Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-γ, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-γ message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-γ expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-γ neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-γ-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-γ induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-γ knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-γ is required for resistance. Nonetheless, infected IFN-γ knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-γ and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-γ production. Thus, we propose that although IFN-γ can drive resistance, the overproduction of IFN-γ during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.

Among the yeast that constitute the normal microbiota of the skin, mucous membranes, and gastrointestinal tract, the species Candida albicans is most frequently associated with opportunistic infections in man. As a disease entity, candidiasis is prevalent in individuals receiving corticosteroids, cytotoxic or immunosuppressive drugs, or radiation therapy and in those with hematologic malignancies or AIDS (1, 2, 3). Such observations indicate that intact cell-mediated immunity is essential for resistance to this fungal infection.

The development of distinct Th responses and cytokine profiles in mice has been shown to critically influence the successful outcome of host defense against microbial and parasitic pathogens (4, 5). While innate defense mechanisms (primarily granulocyte and macrophage mediated) appear to be the initial means by which infections caused by Candida albicans are contained, clearance of infection and long term immunity appear to be determined by the emergence of T cell-mediated immune responses (6). Studies using a systemic model of infection suggest that protection is associated with the expression of a Th1 response, while nonprotective immunity is correlated with a Th2 response (7, 8, 9). In particular, administration of anti-IL-4 Ab or an IL-4-soluble receptor antagonist at the time of infection results in a protective Th1 immune response (10, 11), while, conversely, anti-IFN-γ administration leads to a nonprotective Th2 response (7, 12).

Additionally, studies suggest that development of this protective immunity against C. albicans is linked to the production of endogenous IL-12 during infection (13, 14). IL-12 is a heterodimeric cytokine that exhibits a number of bioactivities that may modulate infectious disease progression, including enhancing NK and T cell cytotoxicity, modulating T cell proliferation, and potentiating Th1 differentiation (15, 16). Moreover, IL-12 influences the production of other immunoregulatory cytokines, particularly IFN-γ, that are thought to help generate protective immunity and to be necessary for microbial killing by macrophages and neutrophils (17, 18).

To gain insight into the functional role of IL-12 in the in vivo development of the cellular immune response, in the present study we examined the effect of exogenous IL-12 administration on the course and outcome of systemic C. albicans infection in mice. We observed that exogenous IL-12, rather than exerting beneficial activity against infection, paradoxically promoted disease progression. We provide evidence that this effect was mediated through the ability of IL-12 to enhance the expression and the production of IFN-γ. We suggest that IFN-γ plays a critical role in pathogenesis despite the evidence that Th1 responses are thought to contribute to protective immunity in this model.

Female C57BL/6 wild-type and IFN-γ knockout mice (The Jackson Laboratory, Bar Harbor, ME), ranging in age from 6 to 8 wk and in weight from 18 to 20 g, were purchased and maintained under American Association for the Accreditation of Laboratory Animal Care-approved conditions. C. albicans (strain 36082, American Type Tissue Collection, Rockville, MD) was obtained as a freeze-dried stock. The culture was rehydrated and grown in a bactopeptone broth (Difco, Detroit, MI) for 1 day at room temperature, then streaked onto a bactopeptone agar plate and grown for 4 or 5 days at ambient temperature. Plates were stored at 4°C. Before infection, a colony from a selected plate was grown in bactopeptone broth overnight, washed three times in physiologic saline (Abbott Laboratories, North Chicago, IL), and measured by spectrophotometry at 540 nm to determine the yeast concentration. For sublethal and lethal challenge studies, mice were infected via bolus tail vein injection of 0.1 ml with 5 × 105 or 1.3 × 106 CFU/mouse of C. albicans, respectively. Survival was monitored for 15 to 30 days.

Recombinant murine IL-12 (rmIL-12),2 produced at Genetics Institute (lots MRB7292 and 021693–1.1) was diluted in sterile physiologic saline and given i.p. in a 0.2-ml volume. In dose-response studies, mice were infected on day 0 and administered IL-12 at a dose range of 0.001 to 0.11 μg/mouse or saline i.p. on days 0 through 4 via a single bolus dose. In all other experiments, mice were chronically infected, then injected with physiologic saline or with 0.1 μg/mouse rmIL-12 i.p. immediately after this sublethal challenge and again the following day. For neutralization studies, endotoxin-free rat IgG1 mAb to IFN-γ, clone XMG1.2 (American Type Tissue Collection, catalogue no. HB10648) was administered via i.p. bolus injection at 0.5 mg/mouse in a volume of 0.2 ml for 2 days. Rat IgG1 (Sigma Chemical Co., St. Louis, MO; catalogue no. I-4131) was rendered endotoxin free by Triton X-114 phase separation (19) and used as a control article for anti-IFN-γ studies. All articles were diluted to appropriate concentrations using sterile physiologic saline.

