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Infection and Immunity, June 2006, p. 3387-3395, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.01985-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

High Levels of Interleukin-10 Impair Resistance to Pulmonary Coccidioidomycosis in Mice in Part through Control of Nitric Oxide Synthase 2 Expression

Maria del Pilar Jimenez,^1, Lorraine Walls,¹ and Joshua Fierer^1,2*

VA Healthcare System,¹ Departments of Medicine and Pathology, University of California San Diego School of Medicine, San Diego, California²

Received 7 December 2005/ Returned for modification 26 January 2006/ Accepted 2 March 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown previously that there is a direct correlation between IL-10 levels and susceptibility to Coccidioides immitis peritonitis in C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice. We now show that B6 mice are also more susceptible to C. immitis pneumonia and that interleukin-10 (IL-10)-deficient (IL-10^–/–) B6 mice are more resistant to C. immitis pneumonia. In addition, we established that high levels of IL-10 are sufficient to make genetically resistant mice susceptible to both C. immitis peritonitis and pneumonia by infecting h.IL-10 transgenic mice. Infected h.IL-10 transgenic mice express lower levels of gamma interferon, IL-12 p40, and inducible nitric oxide synthetase 2 (NOS2) mRNA in their lungs, implicating inducible NOS as a defense mechanism in this disease. We treated DBA/2 mice with aminoguanidine, and they became more susceptible to C. immitis peritonitis and pneumonia. We conclude that high levels of IL-10 are both necessary and sufficient to make mice susceptible to C. immitis, regardless of the genetic background of the mice, and that IL-10 impairs resistance to C. immitis in part by suppressing NO synthesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coccidioidomycosis is one of the endemic mycoses in the United States. It is estimated that there are 100,000 new cases of coccidioidomycosis annually (1). The majority of Coccidioides infections in people produce either no symptoms or a self-limited pneumonia. One characteristic manifestation of primary infection is the syndrome of fever, arthralgias, and erythema nodosum known as Valley Fever, after the San Joaquin Valley in central California (18). People who spontaneously resolve their infections have positive skin tests (delayed hypersensitivity) with fungal antigens. A small percentage of infected patients do not recover spontaneously (53). Most patients who do not recover spontaneously develop extrapulmonary infections, and they usually have negative skin tests and high titers of antibodies, suggesting that they make primarily a Th2 immune response. In contrast, patients who recover spontaneously from the pulmonary infection make only low titers of antibody against the fungus and instead develop delayed-type hypersensitivity (48). Very little is known about what determines whether otherwise-healthy people will recover from infection or will develop progressive disseminated disease, but there is clearly a large genetic component, as Filipinos and African Americans are much more likely to develop disseminated coccidioidomycosis than Caucasians (15, 46, 60).

