFN- (13, 31, 32) and IL-12 but increased production of IL-10 (24, 31, 32).
During infection, IFN- is produced primarily by natural killer (NK) and Th1 lymphocytes. IFN- production is induced by the macrophage- and dendritic cell-derived cytokines IL-12, IL-15, and IL-18, which are produced after engagement of pattern recognition receptors by bacterial products such as LPS. Suppressed secretion of these cytokines is reported to be a hallmark of injury-induced immunoparalysis (20, 28). In contrast, elevated levels of IL-10 may suppress antimicrobial immunity and render the host more susceptible to infections (28). Exogenous administration of IL-10 causes suppression of LPS-induced IFN- and IL-12 production in a manner similar to that observed with LPS tolerance and postinjury immunoparalysis (10, 34). However, the role of IL-10 in causing decreased cytokine production during LPS tolerance is controversial. Some investigators have shown that neutralization of IL-10 will restore cytokine production in LPS-tolerant mice, whereas others have demonstrated that LPS tolerance can be induced in IL-10-deficient mice (2, 9).
Although many investigators consider LPS tolerance to mimic the state of immune dysfunction observed with postinjury immunoparalysis, few studies have measured bacterial clearance and resistance to infection in LPS-tolerant subjects. Prior studies have assessed the resistance of LPS-tolerant mice to infection with Salmonella enterica serovar Typhimurium or polymicrobial sepsis caused by cecal ligation and puncture (7, 14). However, these sources of infection are not common in critically ill patients. Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus spp. more frequently cause nosocomial infection (8, 19, 29, 30). Currently, there are no reports on the resistance of the LPS-tolerant host to infection with these opportunistic organisms.
The present studies were conducted with two primary goals: (i) to determine the ability of LPS-tolerant mice to respond to systemic challenge with the opportunistic pathogen Pseudomonas aeruginosa and (ii) to determine whether neutralization of IL-10 will reverse the LPS-tolerant state. To address these goals, LPS tolerance was induced in wild-type and IL-10-deficient mice. Production of IFN-, IL-12, and IL-10 were measured after LPS or Pseudomonas challenge. We focused on measuring production of IFN-, IL-12, and IL-10 as markers of LPS tolerance because decreased production of these factors has been postulated to be an important factor in the development of postinjury immunosuppression. In further studies, we measured clearance of the opportunistic pathogen P. aeruginosa from blood and lungs of control and LPS-primed mice.
MATERIALS AND METHODS
LPS tolerance model. Female, 6- to 8-week-old wild-type (C57BL/6J) and IL-10 knockout (IL-10 KO) (B6.129P2-Il10tm1cgn) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in a monitored, light-dark-cycled environment and provided with standard lab chow and water ad libitum. Phenol-extracted LPS (E. coli serotype 0111:B4) was purchased from Sigma Chemical (St. Louis, MO). LPS tolerance was induced by intraperitoneal (i.p.) injection of LPS (1 μg in 0.2 ml of normal saline) daily for 2 days. Control mice received normal saline (0.2 ml) in the same regimen. All mice were injected with LPS or saline between 8 a.m. and noon. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch and met National Institute of Health guidelines for the experimental use of animals in research.
Measurement of LPS-induced cytokine production. To measure LPS-induced cytokine concentrations in plasma, mice were challenged with LPS (10 μg in 0.2 ml of normal saline) 2 days after the last priming dose of saline or LPS. Blood was harvested at 8 h after LPS challenge, and plasma cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). In additional experiments, levels of IFN-, IL-12, and IL-10 production in cultured whole blood were measured by harvesting blood at 2 days after saline or LPS priming. Heparinized blood was harvested on day 3 and mixed 1:1 (vol/vol) with RPMI 1640 medium supplemented with 10% fetal bovine plasma, penicillin (10 U/ml), and streptomycin (10 μg/ml). Blood was cultured (37°C, 5% CO2) for 24 h with LPS (100 ng/ml), and supernatants were harvested. Cytokine concentrations in plasma or blood culture supernatants were measured by ELISA.
In further experiments, splenic cytokine production levels in saline- or LPS-primed mice were measured. Mice were challenged with LPS (10 μg, i.p.) 2 days after the last priming dose of saline or LPS. Spleens were harvested 8 h after LPS challenge, and isolated splenocytes were incubated (37°C, 5% CO2) for 24 h. Cytokine concentrations were measured using an ELISA. Spleens were also harvested at designated periods (0, 2, and 6 h) after LPS (10 μg, i.p.) challenge for measurement of cytokine mRNA expression. Cytokine mRNA expression was measured using an RNase protection assay.
Preparation of splenocyte and whole blood cultures. Spleens were aseptically excised from saline- or LPS-primed mice at 8 h after LPS (10 μg) challenge and transferred to 6-well culture plates containing RPMI 1640 medium supplemented with 10% fetal bovine plasma, penicillin (10 U/ml), and streptomycin (10 μg/ml). This medium preparation was used in all experiments unless specified otherwise. Spleens were minced and passed through a sterile mesh, and the erythrocytes were lysed (erythrocyte lysis kit; R&D Systems). The remaining splenocytes were resuspended in medium, and viability was determined to be greater than 95% by trypan blue exclusion. Splenocytes (5 x 106/well) were plated in 96-well plates and cultured (37°C, 5% CO2) for 24 h. The supernatant was harvested for measurement of cytokine levels.
Blood cultures were prepared 2 days after the last priming dose of saline or LPS by harvesting heparinized blood from mice by carotid laceration under 2% isoflurane anesthesia. The heparinized blood was mixed 1:1 (vol/vol) with medium and cultured (37°C, 5% CO2) for 24 h with LPS (100 ng/ml). The cultured blood was centrifuged (1,200 rpm for 10 min), and the supernatant was harvested for cytokine measurements.