Mice were euthanized on day 3 of chronic infection. Kidneys were collected, cut cross-sectionally, and fixed in 10% neutral buffered formalin. Tissue samples were embedded in paraffin, then cut into 5-μm sections and floated onto glass slides. Sections were stained with hematoxylin and eosin (H&E) for morphologic examination or with a periodic acid solution (PAS), using the Hotchkiss McManus method for localization of fungi (20).

Spleens, peripheral lymph nodes, and resident peritoneal cells were obtained from animals at different time points of infection using routine methods and procedures (21). Single cell suspensions were prepared by teasing the organs in tissue culture medium (RPMI 1640 containing 10% heat-inactivated FBS for 30 min at 57°C (HyClone Sterile Systems, Inc., Logan, UT), 16 mM HEPES buffer (Sigma), 200 mM glutamine (Sigma), and 10 μg/ml gentamicin (Sigma)); lymph node cell suspensions were filtered twice through 70-μm pore size nylon cell strainers (Becton Dickinson, Franklin Lakes, NJ) and spleen cells were subjected to distilled water lysis and subsequent passage over glass wool to eliminate RBCs and cellular debris. Peritoneal exudate cells were obtained by lavage with 5 ml of FBS-free culture medium containing 20 U/ml heparin (Lymphomed, Deerfield, IL). All cells were washed with culture medium, then plated in 96- and 24-well plates (Costar, Cambridge, MA) in the presence of medium alone, Con A (Sigma) at a final concentration of 2.5 μg/ml, or heat-killed Candida at 5 × 105 CFU/ml. Splenocytes and lymph node cells were plated at 4 × 105 cells/well in 96-well plates and at 2.2 × 106 cells/well in 24-well plates. Peritoneal cells were plated at 105 cells/well in 96-well plates or at 5.5 × 105 cells/well in 24-well plates. All cell cultures were incubated at 37°C in an atmosphere of 5% CO2. Supernatant fluids were harvested at 24 and 48 h and stored at −80°C.

Cytokine levels in sera or culture supernatant fluids were assessed by ELISAs. Commercially available kits were used to assay for IFN-γ and IL-10 (Endogen, Cambridge, MA). Reagents for IL-4 assays were obtained from PharMingen (San Diego, CA) and were used as follows. Dynatech Immulon-4 ELISA plates (Fisher Scientific, Fairlawn, NJ) were coated with 0.5 μg/ml cytokine-specific capture Ab overnight at 4°C, washed four times with PBS and 0.05% Tween-20, and blocked for 30 min with PBS and 2% BSA at 37°C. After a single washing step, the standards and supernatant fluids were added to the wells and incubated for 2 h at 37°C or overnight at 4°C. Wells were then washed four times and incubated with detector Ab at a concentration of 0.5 μg/ml. The ELISA plates were amplified and developed, after a final wash, using the Vectastain ABS kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions.

Nitrate and nitrite concentrations were used as a relative measure of nitric oxide (NO) synthesis (22) and were measured in the sera using a NO analyzer (Sievers Instruments, Inc., Boulder, CO) according to the manufacturer’s instructions.