In order to study the genetics of resistance to Coccidioides immitis in an experimental animal, we tested several strains of inbred mice for their susceptibility to the fungus; we discovered that DBA/2 mice are quite resistant to C. immitis when arthroconidia are injected intraperitoneally (i.p.), and C57BL/6 (B6), C57BL/10, and BALB/c mice are very susceptible to this infection (31). (Since both DBA/2 and BALB/c mice have the H-2^d major histocompatibility complex [MHC] haplotype, the difference in resistance is not determined by the MHC.) Resistance to C. immitis is the dominant phenotype. Cox et al. showed that DBA/2 mice are more resistant than BALB/c mice to pulmonary infection with Coccidioides (7). By studying C57BL/6 x DBA/2 (BXD) recombinant inbred mice, we learned that resistance in mice is a multigenic trait (14). We also found that DBA/2 mice make less interleukin-10 (IL-10) in response to infection than do susceptible mouse strains, and in the BXD recombinant inbred lines the levels of IL-10 mRNA are directly proportional to the severity of infection measured as CFU of fungi in the lungs 14 days after i.p. infection (12). Most importantly, IL-10-deficient B10 and B6 mice are more resistant to C. immitis than the control parental strains, indicating that high levels of IL-10 are necessary for mediating susceptibility to coccidioidomycosis (12, 13). These results imply that high levels of IL-10 are not simply the result of excess antigen but are also responsible in part for inherited susceptibility to this infection in mice (13, 31). However, we have not established that high levels of IL-10 are sufficient to make mice susceptible to C. immitis. In this study we studied the effect of high levels of IL-10 on resistance to coccidioidomycosis by infecting genetically resistant mice that are transgenic for h.IL-10 under the control of an MHC II promoter (22). We confirmed that high levels of IL-10 are sufficient to make mice susceptible to coccidioidomycosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. Female B6, B6IL-10 (–/–) (32), DBA/2J, and BALB/cJ mice were purchased from Jackson Laboratory (Bar Harbor, ME), and DBF1 mice were purchased from Simonsen Laboratories (Gilroy, CA). We infected only female mice, because male mice are more susceptible to this infection. BALB/c.h.IL-10 (human IL-10) transgenic mice were the gift of Amy Beebe at DNAX (22), and C57BL/6 m.IL-10 (mouse IL-10) transgenics were the gift of Mitchell Kronenberg at La Jolla Allergy and Immunology (24). The h.IL-10 gene is expressed in antigen-presenting cells under the control of the MHC class II EA promoter, and the B6 transgenic expresses IL-10 under the control of the IL-2 promoter. Because BALB/c and B6 are strains are susceptible but resistance to C. immitis is a dominant phenotype (31), we crossed female transgenic mice with male DBA/2 to make F₁ transgenic mice and compared them to normal F₁ mice.

Infection. The RS strain was grown on Mycosel agar with gentamicin, and arthroconidia were harvested by scraping the saline-wetted mat when the culture was mature (58, 59). The mycelia were disrupted by shaking with glass beads. The suspension was passed over a nylon wool column to remove debris. This suspension was kept in saline at 4°C until it was used. An aliquot was cultured to be sure that viability was maintained before each experiment.

We infected mice in a safety hood in a biosafety level 3 laboratory, using a protocol that was approved by the local Biosafety Committee. For intranasal (i.n.) infections, the mice were anesthetized with ketamine (30 mg/ml) plus xylazine (4 mg/ml) given intramuscularly. Different strains of mice require different amounts of ketamine-xylazine to achieve adequate anesthesia. We waited until they did not withdraw from a paw squeeze before we placed 20 µl of a suspension of arthroconidia of the RS strain (59) into their nares. If they were not fully anesthetized after 5 min, we gave additional doses of the anesthetic every 5 min until they did not withdraw. Once the inoculum of approximately 100 arthroconidia was inspired, the unconscious mice were held vertically for about 15 seconds to promote aspiration of the fungus into the lungs. Mice were then kept warm on a heating pad until they were able to walk again. For the i.p. infection, we injected 500 to 1,000 arthroconidia in 0.2 ml of saline. Infected mice were housed five per cage in an isolator box (Germfree Laboratories, Miami, FL). Mice were sacrificed at the indicated times after infection, and their lungs and spleens were removed for quantitative cultures. A piece of the left lung was immediately frozen in liquid nitrogen to serve as a source of RNA. Tissues were ground under the safety hood and then serially diluted in saline before being plated on Mycosel agar as previously described (31). The plates were incubated at 24°C until there was visible growth and then colonies were counted.

mRNA. RNA was isolated from frozen lungs using Ultra Spec and reverse transcribed to cDNA with Superscript 2 (Invitrogen, San Diego, Calif.) as previously described (12). For competitive reverse transcription-PCR (RT-PCR), we pooled equal amounts of RNA from three mice/group that were chosen because they had CFU/lung values that were closest to the median values for the group. The amount of mRNA for several cytokines was measured semiquantitatively using competitive RT-PCR. We expressed the result as molecules of specific mRNA/microgram of total RNA, as we have previously described (10). IL-10 mRNA in transgenic mice was measured using real-time PCR on the ABI Prism 7000 detection system with primers that amplified both mouse and human IL-10 cDNA, because the h.IL-10 transgenic mice make both human and mouse IL-10 and both are functional in mice. The following primers were used: forward, TCTTTCAAACAAAGGACCAGCTG; reverse, AAGGCTTGGCAACCCAAGTA. The primers were 87% homologous with mouse and 95% homologous with human IL-10 cDNA and were able to amplify IL-10 cDNA from both human and mouse cells.