ELISA. IFN- (eBioscience, San Diego, CA), IL-12 p70 (R&D Systems, Minneapolis, MN), and IL-10 (R&D Systems) concentrations in plasma and conditioned medium were determined by ELISA according to the manufacturer's instructions. Briefly, standards or experimental samples were added to microtiter plates coated with monoclonal antibody to the cytokine of interest and incubated for 2 h. After the contents of the plates were washed, horseradish peroxidase-conjugated, cytokine-specific antibody was added to each well, incubated for 2 h, and washed. Substrate solution was added and incubated for 30 min, and the reaction was terminated by the addition of stop solution. Cytokine levels were determined by measuring the optical density at 450 nM with a microtiter plate reader (Dynatech Laboratories, Chantilly, VA).
RPA. Total RNA was isolated from mouse spleens by using TRI Reagent (Molecular Research Center, Cincinnati, OH). An RNase protection assay (RPA) was performed using the Riboquant system (BD Pharmingen) per the manufacturer's instructions. Briefly, radiolabeled RNA probes were synthesized from DNA template sets by using T7 RNA polymerase, [32P]UTP, and pooled nonradiolabeled nucleotides. Isolated total RNAs (20 μg/sample) were hybridized with the purified riboprobes and subjected to RNase digestion. DNA template sets included probes for the L32 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping genes that served as internal controls. Protected RNA species were separated on 5% polyacrylamide sequencing gels by using 0.5x Tris-borate-EDTA running buffer. Gels were run at 50 W constant power for 70 min and dried under vacuum, and the protected fragments were visualized using autoradiography.
Bacterial clearance. Lungs and blood were harvested from mice anesthetized with isoflurane 8 h after intraperitoneal injection of P. aeruginosa (strain ATCC 19660, 5 x 107 CFU). Heparinized blood was aseptically harvested under isoflurane anesthesia after laceration of the carotid artery. Lungs were aseptically excised, weighed, and homogenized using sterile tissue grinders. Serial dilutions of lung homogenates and blood were plated on tryptic soy agar and incubated overnight at 37°C. Colony counts were performed to assess bacterial burden. Pseudomonas aeruginosa was identified by colony morphology. The accuracy of this approach has been confirmed by culturing duplicate samples on Pseudosel agar as well as performing biochemical identification (API-20E system; bioMerieux, Durham, NC).
Data analysis. For comparisons of data from multiple groups, one-way analysis of variance was performed. Comparisons between the groups were carried out using Tukey's posttest analyses. A P value of <0.05 was considered statistically significant.
RESULTS
Plasma concentrations of IFN-, IL-12, and IL-10 in saline- and LPS-primed wild-type and IL-10 KO mice. LPS tolerance was induced by i.p. injection of LPS (1 μg in 0.2 ml of normal saline) daily for 2 days. Control mice received normal saline (0.2 ml) in the same regimen. On the fourth day, mice received intraperitoneal challenge with LPS (10 μg in 0.2 ml of normal saline). Plasma cytokine levels were measured 8 h after LPS challenge (Fig. 1). LPS-primed wild-type mice had decreased concentrations of IFN- in plasma compared to concentrations for saline-primed control wild-type mice (Fig. 1). IL-10 KO mice produced significantly (P < 0.05) higher levels of IFN- than wild-type mice did after LPS challenge. IL-10 KO mice receiving LPS priming had significantly lower LPS-induced IFN- concentrations in plasma than control saline-primed IL-10 KO mice. IL-12 was not detectable in plasma of control or LPS-primed wild-type mice within the limits of our assay at this time point (Fig. 1). However, IL-12 was present in plasma from IL-10 KO mice challenged with LPS (Fig. 1). Plasma IL-12 levels were significantly lower in IL-10 KO mice that received LPS priming than in saline-primed controls. Examination of plasma IL-10 concentrations showed higher IL-10 levels in LPS-primed wild-type mice than in saline-primed controls (Fig. 1). IL-10 was not detected in the plasma of IL-10 KO mice.
In further studies, whole blood was isolated from control and LPS-primed mice and cultured with LPS for 24 h, and cytokine concentrations in supernatants were measured (Fig. 1). Levels of IFN- and IL-12 production were significantly lower in blood from LPS-primed wild-type and IL-10 KO mice than in blood from saline-primed controls (Fig. 1). IL-10 levels were not significantly different between control and LPS-primed wild-type mice. IL-10 was not detectable in whole blood supernatants from IL-10 KO mice.
Expression of IFN-, IL-12, and IL-10 mRNAs in control and LPS-primed wild-type and IL-10 KO mice. Expression levels of mRNAs for IFN-, IL-12p40, and IL-10 were measured in spleens after LPS challenge (Fig. 2). Spleens were harvested at 0, 2, and 6 h after the LPS injection, and expression levels of cytokine mRNAs were measured using an RNase protection assay (Fig. 2). LPS-primed wild-type mice showed increased IL-10 mRNA expression levels at both 2 and 6 h after LPS challenge compared to levels for saline-primed control mice (Fig. 2). IL-10 mRNA was not detectable in IL-10 KO mice. LPS-primed wild-type mice showed decreased levels of IL-12p40 and IFN- mRNA expression compared to levels for control wild-type mice. IL-12p40 and IFN- mRNA expression levels were increased in IL-10 KO mice compared to levels in wild-type mice. At 2 h after LPS challenge, high levels of IFN- and IL-12 were induced in LPS-primed IL-10 KO mice and were comparable to levels observed for control IL-10 KO mice. However, at 6 h after LPS challenge, IFN- and IL-12p40 mRNA expression levels were lower in LPS-primed IL-10 KO mice than in saline-primed controls. IL-12p35 mRNA was constitutively expressed in wild-type and IL-10 KO mice and was not altered by LPS priming (data not shown).