Total RNA was purified from spleens previously snap-frozen in liquid nitrogen. The tissue was thawed in 5.7 M guanidine isothiocyanate (Life Technologies, Gaithersburg, MD), then homogenized with a Tissuemizer electric tissue homogenizer (Tekmar Co., Cincinnati, OH). RNA was isolated from the homogenate by cesium chloride centrifugation followed by phenol chloroform extraction and ethanol precipitation (23) and were stored at −80°C. Single-stranded cDNA was generated from the RNA with oligo(dT) priming and avian myeloblastosis virus reverse transcriptase (Promega Corp. Madison, WI) according to the manufacturer’s protocol. The resulting cDNA was diluted 10-fold for a working solution and used as a template for gene-specific QC-PCR against nonhomologous MIMIC fragments (Clontech Laboratories, Palo Alto, CA). Amplification reactions contained cytokine-specific 5′ and 3′ oligonucleotide primers obtained from Clontech or synthesized at Genetics Institute and are shown in Table I. To control for experimental artifacts, each calculation was normalized to mRNA levels of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Briefly, cDNA samples were added to a 4-fold dilution series of MIMICS with reaction buffer and PCR primers, then layered with mineral oil. Reactions were mixed and heated at 94°C for 3 min, then cooled to 40°C. AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ) was quickly added, and the products were amplified under the following conditions: initial denaturation at 94°C for 1 min, followed by 25 to 35 cycles of denaturation (94°C for 1 min; annealing temperatures are listed in Table I), and extension at 72°C for 7 min. Amplifications were performed in an Omnigene thermal cycler (Hybaid, Middlesex, U.K.). Resulting amplified products were analyzed by 1.2% agarose gel (FMC, Rockland, ME) electrophoresis followed by ethidium bromide staining.

Table I.

Reaction conditions for quantitative, competitive reverse-transcription PCR

GenePrimer SequencesProduct Length (nt)MIMIC Length (nt)[Mg2+] (mM)Annealing Temp., (°C)No. of CyclesCutoff Valuesa
G3PDH        
Upper 5′-TGA AGG TCG GTG TGA ACG GAT TTG GC-3′ 963 600 1.5 60 25 NA 
Lower 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′       
IFNg        
Upper 5′-CAT CTT GGC TTT GCA GCT CTT CCT CAT GGC-3′ 363 500 1.5 60 35 0.2 
Lower 5′-TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC-3′       
IL-4        
Upper 5′-GAG ATC ATC GGC ATT TTG AAC-3′ 306 544 1.5 55 35 2.8 
Lower 5′-GCT CTT TAG GCT TTC CAG GAA GTC-3′       
IL-10        
Upper 5′-CTA TGC TGC CTG CTC TTA CTG A-3′ 622 251 1.5 55 35 0.1 
Lower 5′-TTC AGC AGA CTC AAT ACA CAC T-3′       
iNOS        
Upper 5′-CCC TTC CGA AGT TTC TGG CAG CAG C-3′ 497 400 1.5 60 35 0.1 
Lower 5′-GGC TGT CAG AGC CTC GTG GCT TTG G-3′       
GenePrimer SequencesProduct Length (nt)MIMIC Length (nt)[Mg2+] (mM)Annealing Temp., (°C)No. of CyclesCutoff Valuesa
G3PDH        
Upper 5′-TGA AGG TCG GTG TGA ACG GAT TTG GC-3′ 963 600 1.5 60 25 NA 
Lower 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′       
IFNg        
Upper 5′-CAT CTT GGC TTT GCA GCT CTT CCT CAT GGC-3′ 363 500 1.5 60 35 0.2 
Lower 5′-TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC-3′       
IL-4        
Upper 5′-GAG ATC ATC GGC ATT TTG AAC-3′ 306 544 1.5 55 35 2.8 
Lower 5′-GCT CTT TAG GCT TTC CAG GAA GTC-3′       
IL-10        
Upper 5′-CTA TGC TGC CTG CTC TTA CTG A-3′ 622 251 1.5 55 35 0.1 
Lower 5′-TTC AGC AGA CTC AAT ACA CAC T-3′       
iNOS        
Upper 5′-CCC TTC CGA AGT TTC TGG CAG CAG C-3′ 497 400 1.5 60 35 0.1 
Lower 5′-GGC TGT CAG AGC CTC GTG GCT TTG G-3′       

a Cutoff values shown are less than or equal to the copy number of the respective gene per 1000 copies of G3PDH (spleen only). NA, not applicable.

Survival data were tabulated and analyzed using the Fisher’s protected least significant difference test on Super ANOVA software (Abacus Concepts, Inc., Berkeley, CA).

The effects of IL-12 administration on C57BL/6 mice challenged with C. albicans were determined using several infection and treatment regimens. Animals were monitored for mortality and median survival time (MST). Three different doses of rmIL-12 (0.001, 0.01, and 0.11 μg/mouse) were given to animals with either a chronic or an acute lethal Candida infection. The results of these studies show that infection was exacerbated by treatment with exogenous IL-12 (Fig. 1, A andB). A significant decrease in MST (p < 0.05) was observed in both chronically and lethally infected groups treated with all three doses of IL-12 compared with that in their respective saline-treated, infected controls. However, only in the chronic infections were the MST differences significant (p < 0.05) in a dose-dependent fashion.