NO assays. Three mice were kept in a metabolic cage (Nalgene, Rochester, NY) for 24 h without food but with free access to water. We collected their pooled urine in a tube with gentamicin and ampicillin to prevent bacterial overgrowth and centrifuged the urine to remove debris. Urine samples were then filtered and frozen at –70°C until they were assayed. Each urine sample was diluted between 1:20 and 1:100, and total urinary nitrate excretion was measured using the Griess reaction after reduction of NO₃^– to NO₂ with 1 M Tris, 0.02 mM NADPH, 5 mM glucose-6-phosphate, 10 U/ml of glucose-6-phosphate dehydrogenase, and 1 U/ml of Aspergillus nitrate reductase (Boehringer Mannheim) (20). To correct for the completeness of the collection and for catabolism, we measured urinary creatinine and expressed the ratio of urinary nitrates to urinary creatinine (3).

NOS2 inhibition. We used two inhibitors of inducible nitric oxide synthase (iNOS [NOS2]) (5). We dissolved the L-arginine analogue aminoguanidine (AMG; Sigma) in water to make a 1% (wt/vol) solution. The mice refused to drink the AMG solution, which has a bitter taste, and so we added 75% banana syrup (vol/vol) and 2% glucose to the solution. Control mice received the same solution without the AMG. N⁶(2-iminoethyl) L-lysine dihydrochloride (L-NIL; Alexis Biochemicals, San Diego, Calif.) at 3 mM was dissolved in drinking water. Treatment with the NOS inhibitors began 3 days before mice were infected and continued until the day of sacrifice. Urine was collected within 48 h after infection, well before animals were ill, and between days 13 and 15 after infection. At the later time point infected mice were drinking less and they had lower urine volumes.

Statistics. Colony counts were log transformed, and the geometric means were calculated. The differences between geometric mean colony counts in different groups of mice and other variables were compared using the unpaired t test and an analysis of variance (Graphpad Prism, San Diego, Calif.). We considered a P value of <0.05 to be significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We first compared the natural history of the respiratory infection in B6 and DBA/2 mice infected i.n. with the RS strain. We found that all mice in both groups had bilateral pneumonia and, although there was variation in the distribution of infiltrates from mouse to mouse, there was no consistent bias toward infection of either lung. Therefore, to be consistent from experiment to experiment, we used the right lung for fungal culture and extracted RNA from the left lung. As shown in Fig. 1, the two mouse strains were equally infected on day 10 after infection. By 14 days after infection the median colony count in the lungs of B6 mice had increased more than 10-fold, but it had only increased 2-fold in the DBA/2 mice. Between days 14 and 16 the median colony count in the lung increased another 10-fold in B6 mice but did not increase in DBA/2 mice. The differences in lung counts between the two strains were statistically significantly (P = <0.02) on days 14 and 16 postinfection. The two strains also differed markedly in the prevalence of extrapulmonary dissemination. There was no dissemination on day 10 in either strain, but by day 14 after infection C. immitis had disseminated to spleens of all B6 mice, whereas all the DBA/2 spleens were still sterile. DBA/2 spleens remained sterile on day 16 after infection, while the splenic infection got progressively more severe in the B6 mice. We terminated the experiment on day 16 because B6 mice began to die. We emphasize that the infection did not disseminate from the lungs in DBA/2 mice, but it did in B6 mice, which is similar to the difference in the natural history of human coccidioidomycosis in resistant and susceptible populations (17). These results extend the findings of Cox et al. (7) to show that B6 mice are also more susceptible than DBA/2 mice to pulmonary coccidioidomycosis.

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FIG. 1. DBA/2 mice are more resistant than B6 mice to C. immitis pneumonia. B6 (open bars) and DBA/2 (black bars) mice were infected i.n. as described in Materials and Methods and sacrificed on the indicated days after infection. The geometric mean number of CFU and the standard errors of the means are shown for each group. Ten organisms was the limit of detection for spleen cultures, and so those cultures with no growth are shown at log10 as 1. Differences between the CFU recovered from the lungs of the two mouse strains were not significant on day 10 but highly significant (P = 0.003 and 0.004) on days 14 and 16 after infection. Since the DBA/2 spleens were sterile on all days, we did not calculate P values for those results. There were five mice/group/day.