LPS-induced IFN-, IL-12, and IL-10 production by splenocytes from wild-type and IL-10 KO mice. Splenocytes were obtained from control and LPS-primed mice 8 h after in vivo LPS challenge and cultured for 24 h. Cytokine concentrations in conditioned medium were measured using an ELISA (Fig. 3). Splenocytes isolated from LPS-primed wild-type mice produced smaller amounts of IFN- and IL-12 but secreted increased amounts of IL-10 compared to amounts from splenocytes obtained from saline-primed wild-type mice. Splenocytes isolated from IL-10 KO mice produced significantly (P < 0.05) larger amounts of IFN- than did wild-type mice but no significant difference in IFN- production was observed when comparing splenocytes from saline- and LPS-primed IL-10 KO mice (Fig. 3). Splenocytes from LPS-primed IL-10 KO mice produced significantly higher levels of IL-12 than saline-primed IL-10 KO mice (Fig. 3). IL-10 was not detectable in conditioned media from IL-10 KO mice.
Clearance of Pseudomonas aeruginosa is enhanced in LPS-primed wild-type and IL-10 KO mice. Wild-type and IL-10 KO mice were primed with saline or LPS for 2 days and were challenged with P. aeruginosa (5 x 107 CFU, i.p.) on day 4. Blood and lungs were harvested 6 h after bacterial challenge and plated on tryptic soy agar to measure Pseudomonas CFU (Fig. 4). Bacterial counts in blood and lungs were significantly lower in LPS-primed wild-type mice than in control wild-type mice (Fig. 4). The proportion of mice with detectable Pseudomonas in blood was only three out of nine for LPS-primed wild-type mice, whereas all wild-type control mice (9/9) had positive cultures. This difference was statistically significant (P < 0.05). Bacterial counts in blood and lungs were significantly lower for control IL-10 KO mice than for the control wild-type group. The proportion of IL-10 KO mice (3/9) with positive blood cultures was also significantly lower than the proportion of wild-type mice with positive blood cultures (9/9). Additionally, there were significantly fewer P. aeruginosa CFU in blood and lung cultures from LPS-primed IL-10 KO mice than from control IL-10 KO mice.
Cytokine production by control and LPS-tolerant wild-type and IL-10 KO mice after P. aeruginosa challenge. Control and LPS-primed wild-type and IL-10 KO mice were challenged with P. aeruginosa (5 x 107 CFU), and circulating levels of cytokines were assayed in the plasma 6 h later (Fig. 5). Production levels of IFN- and IL-12 were significantly decreased in LPS-primed wild-type mice compared to levels in control wild-type mice. Control IL-10 KO mice had significantly (P < 0.05) higher plasma levels of IFN- and IL-12 than control wild-type mice. LPS-primed IL-10 KO mice had significantly lower concentrations of IFN- and IL-12 in plasma than control IL-10 KO mice. Levels of IL-10 in the plasma of control and LPS-primed wild-type mice were not significantly different following P. aeruginosa challenge. IL-10 was not measurable in the plasma of IL-10 KO mice.
Clearance of Pseudomonas aeruginosa is enhanced in LPS-primed wild-type mice and in mice treated with anti-IL-10. Wild-type mice were primed with saline or LPS for 2 days. On day 3, mice received an injection of nonspecific immunoglobulin G (IgG) or anti-IL-10 and were challenged with P. aeruginosa (5 x 107 CFU, i.p.) on day 4. Blood and lungs were harvested 6 h after bacterial challenge and plated on tryptic soy agar. Plates were cultured for 24 h, and Pseudomonas counts were performed (Fig. 6). LPS-primed control mice had significantly fewer bacteria in blood and lungs than saline-primed mice (Fig. 6). Treatment of control mice with anti-IL-10 also significantly decreased bacterial counts in blood and lungs compared to counts for control mice receiving nonspecific IgG. LPS-primed mice treated with anti-IL-10 had significantly fewer Pseudomonas CFU in lungs, but not in blood, than did control mice receiving anti-IL-10.
Effect of anti-IL-10 treatment on cytokine production by control and LPS-primed wild-type mice after P. aeruginosa challenge. Wild-type mice were primed with saline or LPS on days 1 and 2, followed by treatment with anti-IL-10 or nonspecific IgG on day 3. All mice were challenged with P. aeruginosa (5 x 107 CFU) on day 4, and concentrations of cytokines in the plasma were measured 6 h later (Fig. 7). LPS priming in mice receiving control IgG caused significant decreases in Pseudomonas-induced IFN- and IL-12 concentrations in plasma, but IL-10 concentrations were increased (Fig. 7). Treatment of saline-primed mice with anti-IL-10 resulted in levels of IFN- and IL-12 in plasma significantly higher than levels for saline-primed mice treated with nonspecific IgG. Anti-IL-10-treated mice primed with LPS had IFN- and IL-12 concentrations in plasma significantly lower than concentrations for anti-IL-10-treated mice primed with saline. IL-10 concentrations in the plasma of mice treated with anti-IL-10 were less than 2 pg/ml. (Fig. 7).