FIGURE 1.

Survival of mice infected with C. albicans and treated with rmIL-12. Groups of C57BL/6 (n = 10) mice were infected i.v. on day 0 with either sublethal challenge (A; 5 × 105 CFU) of C. albicans or lethal challenge of C. albicans (B; 1.3 × 106 CFU), then treated with saline or IL-12 (0.001–0.11 μg/mouse) i.p. from day 0 to day 4. Survival was monitored for 30 days. Inserted tables show the statistical significance (using Fisher’s least significant difference test) between MST of IL-12-treated groups vs that of the saline control group. Results shown are representative of three individual studies.

FIGURE 1.

Survival of mice infected with C. albicans and treated with rmIL-12. Groups of C57BL/6 (n = 10) mice were infected i.v. on day 0 with either sublethal challenge (A; 5 × 105 CFU) of C. albicans or lethal challenge of C. albicans (B; 1.3 × 106 CFU), then treated with saline or IL-12 (0.001–0.11 μg/mouse) i.p. from day 0 to day 4. Survival was monitored for 30 days. Inserted tables show the statistical significance (using Fisher’s least significant difference test) between MST of IL-12-treated groups vs that of the saline control group. Results shown are representative of three individual studies.

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The enhanced susceptibility to infection imposed by IL-12 was also evident in H&E-stained histologic sections of the kidney, which exhibited tissue necrosis and a lack of cellular infiltration in response to IL-12 treatment of infection (Fig. 2,b), compared with infection alone (Fig. 2,a). Correspondingly, PAS-stained kidney sections showed a dramatic increase in fungal burden in IL-12-treated samples, indicated by extensive staining of fungal colonies (Fig. 2,d). PAS-stained kidney sections from an infected untreated animal showed degraded colonies, indicating a resolving lesion and self-limiting infection (Fig. 2, c and d). Furthermore, we observed a mean increase of about 1 log in recovery of yeast from the organ of clearance, the kidney, as determined by colony-forming units per milligram of tissue in mice treated with IL-12 (0.1 μg/mouse). The log colony-forming unit counts were 3.70 ± 1.08 for the saline-treated mice and 6.55 ± 0.33 for mice that received IL-12 (mean ± SD; n = 8; day 5).

FIGURE 2.

Representative micrographs (×40) of H&E- and PAS-stained paraffin sections of Candida-infected kidney. Kidneys were removed from mice infected i.v. with a sublethal challenge (5 × 105 CFU) and given daily i.p. injections of rmIL-12 (0.1 μg/mouse) for 2 days. Mice were euthanized on day 3 to harvest tissue. Photomicrographs a and b are H&E-stained 5-μm sections. a, Kidney from an infected animal that did not receive IL-12. b, Kidney section from an infected and IL-12-treated animal. Photomicrographs c and d are stained with PAS; the vivid pink color (PAS positive) indicates the presence of viable yeast colonies. c, Section of kidney from an infected animal that did not receive IL-12. d, Tissue from an infected plus IL-12-treated animal.

FIGURE 2.

Representative micrographs (×40) of H&E- and PAS-stained paraffin sections of Candida-infected kidney. Kidneys were removed from mice infected i.v. with a sublethal challenge (5 × 105 CFU) and given daily i.p. injections of rmIL-12 (0.1 μg/mouse) for 2 days. Mice were euthanized on day 3 to harvest tissue. Photomicrographs a and b are H&E-stained 5-μm sections. a, Kidney from an infected animal that did not receive IL-12. b, Kidney section from an infected and IL-12-treated animal. Photomicrographs c and d are stained with PAS; the vivid pink color (PAS positive) indicates the presence of viable yeast colonies. c, Section of kidney from an infected animal that did not receive IL-12. d, Tissue from an infected plus IL-12-treated animal.