To begin to delineate the differences between the immune responses of the two mouse strains to this infection, we compared the levels of IL-10 and IL-4 mRNA in their lungs on days 10 and 16 after i.n. infection (Fig. 2). There were very low levels of IL-4 and IL-10 mRNA in uninfected lungs. By day 10 after infection the IL-10 mRNA levels had risen 500-fold in B6 mice but less than 10-fold in DBA/2 mice, even though both strains of mice had the same numbers of CFU/lung on that day (Fig. 1). Similarly, on day 10 after infection IL-4 mRNA levels had risen nearly 1,000-fold in B6 mice and only 25-fold in DBA/2 mice. IL-10 levels did not change on day 16 after infection in either strain, but in B6 mice IL-4 mRNA levels decreased on day 16 so that there was no statistically significant difference between IL-4 mRNA levels in the two strains of mice on that day, at the time that B6 mice were dying of infection. The kinetics of the IL-4 response in the B6 mice is similar to what Magee and Cox found in BALB/c mice, another highly susceptible strain (37).

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FIG. 2. IL-4 and IL-10 mRNA levels increase in the lungs of mice with C. immitis pneumonia. mRNA in the lungs of infected and uninfected mice was measured semiquantitatively using competitive RT-PCR. Each time point represents equal amounts of RNA pooled from three mice per group (see Materials and Methods), and the control mRNA is from the lungs of two healthy uninfected mice. The time course of infection for this experiment is shown in Fig. 1. These measurements were repeated on the same samples with similar results.

Based on our findings of high levels of IL-10 mRNA in the lung after i.n. infection, which were similar to what we found in the peritonitis model (12), we decided to determine if the absence of IL-10 would make mice more resistant to C. immitis pneumonia by infecting B6 IL-10^–/– mice i.n. We used colony counts in the lungs and spleens on day 15 after infection as a measure of susceptibility, as most of the B6 mice were still alive at that time but the infection had progressed far enough so that we were able to clearly distinguish between genetically susceptible and resistant mouse strains. As shown in Fig. 3, the median colony count in lungs from B6.IL-10^–/– mice was 10-fold lower than the counts from the lungs of B6 mice and similar to the median count in DBA lungs. Even more important, the infection did not disseminate to the spleens of either the IL-10^–/– or the DBA mice. These results suggest that high levels of IL-10 impair the ability of B6 mice to contain the infection in the lung and permit hematogenous dissemination.

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FIG. 3. IL-10–/– mice are more resistant to C. immitis pneumonia. Mice were infected i.n. with 100 arthroconidia as described in Materials and Methods and sacrificed 15 days later. The difference between the median CFU/lung in B6 and B6.IL-10–/– mice was not quite statistically significant (P = 0.056) because of the large standard deviation in the IL-10–/– group, but the difference in spleen CFU was significant (P = 0.034), suggesting that the IL-10–/– mice were less likely to have a disseminated infection. DBA/2 mice were included only as a resistant control, and no statistical comparisons were made between IL-10–/– and DBA/2 mice in this experiment. There were 10 mice/group.