DISCUSSION
It has been proposed that the widely used model of LPS tolerance mimics the state of immunoparalysis commonly observed to occur in critically ill patients (10, 24). This is because many of the immunological alterations observed during postinjury immunosuppression, such as decreased production of IFN- and IL-12 (16, 17, 20, 28), impaired antigen presentation (1, 11), and increased production of IL-10 (24, 27), are also characteristic of LPS tolerance (13, 34, 35). We have reported that mice recovering from injury caused by burns or cecal ligation and puncture exhibit suppressed production of IFN- and IL-12 as well as increased IL-10 secretion (17, 28). These cytokine alterations correlated with a decreased ability to clear P. aeruginosa (17). The present study shows that LPS-tolerant mice are more efficient than control mice in clearing P. aeruginosa from lungs and blood following systemic bacterial challenge. This is despite the observation that IL-12 and IFN- production are suppressed and IL-10 secretion is increased in these mice. These findings suggest that endotoxin tolerance does not replicate the immunosuppressive state typical of septic patients and may represent a state of altered proinflammatory cytokine production without adverse, or perhaps even with beneficial, effect on antimicrobial immune function. The mechanisms of improved bacterial clearance in LPS-tolerant mice are not clear at this juncture. However, some reports indicate that phagocytic function may be improved in mice receiving prior LPS exposure (23). Nevertheless, the mechanisms of the enhanced bacterial clearance remain to be elucidated and warrant further study.
This study also demonstrates that IL-10-deficient mice are more effective in clearing P. aeruginosa than wild-type mice and that IL-10-deficient mice are particularly efficient in removing P. aeruginosa from blood and lungs after systemic bacterial challenge. Sewnath and colleagues (26) previously showed that IL-10 KO mice remove E. coli more efficiently than wild-type mice but have higher mortality during E. coli peritonitis. The increased mortality of the IL-10 KO mice was attributed to inflammation-induced tissue injury and organ dysfunction. The present study confirms enhanced bacterial clearance and increased systemic cytokine production in IL-10-deficient mice following systemic challenge with P. aeruginosa. Interestingly, LPS-tolerant IL-10 KO mice clear P. aeruginosa more efficiently than control IL-10 KO mice but produce proinflammatory cytokines at levels that are significantly lower than those produced by saline-primed IL-10 KO mice and that are typical of wild-type mice. This observation was confirmed for LPS-primed mice that received treatment with antibody against IL-10. These findings imply that IL-10 has an inhibitory effect on bacterial clearance that is independent of the ability to regulate production of IFN- and IL-12. Previous investigators have shown that IL-10 inhibits the phagocytic function of neutrophils and macrophages (12, 21). In additional studies, Steinhauser and colleagues (27) demonstrated that IL-10 directly contributes to impaired P. aeruginosa clearance in the lungs of mice previously exposed to cecal ligation and puncture. Interestingly, we observed that LPS-tolerant wild-type mice, in which IL-10 production is generally increased, have an improved ability to clear P. aeruginosa from blood and lungs. One interpretation of these findings is that induction in LPS tolerance causes specific changes, perhaps enhanced phagocytosis, which result in improved bacterial clearance despite the increase in IL-10 production. Removal of IL-10 may eliminate an additional inhibitory factor and further enhance bacterial clearance.
These studies confirm that IL-10 is an important regulator of LPS-induced production of IFN- and IL-12. IFN- and IL-12 were produced at higher levels by IL-10 KO mice than by wild-type controls. This finding is in agreement with previous studies which show that IL-10 is a potent negative regulator of IL-12 and IFN- production (5, 6). The role of IL-10 in causing suppressed IFN- production during LPS tolerance appears organ specific. LPS tolerance-induced suppression of IFN- and IL-12 production was reversed in the spleens but not in the blood of IL-10 KO mice. Therefore, IL-10 may be important for the regulation of IFN- and IL-12 production in the spleen during LPS tolerance but not for leukocytes circulating in blood or other tissues that contribute to plasma IFN- and IL-12. This complex picture may explain conflicting results from other studies aimed at understanding the role of IL-10 in the development of LPS tolerance. Berg et al. (2) reported that LPS tolerance can be induced in IL-10-deficient mice, and Wysocka et al. (36) showed that IL-12 production is suppressed in LPS-tolerant IL-10 KO mice. Both investigators concluded that IL-10 does not play a major role in the development of LPS tolerance. However, other investigators have shown that IL-10 neutralization during LPS priming interferes with the development of LPS tolerance (18, 22). Taken together, these findings show that IL-10 may partially mediate cytokine suppression in some tissues during LPS tolerance. Numerous additional mechanisms of suppressed cytokine production in the LPS-tolerant host have also been demonstrated. Among these are generation of p50/p50 NF-B homodimers, suppressed mitogen-activated protein kinase signaling, and upregulation of SOCS proteins (9, 38). Our findings indicate that IL-10 appears to make a limited, tissue-specific contribution to the overall regulation of IFN- and IL-12 production during LPS tolerance.
In summary, this report shows that the model of LPS tolerance may not mimic postinflammatory immunoparalysis, because LPS-tolerant mice have an increased ability to clear P. aeruginosa despite diminished production of IFN- and IL-12 but increased secretion of IL-10. LPS-tolerant, IL-10-deficient mice were the most efficient in clearing bacteria from blood and lungs, which suggests that IL-10 has a direct negative impact on bacterial clearance mechanisms. The role of IL-10 in causing suppressed IFN- and IL-12 production during LPS tolerance is organ specific and only partially accounts for suppressed cytokine production in the LPS-tolerant host.
ACKNOWLEDGMENTS
These studies were supported by NIH grant R01 GM66885 and grant 8780 and 8650 from the Shriners of North America.
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Shriners Hospital for Children, Galveston, Texas
ABSTRACT
Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN- and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN- and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.