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To correlate the outcome of a chronic Candida challenge with the nature of the immune responses generated in IL-12-treated mice, circulating levels of cytokines were measured by ELISA. In addition, IL-4 production was assayed from in vitro mitogen- and Ag-stimulated supernatant fluids derived from spleen, lymph node, and peritoneal cell populations. The experimental groups of mice were naive, IL-12 treated, Candida infected, and Candida infected plus IL-12. Our data confirm that IL-12 is a potent inducer of IFN-γ, as demonstrated by the experimental group of animals receiving IL-12 treatment alone, which had concentrations as high as 1800 pg/ml of IFN-γ in the serum on day 2 (Fig. 3). Infected control animals had lower, but detectable, amounts of serum IFN-γ on day 1 of infection, which then returned to baseline the following day. Infected animals treated with IL-12 had the highest level of circulating IFN-γ (>3500 pg/ml on day 1), and this was sustained for at least 2 days longer than that in either the IL-12 alone or infection alone groups.

FIGURE 3.

Determination of serum IFN-γ concentrations by ELISA. Serum was collected from C57BL/6 mice on the days indicated. Treatment groups of 10 mice each were saline treated, IL-12 treated (0.1 μg/mouse), sublethally infected (5 × 105 CFU) with Candida, or infected with Candida and treated with IL-12 (0.1 μg/mouse). The assay’s limit of detection was 1 ng/ml. * indicates that the level was below detectable limits. Each bar represents the mean ± SD of the values obtained from replicate cultures. The results shown are representative of three experiments.

FIGURE 3.

Determination of serum IFN-γ concentrations by ELISA. Serum was collected from C57BL/6 mice on the days indicated. Treatment groups of 10 mice each were saline treated, IL-12 treated (0.1 μg/mouse), sublethally infected (5 × 105 CFU) with Candida, or infected with Candida and treated with IL-12 (0.1 μg/mouse). The assay’s limit of detection was 1 ng/ml. * indicates that the level was below detectable limits. Each bar represents the mean ± SD of the values obtained from replicate cultures. The results shown are representative of three experiments.

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The cytokines IL-4 and IL-10 have been shown to be associated with increased susceptibility to Candida infections (10, 24, 25). Our IL-10 results are in agreement with the literature. Infected mice receiving IL-12, the most susceptible group, had approximately a twofold higher circulating level of IL-10 on days 2 and 3 (data not shown). However, in our model no IL-4 was detected serologically or in the culture supernatant fluids of Ag-stimulated spleen, lymph node, and peritoneal cell populations; all values were <30 pg/ml (data not shown).

To strengthen the data for cytokine products, we examined the pattern of cytokine gene expression. QC-PCR analysis was performed on cDNA generated from the total RNA of pooled spleens taken from the four experimental groups on days 1, 2, 3, and 7 (Fig. 4). By day 1, the day after infection and IL-12 treatment, RNA message levels for IFN-γ increased substantially in infected mice (more than twofold) and IL-12-treated mice (more than fivefold). The highest levels of IFN-γ transcripts were detected in the infected animals that had received IL-12 and were eightfold higher than naive levels. In addition, this group maintained this increased level of IFN-γ mRNA on day 2, whereas groups that were infected or IL-12-treated alone had returned to baseline (Fig. 4,A). Thus, consistent with our IFN-γ product data (Fig. 3), elevated mRNA levels for IFN-γ are transient in IL-12-treated and infection alone groups and are both higher and sustained when infected animals are treated with IL-12.

FIGURE 4.

Determination of mRNA levels by QC-PCR. Total RNA was extracted from freshly isolated C57BL/6 spleen cells on days 1, 2, 3, and 7 of Candida infection (CAN), infection plus IL-12 treatment (CAN+IL-12), or IL-12 treatment alone (IL-12). cDNA was generated from the purified RNA and used in the QC-PCR methods detailed in Materials and Methods. Transcript levels were determined and expressed as copies per 1000 copies of the housekeeping gene G3PDH. Results shown are representative of three independent experiments. A, IFN-γ; B, IL-10; C, iNOS.

FIGURE 4.

Determination of mRNA levels by QC-PCR. Total RNA was extracted from freshly isolated C57BL/6 spleen cells on days 1, 2, 3, and 7 of Candida infection (CAN), infection plus IL-12 treatment (CAN+IL-12), or IL-12 treatment alone (IL-12). cDNA was generated from the purified RNA and used in the QC-PCR methods detailed in Materials and Methods. Transcript levels were determined and expressed as copies per 1000 copies of the housekeeping gene G3PDH. Results shown are representative of three independent experiments. A, IFN-γ; B, IL-10; C, iNOS.