We then asked whether high levels of IL-10 were sufficient to make resistant mice more susceptible. Because of the biohazard we housed infected mice in an isolation glove box, and so it was not feasible to give them daily injections of IL-10. As an alternative to administering exogenous IL-10, we infected IL-10 transgenic mice after they were crossed to the resistant DBA/2 strain, so that the transgenic mice would be on a genetically resistant background (31). We first infected mice that express the human IL-10 transgene (h.IL-10) under the control of the mouse MHC class II promoter (22). We anticipated this strategy would work because resistance to C. immitis is dominant (31) and, although the transgenic F₁ mice expressed h.IL-10 from only one chromosome, others have shown that h.IL-10 F₁ transgenic mice are still more susceptible to Listeria monocytogenes and Leishmania major (22). As shown in Fig. 4A, transgenic F₁ mice were more susceptible to C. immitis peritonitis than (DBA/2 x BALB/c)F₁ animals, with median lung and splenic fungal counts more than 1.5 logs higher in the transgenic mice than in the DB/c F1 controls. We then infected the mice i.n. and found that transgenic F₁ mice were also more susceptible when infected by that route (Fig. 4B). The median colony count in the lungs of transgenic F1 mice was nearly 1 log higher than the median for control DB/c F₁ mice (P = 0.02). In addition, the fungus disseminated to the spleens of all the transgenic F₁ mice but not to the spleens of the control DB/c F₁ mice. Interestingly, when we did the same experiment using m.IL-10^+/– transgenic mice (on a DBA/2 x B6 background), which express m.IL-10 under the control of the IL-2 promoter (24), those IL-10 transgenic mice were not more susceptible to C. immitis peritonitis (data not shown). To try to understand why there was a difference between the two strains of transgenic mice, we measured total IL-10 mRNA in the infected lungs using RT-PCR. Since human and mouse IL-10 cDNA are so homologous, we were able to design PCR primers that amplified both human and mouse cDNA, which allowed us to measure total IL-10 mRNA in mice expressing both the mouse and the human transgene. As shown Fig. 5, the IL-10 mRNA in (BALB/c x DBA/2)F₁ (controls) only increased 2-fold, while the transgenic mice with the IL-10 gene under control of the MHC promoter had nearly a 15-fold increase in IL-10 mRNA. In contrast, the transgenic mice that express IL-10 under the control of the IL-2 promoter had only a twofold increase in IL-10 mRNA, even less than the increase in the F₁ controls for that experiment.

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FIG. 4. High levels of IL-10 are sufficient to increase the severity of coccidioidomycosis. A. DB/c F1 h.IL-10+/– transgenic mice and control DB/c F1 mice were infected i.p. with 310 arthroconidia and sacrificed 14 days later. Spleens and lungs were removed for quantitative culture. The F1 mice expressing the transgene had more organisms in their lungs and spleens than the genetically resistant DB/c F1 control mice (P = <0.01). BALB/c mice were included as susceptible controls, and no statistical comparisons were made between them and the other two groups of mice. B. The same groups of mice were infected i.n. with 50 arthroconidia and were sacrificed 15 days after infection for quantitative mycology. The difference between median lung CFU in DB/c h.IL-10+/– and DB/c F1 mice was nearly 10-fold (P = 0.02), and only the IL-10 transgenic mice had C. immitis in their spleens. There were 10 mice/group.

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FIG. 5. F1 mice that express the h.IL-10 transgene under the control of the MHC II promoter (A) make more IL-10 than mice that have a transgene under control of the IL-2 promoter (B). We analyzed RNA from the lungs of three mice infected i.n. with C. immitis 15 days earlier. The change in IL-10 mRNA expression was calculated in relation to expression of IL-10 in uninfected lungs. The mean increase in mRNA ± the standard error of the mean is shown for each group.

We then compared the levels of other selected cytokine mRNA in infected lungs from h.IL-10 F₁ mice and the appropriate control mice. As shown in Table 1, the control mice had nearly 10-fold more gamma interferon (IFN-) and IL-12 p40 mRNA on day 15 after infection and 25 times more NOS2 mRNA than h.IL-10^+/– transgenic mice. There was no significant difference in IL-4, IL-2, or tumor necrosis factor alpha (TNF-) mRNA levels in the two strains after infection.

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TABLE 1. Comparison of cytokine mRNA levels in the lungs of transgenic h.IL-10+/– and control mice 15 days after i.n. infection