INTRODUCTION
Many investigators have reported that severe trauma or critical illness can precipitate a state of impaired immune function that predisposes the host to infectious complications (3, 25, 33, 37). This syndrome is often termed immunoparalysis or postinflammatory immunosuppression and is characterized by decreased production of gamma interferon (IFN-) and interleukin-12 (IL-12) as well as increased secretion of the anti-inflammatory cytokine IL-10 in response to an infectious challenge. Some researchers have proposed that the state of endotoxin (lipopolysaccharide [LPS]) tolerance is a model of immunoparalysis because LPS-tolerant animals exhibit a pattern of cytokine production that is nearly identical to that observed during postinflammatory immunosuppression (10, 28, 31). LPS tolerance is induced by exposure to sublethal doses of LPS, which results in a suppressed proinflammatory response and improved survival after challenge with a normally lethal dose of LPS (4, 15). LPS tolerance is characterized by decreased LPS-induced production of proinflammatory cytokines, such as tumor necrosis factor alpha and IL-1, as well as reduced secretion of IFN- (13, 31, 32) and IL-12 but increased production of IL-10 (24, 31, 32).
During infection, IFN- is produced primarily by natural killer (NK) and Th1 lymphocytes. IFN- production is induced by the macrophage- and dendritic cell-derived cytokines IL-12, IL-15, and IL-18, which are produced after engagement of pattern recognition receptors by bacterial products such as LPS. Suppressed secretion of these cytokines is reported to be a hallmark of injury-induced immunoparalysis (20, 28). In contrast, elevated levels of IL-10 may suppress antimicrobial immunity and render the host more susceptible to infections (28). Exogenous administration of IL-10 causes suppression of LPS-induced IFN- and IL-12 production in a manner similar to that observed with LPS tolerance and postinjury immunoparalysis (10, 34). However, the role of IL-10 in causing decreased cytokine production during LPS tolerance is controversial. Some investigators have shown that neutralization of IL-10 will restore cytokine production in LPS-tolerant mice, whereas others have demonstrated that LPS tolerance can be induced in IL-10-deficient mice (2, 9).
Although many investigators consider LPS tolerance to mimic the state of immune dysfunction observed with postinjury immunoparalysis, few studies have measured bacterial clearance and resistance to infection in LPS-tolerant subjects. Prior studies have assessed the resistance of LPS-tolerant mice to infection with Salmonella enterica serovar Typhimurium or polymicrobial sepsis caused by cecal ligation and puncture (7, 14). However, these sources of infection are not common in critically ill patients. Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus spp. more frequently cause nosocomial infection (8, 19, 29, 30). Currently, there are no reports on the resistance of the LPS-tolerant host to infection with these opportunistic organisms.
The present studies were conducted with two primary goals: (i) to determine the ability of LPS-tolerant mice to respond to systemic challenge with the opportunistic pathogen Pseudomonas aeruginosa and (ii) to determine whether neutralization of IL-10 will reverse the LPS-tolerant state. To address these goals, LPS tolerance was induced in wild-type and IL-10-deficient mice. Production of IFN-, IL-12, and IL-10 were measured after LPS or Pseudomonas challenge. We focused on measuring production of IFN-, IL-12, and IL-10 as markers of LPS tolerance because decreased production of these factors has been postulated to be an important factor in the development of postinjury immunosuppression. In further studies, we measured clearance of the opportunistic pathogen P. aeruginosa from blood and lungs of control and LPS-primed mice.
MATERIALS AND METHODS
LPS tolerance model. Female, 6- to 8-week-old wild-type (C57BL/6J) and IL-10 knockout (IL-10 KO) (B6.129P2-Il10tm1cgn) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in a monitored, light-dark-cycled environment and provided with standard lab chow and water ad libitum. Phenol-extracted LPS (E. coli serotype 0111:B4) was purchased from Sigma Chemical (St. Louis, MO). LPS tolerance was induced by intraperitoneal (i.p.) injection of LPS (1 μg in 0.2 ml of normal saline) daily for 2 days. Control mice received normal saline (0.2 ml) in the same regimen. All mice were injected with LPS or saline between 8 a.m. and noon. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch and met National Institute of Health guidelines for the experimental use of animals in research.
Measurement of LPS-induced cytokine production. To measure LPS-induced cytokine concentrations in plasma, mice were challenged with LPS (10 μg in 0.2 ml of normal saline) 2 days after the last priming dose of saline or LPS. Blood was harvested at 8 h after LPS challenge, and plasma cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). In additional experiments, levels of IFN-, IL-12, and IL-10 production in cultured whole blood were measured by harvesting blood at 2 days after saline or LPS priming. Heparinized blood was harvested on day 3 and mixed 1:1 (vol/vol) with RPMI 1640 medium supplemented with 10% fetal bovine plasma, penicillin (10 U/ml), and streptomycin (10 μg/ml). Blood was cultured (37°C, 5% CO2) for 24 h with LPS (100 ng/ml), and supernatants were harvested. Cytokine concentrations in plasma or blood culture supernatants were measured by ELISA.
In further experiments, splenic cytokine production levels in saline- or LPS-primed mice were measured. Mice were challenged with LPS (10 μg, i.p.) 2 days after the last priming dose of saline or LPS. Spleens were harvested 8 h after LPS challenge, and isolated splenocytes were incubated (37°C, 5% CO2) for 24 h. Cytokine concentrations were measured using an ELISA. Spleens were also harvested at designated periods (0, 2, and 6 h) after LPS (10 μg, i.p.) challenge for measurement of cytokine mRNA expression. Cytokine mRNA expression was measured using an RNase protection assay.
Preparation of splenocyte and whole blood cultures. Spleens were aseptically excised from saline- or LPS-primed mice at 8 h after LPS (10 μg) challenge and transferred to 6-well culture plates containing RPMI 1640 medium supplemented with 10% fetal bovine plasma, penicillin (10 U/ml), and streptomycin (10 μg/ml). This medium preparation was used in all experiments unless specified otherwise. Spleens were minced and passed through a sterile mesh, and the erythrocytes were lysed (erythrocyte lysis kit; R&D Systems). The remaining splenocytes were resuspended in medium, and viability was determined to be greater than 95% by trypan blue exclusion. Splenocytes (5 x 106/well) were plated in 96-well plates and cultured (37°C, 5% CO2) for 24 h. The supernatant was harvested for measurement of cytokine levels.