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Transcript analysis for IL-10 mRNA showed more than a threefold up-regulation of message in the infected mice treated with IL-12 compared with that in the infection alone group on day 2 (Fig. 4 B). These data were consistent with the enhanced level of IL-10 observed in the serum (data not shown). Also, message analysis for IL-4 confirmed the observed lack of IL-4 synthesis; specifically, no message >2.8 copies of IL-4/1000 copies of G3PDH was detected (data not shown).

NO, strongly influenced by IFN-γ levels, has been implicated in host defense of both intra- and extracellular pathogens (26, 27, 28). We investigated the possibility that IL-12-induced IFN-γ was affecting iNOS regulation and subsequent release of NO oxidative end products. Figure 4 C shows that iNOS message was enhanced in IL-12-treated, infected mice. Subsequently, using an automated NO analyzer to measure nitrite and nitrate in the sera, we were able to detect nitrate in saline- or IL-12-treated animals on day 2 at concentrations of 26.5 and 12.5 μM, respectively. Infected mice had a concentration of 85.5 μM, while the infected animals treated with IL-12 had a fivefold higher serum nitrate concentration of 407.5 μM.

To ascertain whether gene expression at the major site of infection was different from what was observed systemically, transcripts from the kidney were evaluated. Table II shows the number of transcript copies per 1000 copies of G3PDH for IFN-γ, iNOS, IL-4, and IL-10. As seen systemically, the highest levels of IFN-γ and iNOS were detected in infected plus IL-12-treated animals, whereas IL-4 was below the limit of detection. In contrast to spleen message analysis, IL-10 was below the level of detection in the kidney.

Table II.

Quantification of cytokine mRNA levels in kidneya

DayIFN-γIL-4iNOSIL-10
Naive 0.4 <1.2 <0.1 <0.4 
IL-12  2.9 <1.8 <0.1 <0.8 
Candida  28.7 <1.2 <0.4 
Candida+ IL-12  45.9 <1.8 <0.7 
IL-12 1.8 <4.6 <0.7 
Candida  0.7 <1.8 <0.7 
Candida+ IL-12  2.9 <1.8 16.8 <0.7 
IL-12 0.7 <1.2 <0.1 <0.4 
Candida  1.1 <1.8 <0.1 <0.7 
Candida+ IL-12  2.9 <1.8 6.7 <0.7 
DayIFN-γIL-4iNOSIL-10
Naive 0.4 <1.2 <0.1 <0.4 
IL-12  2.9 <1.8 <0.1 <0.8 
Candida  28.7 <1.2 <0.4 
Candida+ IL-12  45.9 <1.8 <0.7 
IL-12 1.8 <4.6 <0.7 
Candida  0.7 <1.8 <0.7 
Candida+ IL-12  2.9 <1.8 16.8 <0.7 
IL-12 0.7 <1.2 <0.1 <0.4 
Candida  1.1 <1.8 <0.1 <0.7 
Candida+ IL-12  2.9 <1.8 6.7 <0.7 
a

Total RNA was extracted from kidneys, snap frozen on days indicated and then analyzed using QC-PCR to measure cytokine message. The table shows number of transcript copies per 1000 copies of the housekeeping gene G3PDH.

Neutralization of IFN-γ has been shown to exacerbate disease in other models of candidiasis (7, 12); however, in the present study increased levels of IFN-γ were associated with progressive disease. We, therefore, examined the effect of IFN-γ neutralization on the IL-12-induced susceptibility to infection. Animals were chronically infected and coadministered IL-12 and anti-IFN-γ with appropriate controls, then monitored for survival for 15 days. As shown in Figure 5, the mice receiving neutralizing IFN-γ Ab had an MST equivalent to those with infection alone at 13.6 and 13.9 days, respectively. These results suggest that the high level of IFN-γ induced by IL-12 contributed to lethality, while blocking IFN-γ-offered protection. Although there appeared to be complete resistance to disease in the infected group receiving anti-IFN-γ alone, the difference in MST was not statistically significant (p > 0.05) compared with that in the groups with infection alone or infection plus a control Ab.

FIGURE 5.