Because NOS2 mRNA levels were so much lower in the h.IL-10 transgenic mice and NOS2 has been shown to contribute to resistance to several pathogens, we asked whether NO played a role in resistance to C. immitis (2, 11, 43). To confirm that the increased NOS2 mRNA levels reflected increased enzymatic activity, we infected DBA/2 mice and measured the urinary excretion of nitrates, a measure of overall activity of NOS in the host (19). We also determined the effect of the NOS inhibitor AMG on urine nitrate levels and the effect of AMG treatment on growth of the fungus. As shown in Fig. 6, i.p.-infected DBA/2 mice excreted very high levels of urinary nitrate, and AMG treatment lowered these levels by 85%. AMG treatment also exacerbated the infection, since 3/10 AMG-treated mice died from an infectious dose that never kills normal DBA/2 mice, and the surviving AMG-treated mice had about a 10-fold increase in geometric mean numbers of viable organisms in their lungs (5.53 versus 4.6; P = 0.0005) and spleens (4.9 versus 4.0; P = 0.0024) (Fig. 7). We then repeated the experiment using i.n. infection, and again AMG treatment significantly increased (P = <0.003) colony counts in the lungs 14 days after infection (Fig. 8). However, there was no dissemination to the spleens in either group, indicating that suppression of NO production either was not complete enough or that there are additional defense mechanisms in DBA/2 mice that are able to prevent dissemination.

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FIG. 6. Orally administered AMG reduces the urinary excretion of nitrates in infected DBA/2 mice. We put 1% AMG in the drinking water of DBA/2 mice beginning 48 h prior to infection as described in Materials and Methods. Each symbol represents urine from pools of three mice on either day 13 or 14 after i.p. infection. The concentration of creatinine was used to correct for differences in the completeness of the collection and the concentration of the urines. Nitrate excretion is expressed as the molar ratio of nitrates/creatinine. AMG reduced that ratio by three- to fivefold.

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FIG. 7. Oral AMG increases the severity of C. immitis peritonitis in DBA/2. A. CFU/lung 14 days after i.p. infection in mice treated with 1% AMG in their drinking water and untreated controls. B. CFU/spleen in the same mice. The differences between the means of the groups are shown. These data are the composite of two separate experiments, and the total number of mice in each group is in parentheses.

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FIG. 8. AMG increases the severity of C. immitis pneumonia in DBA/2 mice infected by inhalation. Mice were treated with 1% AMG in their drinking water and infected by i.n. inoculation with 50 C. immitis arthroconidia. We sacrificed the mice 14 days later and performed quantitative cultures of their lungs. Only mice with positive lung cultures were included, because we did not succeed in infecting all the mice. There were 10 mice in each group. The geometric mean ± standard error of the mean is shown. Means were compared using the unpaired t test.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A Th1 cell-mediated immune response is required for recovery from infections with Coccidioides sp., whereas the development of a Th2-driven humoral immune response is a bad prognostic feature (reviewed in reference 7a). Thus, immunity to coccidioidomycosis is similar to immunity to leprosy and leishmaniasis in people and mice (reviewed in reference 35a). What is unusual about coccidioidomycosis is that there is a strong association between ethnicity and susceptibility; African Americans and Filipinos are about 10 times more likely to have clinically evident extrapulmonary dissemination of the infection and high titers of antibodies than Caucasians (21, 28, 47). This suggests that there is a genetic predisposition to severe coccidioidomycosis in humans.

Inbred mice also vary in their susceptibility to C. immitis. In previous studies we showed that B6 mice were more susceptible then DBA/2 mice to C. immitis peritonitis, and in this study we extended that observation to show that B6 mice are also more susceptible to i.n. infection with C. immitis RS, but B6 IL-10^–/– mice are resistant. We repeated those experiments with i.n. infection because there is some evidence that the immune response to inhaled pathogens is different than the response to the same pathogen injected intravenously or i.p., possibly because pulmonary macrophages have a fairly distinct phenotype that is largely antiinflammatory (6, 34, 54).

We found that, as in the peritonitis model, i.n.-infected B6 mice had higher levels of IL-10 and IL-4 mRNA in their lungs, even before the infection had disseminated to their spleens. The question remained whether high levels of IL-10 are sufficient to make mice more susceptible to C. immitis infection. To answer this question we bred DB/c F₁ h.IL-10^+/– mice that expressed higher levels of biologically active IL-10. In fact, even though these mice had only one transgenic chromosome, they had nearly 15 times more IL-10 mRNA (Fig. 5) and they were more susceptible to this infection. We also infected a different IL-10 transgenic strain that expresses the m.IL-10 transgene under the control of the IL-2 promoter; those mice increased IL-10 mRNA production only twofold, and they were not more susceptible to C. immitis. Others have shown that the mice with IL-10 expressed from the IL-2 promoter are more susceptible to tuberculosis, but they were studied as homozygous animals (two copies of the transgene) (56).