Blood cultures were prepared 2 days after the last priming dose of saline or LPS by harvesting heparinized blood from mice by carotid laceration under 2% isoflurane anesthesia. The heparinized blood was mixed 1:1 (vol/vol) with medium and cultured (37°C, 5% CO2) for 24 h with LPS (100 ng/ml). The cultured blood was centrifuged (1,200 rpm for 10 min), and the supernatant was harvested for cytokine measurements.
ELISA. IFN- (eBioscience, San Diego, CA), IL-12 p70 (R&D Systems, Minneapolis, MN), and IL-10 (R&D Systems) concentrations in plasma and conditioned medium were determined by ELISA according to the manufacturer's instructions. Briefly, standards or experimental samples were added to microtiter plates coated with monoclonal antibody to the cytokine of interest and incubated for 2 h. After the contents of the plates were washed, horseradish peroxidase-conjugated, cytokine-specific antibody was added to each well, incubated for 2 h, and washed. Substrate solution was added and incubated for 30 min, and the reaction was terminated by the addition of stop solution. Cytokine levels were determined by measuring the optical density at 450 nM with a microtiter plate reader (Dynatech Laboratories, Chantilly, VA).
RPA. Total RNA was isolated from mouse spleens by using TRI Reagent (Molecular Research Center, Cincinnati, OH). An RNase protection assay (RPA) was performed using the Riboquant system (BD Pharmingen) per the manufacturer's instructions. Briefly, radiolabeled RNA probes were synthesized from DNA template sets by using T7 RNA polymerase, [32P]UTP, and pooled nonradiolabeled nucleotides. Isolated total RNAs (20 μg/sample) were hybridized with the purified riboprobes and subjected to RNase digestion. DNA template sets included probes for the L32 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping genes that served as internal controls. Protected RNA species were separated on 5% polyacrylamide sequencing gels by using 0.5x Tris-borate-EDTA running buffer. Gels were run at 50 W constant power for 70 min and dried under vacuum, and the protected fragments were visualized using autoradiography.
Bacterial clearance. Lungs and blood were harvested from mice anesthetized with isoflurane 8 h after intraperitoneal injection of P. aeruginosa (strain ATCC 19660, 5 x 107 CFU). Heparinized blood was aseptically harvested under isoflurane anesthesia after laceration of the carotid artery. Lungs were aseptically excised, weighed, and homogenized using sterile tissue grinders. Serial dilutions of lung homogenates and blood were plated on tryptic soy agar and incubated overnight at 37°C. Colony counts were performed to assess bacterial burden. Pseudomonas aeruginosa was identified by colony morphology. The accuracy of this approach has been confirmed by culturing duplicate samples on Pseudosel agar as well as performing biochemical identification (API-20E system; bioMerieux, Durham, NC).
Data analysis. For comparisons of data from multiple groups, one-way analysis of variance was performed. Comparisons between the groups were carried out using Tukey's posttest analyses. A P value of <0.05 was considered statistically significant.
RESULTS
Plasma concentrations of IFN-, IL-12, and IL-10 in saline- and LPS-primed wild-type and IL-10 KO mice. LPS tolerance was induced by i.p. injection of LPS (1 μg in 0.2 ml of normal saline) daily for 2 days. Control mice received normal saline (0.2 ml) in the same regimen. On the fourth day, mice received intraperitoneal challenge with LPS (10 μg in 0.2 ml of normal saline). Plasma cytokine levels were measured 8 h after LPS challenge (Fig. 1). LPS-primed wild-type mice had decreased concentrations of IFN- in plasma compared to concentrations for saline-primed control wild-type mice (Fig. 1). IL-10 KO mice produced significantly (P < 0.05) higher levels of IFN- than wild-type mice did after LPS challenge. IL-10 KO mice receiving LPS priming had significantly lower LPS-induced IFN- concentrations in plasma than control saline-primed IL-10 KO mice. IL-12 was not detectable in plasma of control or LPS-primed wild-type mice within the limits of our assay at this time point (Fig. 1). However, IL-12 was present in plasma from IL-10 KO mice challenged with LPS (Fig. 1). Plasma IL-12 levels were significantly lower in IL-10 KO mice that received LPS priming than in saline-primed controls. Examination of plasma IL-10 concentrations showed higher IL-10 levels in LPS-primed wild-type mice than in saline-primed controls (Fig. 1). IL-10 was not detected in the plasma of IL-10 KO mice.
In further studies, whole blood was isolated from control and LPS-primed mice and cultured with LPS for 24 h, and cytokine concentrations in supernatants were measured (Fig. 1). Levels of IFN- and IL-12 production were significantly lower in blood from LPS-primed wild-type and IL-10 KO mice than in blood from saline-primed controls (Fig. 1). IL-10 levels were not significantly different between control and LPS-primed wild-type mice. IL-10 was not detectable in whole blood supernatants from IL-10 KO mice.