Survival of infected mice treated with neutralizing IFN-γ Ab. Groups of 10 C57BL/6 mice were i.v. infected with a sublethal dose of Candida (5 × 105 CFU) and administered IL-12 (0.1 μg/mouse) or saline for 2 days. Anti-IFN-γ (XMG1.2) or rat IgG, as a control, was administered at 0.5 mg/mouse 1 day before infection and every other day postinfection for 2 wk. Survival was monitored for 15 days. Results shown are representative of two individual studies.

FIGURE 5.

Survival of infected mice treated with neutralizing IFN-γ Ab. Groups of 10 C57BL/6 mice were i.v. infected with a sublethal dose of Candida (5 × 105 CFU) and administered IL-12 (0.1 μg/mouse) or saline for 2 days. Anti-IFN-γ (XMG1.2) or rat IgG, as a control, was administered at 0.5 mg/mouse 1 day before infection and every other day postinfection for 2 wk. Survival was monitored for 15 days. Results shown are representative of two individual studies.

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To ensure the complete absence of endogenous IFN-γ during infection, animals deficient in IFN-γ were examined as well. As shown in Figure 6, IFN-γ knockout mice are actually more susceptible to infection than their counterpart wild types, as demonstrated by respective survival rates of 0 and 70% on day 30. This was not the result we initially anticipated based on our in vivo blocking studies. Instead, the IFN-γ knockout data suggest a protective role for endogenous IFN-γ. However, infected, IL-12-treated knockout mice had an MST equivalent to that in mice with infection alone (20.1 and 19.3 days, respectively). These results confirmed our IFN-γ neutralization data, indicating that IL-12 enhances susceptibility via IFN-γ production. Thus, collectively the IFN-γ knockout data imply that endogenous IFN-γ is needed for protection, while enhanced IFN-γ production via IL-12 administration can exacerbate Candida infections.

FIGURE 6.

Survival of IFN-γ knockout mice and their wild-type counterparts. Groups of 10 IFN-γ knockout mice and C57BL/6 wild-type counterparts were infected with 5 × 105 CFU Candida and administered 0.1 μg/mouse rmIL-12 i.p. for 2 days. Survival was monitored for 30 days. The data shown are representative of three separate experiments.

FIGURE 6.

Survival of IFN-γ knockout mice and their wild-type counterparts. Groups of 10 IFN-γ knockout mice and C57BL/6 wild-type counterparts were infected with 5 × 105 CFU Candida and administered 0.1 μg/mouse rmIL-12 i.p. for 2 days. Survival was monitored for 30 days. The data shown are representative of three separate experiments.

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The capacity to control disease lies, in part, in the ability of the host to generate an appropriate cell-mediated immune response. More specifically, Th cell interaction with invading micro-organisms influences cytokine networks. Murine candidiasis studies have suggested that resistance is linked to a Th1 response and the production of IFN-γ, while the failure to elicit protective immunity is associated with a Th2 response and the production of IL-4 and IL-10 (6, 14, 24). Accumulating evidence in this and other infectious disease models indicates that IL-12 is crucial for the development of protective Th1 responses and that this cytokine is particularly potent in its ability to induce the production of IFN-γ, a major mediator of antimicrobial activity (29, 30, 31, 32). The present study examined the effects of exogenous IL-12 administration on the progression of disease and the cellular immune response during a chronic systemic infection. Surprisingly, we see that the administration of IL-12 profoundly modified the course and the outcome of infection by adversely affecting the development of protective immunity and exacerbating disease.

Several important observations are relevant to the present results; most strikingly, resistant mice that normally survive at least 3 wk with chronic systemic disease succumbed within a few days when given IL-12 therapy (Fig. 1,A). Treatment with IL-12 also accelerated mortality in mice given an acute lethal challenge of C. albicans (Fig. 1,B). This notable decrease in host resistance was associated with the absence of an inflammatory response and increased fungal load in the target organs, the kidneys (Fig. 2), as well as with elevated and sustained production of IFN-γ in vivo (Fig. 3).

NO production was correlated with the level of IFN-γ. This result was expected, since IFN-γ is a known inducer of iNOS expression (33, 34). Specifically, the IL-12-treated, infected group had the highest IFN-γ concentration as well as the highest levels of iNOS mRNA and serum nitrate. Initially, it was surprising that this group was also the most susceptible, considering that NO has been implicated in microbial killing (34, 35). Recently, however, it has been suggested that NO is not directly involved in candidacidal activity (33). Instead, NO may still be associated with candidastatic mechanisms that allow other macrophage candidacidal pathways to function (33). This evidence suggests that nitrite/nitrate levels may not accurately predict susceptibility or resistance.