Moore et al. (41) recently reviewed the subject of IL-10 and susceptibility to infections. IL-10 is vitally important for modulating the inflammatory response to microbial products that can otherwise result in septic shock and other pathologies (33). However, too much IL-10 can compromise the host's ability to resist bacterial, viral, fungal, and protozoan infections (26, 30, 49, 57). Resistance to infections is almost always increased when IL-10 is eliminated, whether with neutralizing antibody or by mutating IL-10 genes. Even normally resistant mice benefit from having less IL-10 when they are infected experimentally (9). Conversely, artificially high levels of IL-10, whether the cytokine is administered exogenously or endogenously synthesized by transgenic mice, are often deleterious (22). Interestingly, humans with tuberculosis who make high levels of IL-10 are anergic and remain so even after successful treatment with antituberculosis drugs (4).

IL-10 is a major immunoregulatory cytokine, which accounts for its activity in so many diverse experimental infections. IL-10 down-regulates the effector functions of macrophages and polymorphonuclear leukocytes (42), including release of reactive oxygen intermediates and proinflammatory cytokines, such as IL-12, TNF-, IL-6, and IL-1 (50, 51). Similarly, mice that lack the IL-10 transcription factor STAT3 in their macrophages have an overexuberant inflammatory response to infection (39). IL-10 also affects the acquired immune response by down-regulating the expression of MHC class II and costimulatory molecules on antigen-presenting cells (29). The suppression of IL-12 secretion by accessory cells probably contributes to the Th2 bias that is attributed to IL-10 (55). Recently it was shown that dendritic cells from IL-10^–/– and normal B6 mice activate different programs of protein expression after they are pulsed with chlamydial elementary bodies, but how that biases the stimulated T cells to IFN- production is not yet known (25). IL-10 also promotes the generation of regulatory T cells (Tr1) that in turn suppress antigen-specific immune responses in part through the secretion of IL-10 (23).

An interesting aspect of the relationship between IL-10 and resistance to murine coccidioidomycosis is the genetic control over the IL-10 response; infected B6 and BALB/c mice make more IL-10 than do DBA/2 mice (12). The IL-10 response of mice to Candida albicans infection is also under genetic control, but in that fungal infection DBA/2 mice make more IL-10 and are more susceptible than B6 mice (51). Thus, there are similarities between our studies with C. immitis and the C. albicans studies, but the two fungi induce the opposite IL-10 response in the same pair of mice; C. immitis stimulates more IL-10 in B6 mice while C. albicans stimulates more IL-10 in DBA/2 mice. The fungal products of C. immitis and C. albicans that are responsible for stimulating IL-10 production in mice are not known in either case.

Resistance to most pathogens involves a rapid, innate immune response that is followed by an antigen-specific adaptive immune response (40). This is of course an oversimplification, since the innate immune response usually continues through the course of infection, and the nature of the adaptive immune response is strongly influenced by the innate response (44). Resistance to C. immitis requires a well-coordinated immune response that has several crucial components. Magee and Cox have shown that IFN- and IL-12 are both necessary for an effective immune response in mice (37, 38). The early production of IL-10 could interfere with the production or the effects (or both) of those cytokines. Infected B6 IL-10^–/– mice made more IL-12 p40 mRNA than B6 mice, showing that they were capable of making even more IL-12 p40 in response to infection. Conversely, the infected h.IL-10 transgenic mice had less IL-12 p40 mRNA than the F₁ controls, indicating that IL-10 can suppress transcription of IL-12 p40 even in genetically resistant mice. This could mean that in murine coccidioidomycosis IL-10 acts in part to suppress transcription of IL-12 p40, and IL-12 is pivotal in directing the immune response toward a Th1 T-cell response (55). IL-10 produced during the early phase of infection can act directly on T cells, inhibiting chemotaxis of CD4⁺ cells and IL-2 production, and it can act on antigen-presenting cells to bias them toward T regulatory cells (41).