Expression of IFN-, IL-12, and IL-10 mRNAs in control and LPS-primed wild-type and IL-10 KO mice. Expression levels of mRNAs for IFN-, IL-12p40, and IL-10 were measured in spleens after LPS challenge (Fig. 2). Spleens were harvested at 0, 2, and 6 h after the LPS injection, and expression levels of cytokine mRNAs were measured using an RNase protection assay (Fig. 2). LPS-primed wild-type mice showed increased IL-10 mRNA expression levels at both 2 and 6 h after LPS challenge compared to levels for saline-primed control mice (Fig. 2). IL-10 mRNA was not detectable in IL-10 KO mice. LPS-primed wild-type mice showed decreased levels of IL-12p40 and IFN- mRNA expression compared to levels for control wild-type mice. IL-12p40 and IFN- mRNA expression levels were increased in IL-10 KO mice compared to levels in wild-type mice. At 2 h after LPS challenge, high levels of IFN- and IL-12 were induced in LPS-primed IL-10 KO mice and were comparable to levels observed for control IL-10 KO mice. However, at 6 h after LPS challenge, IFN- and IL-12p40 mRNA expression levels were lower in LPS-primed IL-10 KO mice than in saline-primed controls. IL-12p35 mRNA was constitutively expressed in wild-type and IL-10 KO mice and was not altered by LPS priming (data not shown).
LPS-induced IFN-, IL-12, and IL-10 production by splenocytes from wild-type and IL-10 KO mice. Splenocytes were obtained from control and LPS-primed mice 8 h after in vivo LPS challenge and cultured for 24 h. Cytokine concentrations in conditioned medium were measured using an ELISA (Fig. 3). Splenocytes isolated from LPS-primed wild-type mice produced smaller amounts of IFN- and IL-12 but secreted increased amounts of IL-10 compared to amounts from splenocytes obtained from saline-primed wild-type mice. Splenocytes isolated from IL-10 KO mice produced significantly (P < 0.05) larger amounts of IFN- than did wild-type mice but no significant difference in IFN- production was observed when comparing splenocytes from saline- and LPS-primed IL-10 KO mice (Fig. 3). Splenocytes from LPS-primed IL-10 KO mice produced significantly higher levels of IL-12 than saline-primed IL-10 KO mice (Fig. 3). IL-10 was not detectable in conditioned media from IL-10 KO mice.
Clearance of Pseudomonas aeruginosa is enhanced in LPS-primed wild-type and IL-10 KO mice. Wild-type and IL-10 KO mice were primed with saline or LPS for 2 days and were challenged with P. aeruginosa (5 x 107 CFU, i.p.) on day 4. Blood and lungs were harvested 6 h after bacterial challenge and plated on tryptic soy agar to measure Pseudomonas CFU (Fig. 4). Bacterial counts in blood and lungs were significantly lower in LPS-primed wild-type mice than in control wild-type mice (Fig. 4). The proportion of mice with detectable Pseudomonas in blood was only three out of nine for LPS-primed wild-type mice, whereas all wild-type control mice (9/9) had positive cultures. This difference was statistically significant (P < 0.05). Bacterial counts in blood and lungs were significantly lower for control IL-10 KO mice than for the control wild-type group. The proportion of IL-10 KO mice (3/9) with positive blood cultures was also significantly lower than the proportion of wild-type mice with positive blood cultures (9/9). Additionally, there were significantly fewer P. aeruginosa CFU in blood and lung cultures from LPS-primed IL-10 KO mice than from control IL-10 KO mice.
Cytokine production by control and LPS-tolerant wild-type and IL-10 KO mice after P. aeruginosa challenge. Control and LPS-primed wild-type and IL-10 KO mice were challenged with P. aeruginosa (5 x 107 CFU), and circulating levels of cytokines were assayed in the plasma 6 h later (Fig. 5). Production levels of IFN- and IL-12 were significantly decreased in LPS-primed wild-type mice compared to levels in control wild-type mice. Control IL-10 KO mice had significantly (P < 0.05) higher plasma levels of IFN- and IL-12 than control wild-type mice. LPS-primed IL-10 KO mice had significantly lower concentrations of IFN- and IL-12 in plasma than control IL-10 KO mice. Levels of IL-10 in the plasma of control and LPS-primed wild-type mice were not significantly different following P. aeruginosa challenge. IL-10 was not measurable in the plasma of IL-10 KO mice.
Clearance of Pseudomonas aeruginosa is enhanced in LPS-primed wild-type mice and in mice treated with anti-IL-10. Wild-type mice were primed with saline or LPS for 2 days. On day 3, mice received an injection of nonspecific immunoglobulin G (IgG) or anti-IL-10 and were challenged with P. aeruginosa (5 x 107 CFU, i.p.) on day 4. Blood and lungs were harvested 6 h after bacterial challenge and plated on tryptic soy agar. Plates were cultured for 24 h, and Pseudomonas counts were performed (Fig. 6). LPS-primed control mice had significantly fewer bacteria in blood and lungs than saline-primed mice (Fig. 6). Treatment of control mice with anti-IL-10 also significantly decreased bacterial counts in blood and lungs compared to counts for control mice receiving nonspecific IgG. LPS-primed mice treated with anti-IL-10 had significantly fewer Pseudomonas CFU in lungs, but not in blood, than did control mice receiving anti-IL-10.
Effect of anti-IL-10 treatment on cytokine production by control and LPS-primed wild-type mice after P. aeruginosa challenge. Wild-type mice were primed with saline or LPS on days 1 and 2, followed by treatment with anti-IL-10 or nonspecific IgG on day 3. All mice were challenged with P. aeruginosa (5 x 107 CFU) on day 4, and concentrations of cytokines in the plasma were measured 6 h later (Fig. 7). LPS priming in mice receiving control IgG caused significant decreases in Pseudomonas-induced IFN- and IL-12 concentrations in plasma, but IL-10 concentrations were increased (Fig. 7). Treatment of saline-primed mice with anti-IL-10 resulted in levels of IFN- and IL-12 in plasma significantly higher than levels for saline-primed mice treated with nonspecific IgG. Anti-IL-10-treated mice primed with LPS had IFN- and IL-12 concentrations in plasma significantly lower than concentrations for anti-IL-10-treated mice primed with saline. IL-10 concentrations in the plasma of mice treated with anti-IL-10 were less than 2 pg/ml. (Fig. 7).