The most dramatic outcome in our studies was that neutralization of IFN-γ in systemically infected animals treated with IL-12 resulted in an enhanced resistance (Fig. 5). Nevertheless, our results obtained from the IFN-γ knockout animal studies are in agreement with those reported by Romani et al. (7) and suggest that IFN-γ does play a role in protection against Candida infections. We hypothesize that overproduction of IFN-γ, mediated through exogenous IL-12 administration, results in enhanced susceptibility to the fungus. This hypothesis is supported by Garner et al. (36), who have shown that administration of IFN-γ during murine candidiasis increased morbidity and mortality as well as yeast colonization in the kidneys. More recently, Qian and Cutler (37), using BALB/c mice that were genetically deficient in IFN-γ demonstrated that this cytokine was not essential for resistance against disseminated disease. While the reasons for the differences in their data and those of the current study are not completely understood, they may be in part due to the background strain of the mice used (BALB/c vs C57BL/6), the challenge dose given (2.5–5.0 × 105 vs 5.0–13.0 × 105 CFU), and the strain of Candida albicans inoculated (Ca-1 serotype A vs American Type Culture Collection 36082). Nonetheless, their studies do not eliminate IFN-γ having a contributing role in the host response or pathogenesis of this infection.

Previous studies (7, 12, 13, 14) have suggested that endogenous production of both IL-12 and IFN-γ may be associated with protective immunity in mice with candidiasis. We now see that IL-12, when administered exogenously, may exert a variety of opposing biologic effects that determine the final outcome of infection. We suggest that IFN-γ and IL-12 may influence the complex interactions that occur between the innate and acquired components of the immune response during this infection. While endogenous IL-12 may be beneficial in promoting protective Th-mediated immunity, exogenous administration of this cytokine and the resultant increase in IFN-γ production may have detrimental effects on the inflammatory response, namely macrophages and neutrophils, which are critical for recovery from candidemia (38, 39). However, to date, no direct effects of IL-12 have been observed on either neutrophil (M. Klempner, unpublished observations) or macrophage function in vitro. It is possible that IL-12, via IFN-γ, may be affecting the innate immune response by impairing the production of certain cytokines, such as IL-1 (40), which has been shown to protect mice against a lethal C. albicans infection (41, 42), and of chemokines such as IL-8 and JE/MCP-1 (43, 44, 45), which may be important in recruiting macrophages and neutrophils to local sites of infection. This may explain our observation of the lack of an inflammatory response in infected kidneys of IL-12-treated mice in contrast to that in mice resolving their infection (Fig. 2, a andb). Our observations suggesting a dichotomy in immune responses in the Candida model due to a cytokine are not unique to IL-12 and have been recently demonstrated with TGF-β. Endogenous TGF-β, like IL-12, appears to be necessary for the development of protective Th cell-mediated immunity, while exogenous administration appears to exacerbate disease (46). The seemingly paradoxical effect of IFN-γ in candidiasis appears to be unique, in that similar observations have not been demonstrated in other experimental models of fungal infection. Studies on infections caused by Cryptococcus neoformans (47), Coccidioides immitis (48), and Histoplasma capsulatum (49) have all suggested a protective role for both IL-12 and IFN-γ.

In conclusion, while native IL-12 may be required for the generation of a protective immune response against candidiasis in vivo, the present study shows that its exogenous administration can exacerbate disease by an apparent IFN-γ-dependent mechanism. Understanding how IL-12 is capable of inducing such opposing immunologic effects will be important for our understanding in the utilization of IL-12 and other cytokines in the therapy of infectious diseases in the future.

The authors thank Dr. Robert Schaub for his support and helpful discussions during the course of this work and for reviewing the manuscript. We also thank Jana Subramanyam, Tunu Misra, Jamie Erikson, and Sharon Hunter for their technical assistance, and Ron Dattoli for animal care and maintenance.

2

Abbreviations used in this paper: rmIL-12, recombinant murine interleukin-12; H&E, hematoxylin and eosin; PAS, periodic acid solution; NO, nitric oxide; QC-PCR, quantitative competitive polymerase chain reaction; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; MST, median survival time; iNOS, inducible nitric oxide synthase.

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