High levels of IL-4 are also detrimental to mice infected with C. immitis (37). IL-4 is a cytokine that biases the immune response to Th2 (16), and IL-4 levels are at least transiently higher in genetically susceptible mice than in resistant mice (Fig. 2) (37). IL-4 is a potent inducer of IL-10 in T cells (35). However, BALB/c mice treated with neutralizing antibody against IL-4 showed only a modest increase in resistance to C. immitis (37), and IL-4^–/– mice are only slightly more resistant to C. immitis infections (12). Of course, there is no reason why IL-4 and IL-10 cannot both down-regulate the immune response to C. immitis in susceptible mice. Interestingly, even in L. major infections in mice, where the amount of IL-4 made is genetically determined and is crucial for determining whether or not the lesions heal, IL-10 has been shown to be important for promoting susceptibility, and IL-4/IL-10 double-knockout mice are more resistant than either single knockout (45). Similarly, mice that are doubly deficient in IL-10 and IL-4 do not make a Th2 response to Schistosoma mansoni eggs, whereas mice deficient in either cytokine still make a weak Th2 response and have more pulmonary granulomas (61).

Part of the deleterious effect of IL-10 in C. albicans infection is due to down-regulation of NO production, inhibiting the candicidal activity of macrophages (51). We too found that IL-10 overproduction down-regulated NOS2 expression transcriptionally. IL-10 modulates macrophage expression of NOS2 (51). When we found that DBA/2 mice had higher lung levels of NOS2 mRNA than B6 mice and that h.IL-10 transgenic mice had lower levels of NOS2 mRNA, we postulated induction of NOS2 was an important mechanism for controlling the growth of this fungus. NO is a well-known antimicrobial agent that is active against a variety of bacteria, parasites, and even fungi (8). To determine the role of NO in coccidioidomycosis, we treated DBA/2 mice with the NOS inhibitor AMG. We did not use NOS2^–/– mice, because they are on a B6 background, which is a susceptible strain. DBA/2 mice treated with oral AMG had approximately 10-fold more C. immitis in both their lungs and spleens, and 3/10 AMG-treated mice died of infection. Since AMG is not a specific inhibitor of NOS2, we cannot be certain that the exacerbation of the infection was due solely to inhibition of NOS2. We did attempt to treat mice orally with the more specific NOS2 inhibitor L-NIL at a 3 mM concentration in drinking water. However, this dose achieved only a 50% reduction in excretion of urinary nitrate in infected mice, and there was only about a threefold increase in lung CFU (data not shown) that barely reached statistical significance (P = 0.05). Nevertheless, the trend was toward confirmation of the AMG result, and we think this provides additional support for the idea that NOS2 contributes to resistance to C. immitis. AMG- and L-NIL-treated DBA/2 mice were still not as susceptible as B6 mice, which could mean that DBA/2 mice have other important resistance mechanisms that are not inhibited by those drugs, or it could reflect our inability to completely inhibit NO synthesis with oral inhibitors. AMG treatment has been shown to exacerbate several other granulomatous infections in mice, including Salmonella enterica and New World trypanosomiasis (36, 52, 62). In some cases AMG also decreases IL-10 release from activated macrophages (27). Whether AMG has a similar effect on macrophage inhibition of C. immitis remains to be established. L-NIL-treated mice were not as susceptible, but with the dose we used of oral L-NIL we were unable to inhibit NO production by more than 50%, which could explain why it was less effective than AMG.

 
We thank Amy Beebe of DNAX and Mitchell Kronenberg of the La Jolla Institute of Allergy and Immunology for providing breeding pairs of IL-10 transgenic mice.

This work was supported by a grant from the Research Service of the Department of Veterans Affairs.

 
* Corresponding author. Mailing address: Infectious Diseases Section (111F), VA San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161. Phone: (858) 552-7446. Fax: (858) 552-4398. E-mail: jfierer{at}ucsd.edu.

Editor: A. Casadevall

Present address: Grupo de Micología Médica y Experimental, Corporación para Investigaciones Biológicas, Medellín, Columbia.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, June 2006, p. 3387-3395, Vol. 74, No. 6
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