DISCUSSION
It has been proposed that the widely used model of LPS tolerance mimics the state of immunoparalysis commonly observed to occur in critically ill patients (10, 24). This is because many of the immunological alterations observed during postinjury immunosuppression, such as decreased production of IFN- and IL-12 (16, 17, 20, 28), impaired antigen presentation (1, 11), and increased production of IL-10 (24, 27), are also characteristic of LPS tolerance (13, 34, 35). We have reported that mice recovering from injury caused by burns or cecal ligation and puncture exhibit suppressed production of IFN- and IL-12 as well as increased IL-10 secretion (17, 28). These cytokine alterations correlated with a decreased ability to clear P. aeruginosa (17). The present study shows that LPS-tolerant mice are more efficient than control mice in clearing P. aeruginosa from lungs and blood following systemic bacterial challenge. This is despite the observation that IL-12 and IFN- production are suppressed and IL-10 secretion is increased in these mice. These findings suggest that endotoxin tolerance does not replicate the immunosuppressive state typical of septic patients and may represent a state of altered proinflammatory cytokine production without adverse, or perhaps even with beneficial, effect on antimicrobial immune function. The mechanisms of improved bacterial clearance in LPS-tolerant mice are not clear at this juncture. However, some reports indicate that phagocytic function may be improved in mice receiving prior LPS exposure (23). Nevertheless, the mechanisms of the enhanced bacterial clearance remain to be elucidated and warrant further study.
This study also demonstrates that IL-10-deficient mice are more effective in clearing P. aeruginosa than wild-type mice and that IL-10-deficient mice are particularly efficient in removing P. aeruginosa from blood and lungs after systemic bacterial challenge. Sewnath and colleagues (26) previously showed that IL-10 KO mice remove E. coli more efficiently than wild-type mice but have higher mortality during E. coli peritonitis. The increased mortality of the IL-10 KO mice was attributed to inflammation-induced tissue injury and organ dysfunction. The present study confirms enhanced bacterial clearance and increased systemic cytokine production in IL-10-deficient mice following systemic challenge with P. aeruginosa. Interestingly, LPS-tolerant IL-10 KO mice clear P. aeruginosa more efficiently than control IL-10 KO mice but produce proinflammatory cytokines at levels that are significantly lower than those produced by saline-primed IL-10 KO mice and that are typical of wild-type mice. This observation was confirmed for LPS-primed mice that received treatment with antibody against IL-10. These findings imply that IL-10 has an inhibitory effect on bacterial clearance that is independent of the ability to regulate production of IFN- and IL-12. Previous investigators have shown that IL-10 inhibits the phagocytic function of neutrophils and macrophages (12, 21). In additional studies, Steinhauser and colleagues (27) demonstrated that IL-10 directly contributes to impaired P. aeruginosa clearance in the lungs of mice previously exposed to cecal ligation and puncture. Interestingly, we observed that LPS-tolerant wild-type mice, in which IL-10 production is generally increased, have an improved ability to clear P. aeruginosa from blood and lungs. One interpretation of these findings is that induction in LPS tolerance causes specific changes, perhaps enhanced phagocytosis, which result in improved bacterial clearance despite the increase in IL-10 production. Removal of IL-10 may eliminate an additional inhibitory factor and further enhance bacterial clearance.
These studies confirm that IL-10 is an important regulator of LPS-induced production of IFN- and IL-12. IFN- and IL-12 were produced at higher levels by IL-10 KO mice than by wild-type controls. This finding is in agreement with previous studies which show that IL-10 is a potent negative regulator of IL-12 and IFN- production (5, 6). The role of IL-10 in causing suppressed IFN- production during LPS tolerance appears organ specific. LPS tolerance-induced suppression of IFN- and IL-12 production was reversed in the spleens but not in the blood of IL-10 KO mice. Therefore, IL-10 may be important for the regulation of IFN- and IL-12 production in the spleen during LPS tolerance but not for leukocytes circulating in blood or other tissues that contribute to plasma IFN- and IL-12. This complex picture may explain conflicting results from other studies aimed at understanding the role of IL-10 in the development of LPS tolerance. Berg et al. (2) reported that LPS tolerance can be induced in IL-10-deficient mice, and Wysocka et al. (36) showed that IL-12 production is suppressed in LPS-tolerant IL-10 KO mice. Both investigators concluded that IL-10 does not play a major role in the development of LPS tolerance. However, other investigators have shown that IL-10 neutralization during LPS priming interferes with the development of LPS tolerance (18, 22). Taken together, these findings show that IL-10 may partially mediate cytokine suppression in some tissues during LPS tolerance. Numerous additional mechanisms of suppressed cytokine production in the LPS-tolerant host have also been demonstrated. Among these are generation of p50/p50 NF-B homodimers, suppressed mitogen-activated protein kinase signaling, and upregulation of SOCS proteins (9, 38). Our findings indicate that IL-10 appears to make a limited, tissue-specific contribution to the overall regulation of IFN- and IL-12 production during LPS tolerance.
In summary, this report shows that the model of LPS tolerance may not mimic postinflammatory immunoparalysis, because LPS-tolerant mice have an increased ability to clear P. aeruginosa despite diminished production of IFN- and IL-12 but increased secretion of IL-10. LPS-tolerant, IL-10-deficient mice were the most efficient in clearing bacteria from blood and lungs, which suggests that IL-10 has a direct negative impact on bacterial clearance mechanisms. The role of IL-10 in causing suppressed IFN- and IL-12 production during LPS tolerance is organ specific and only partially accounts for suppressed cytokine production in the LPS-tolerant host.
ACKNOWLEDGMENTS
These studies were supported by NIH grant R01 GM66885 and grant 8780 and 8650 from the Shriners of North America.
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