Altered Immune Responses and Susceptibility to Leishmania major and Staphylococcus aureus Infection in IL-18-Deficient Mice

Generation of IL-18^−/− mice

IL-18 genomic DNA was screened from a 129/sv mouse genomic library (Stratagene, Cambridge, U.K.), subcloned into pBluescript vector (Stratagene, La Jolla, CA), and characterized by restriction enzyme mapping and DNA sequencing. A targeting construct was made to replace exons 4, 5, and 6 with a bacterial β-galactosidase and neomycin resistance genes as reporter gene and selection marker, respectively. In the targeting construct, the introduced genes and selection marker were flanked by half of exons 3 and 5 for deletion of the start codon of the IL-18 gene. An HSV-tk cassette was introduced at the 5′ end of targeting construct. Embryonic stem (ES) cells (E14) were transfected with this linearized targeting vector and selected with G418 and gancyclovir. Double-resistant clones were screened for homologous recombination by Southern blot using a 3′ end external probe and confirmed by a 5′ end external probe and a neomycin-resistant gene probe. The targeted ES clones were microinjected into the blastocysts of C57BL/6 mice to create chimeras. The chimeric mice were then mated with female CD1 mice to produce heterozygotes, which were then intercrossed as described previously (22) to obtain homozygous, heterozygous, and wild-type littermates for experiments. Mice were screened by Southern blot analysis as described above and by RT-PCR.

RT-PCR

Peritoneal cells were collected from IL-18 homozygous, heterozygous, or wild-type mice and cultured at 37°C in RPMI medium in 25-cm² tissue culture flasks (Costar, Cambridge, MA) at 1 × 10⁶/ml in the presence of IFN-γ (100 U/ml; a gift from Dr. G. Adolf, Bender Wien, Austria) and LPS (10 ng/ml; Sigma, Poole, U.K.). Cells were collected 16 h after activation, and total RNA was isolated with RNAzol B (Biogenesis, Poole, U.K.). The mRNA was reverse transcripted with SuperScript II (Life Technologies/BRL, Paisley, U.K.). PCR was conducted under standard conditions. Primer pairs were: IL-18: sense, 5′-TTACAGGAGAGGGTAGACATTTTACTATCC-3′; antisense, 5′-CAGCATCAGGACAAAGAAAGCCGCCTCAAA-3′); and β-actin, sense, 5′-GGGCTATGCTCTCCCTCACGCCATCCTGCG-3′; anti-sense, 5′-TTGGCATAGAGGTCTTTACGGATGTCAACG-3′. The PCR products were visualized by electrophoresis of 15 μl of the reaction product.

L. major infection in vivo

Groups of homozygous and heterozygous mice (female, 6–8 wk old) were infected in the right hind footpad with 1 × 10⁶ stationary phase promastigotes of L. major (LV39). Lesion development was measured at regular intervals with a constant pressure, spring-loaded, dial caliper (Kroeplin, Munich, Germany) and was expressed as the footpad thickness increase in the infected right hind foot compared with that in the uninfected left hind foot. At the end of experiments, mice were sacrificed, and footpads were removed. The number of parasites present in the infected footpad was quantified using a limiting dilution method previously described (23). Draining lymph nodes and spleens were harvested at various times during the infection for determination of in vitro proliferation and cytokine production in response to killed L. major Ags (23). Cytokines in the culture supernatant or in the serum were determined by ELISA using paired Ab (PharMingen, San Diego, CA) as previously described (24). The lower limit of detection in each assay was as follows: TNF-α, 10 pg/ml; IFN-γ, 30 pg/ml; IL-4, 40 pg/ml; and IL-12 (p40 and p70), 20 pg/ml.

S. aureus infection

S. aureus LS-1 was originally isolated from a swollen joint of a New Zealand Black/White mouse with spontaneous arthritis (a gift from Dr. T. Bremell, University of Lund, Lund, Sweden) (25). This bacterial strain produces large amounts of TSST-1, coagulase, and catalase. The bacteria were kept frozen at −20°C in a PBS solution (0.13 M NaCl, 10 mM sodium phosphate (pH 7.4), 5% BSA, and 10% DMSO) until use. Before use, the bacterial solution was thawed and washed in PBS. Viable counting was used to check the number of bacteria as colony-forming units per milliliter by blood agar plate culture. Groups of male homozygous or wild-type mice were injected i.v. with 1 × 10⁷ live bacteria in PBS. Mice were observed daily for up to 7 days; the number of limbs involved was determined, and footpad/intermalleolar diameters were measured with a dial caliper (Kroeplin). An articular index was derived (three or fewer points per limb): 1, erythema alone; 2, swelling and erythema; and 3, erythema, swelling, and extension/loss of function. The severity of septicemia was judged clinically by characteristic changes (24) in coat appearance, posture, presence of spontaneous movement, and mucocutaneous abscess formation, from which a septic index (severity score; maximum score, 4/mouse) was derived for each animal by two observers in blinded fashion. Viable S. aureus tissue distribution was estimated by whole organ culture as described previously (21). Kidneys and paws were removed and homogenized before dilution in PBS. Blood (100 μl) was also diluted in PBS. Each dilution (50 μl) was plated onto prewarmed blood agar and cultured for 18 h at 37°C to determine the number of CFU present, expressed per milliliter of blood, or per whole organ. Serum was also collected and stored at −70°C until cytokine assay by ELISA. The spleens were removed for determination of proliferation and cytokine production in response to killed S. aureus, TSST-1, or Staphylococcal enterotoxin A (SEA).

Spleen cell proliferation and cytokine production

Splenic single cell suspensions, from L. major-infected or S. aureus-infected mice were cultured at 2 × 10⁶ cells/ml for up to 96 h in RPMI (Life Technologies/BRL, Gaithersburg, MD). The medium was supplemented with 10% FCS, 2 mM l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (all from Life Technologies/BRL), and 50 μM 2-mercaptoenthanol (Sigma). Cells were stimulated with killed L. major (23) for L. major-infected mice or with killed S. aureus, SEA, or TSST-1 (last two obtained from Sigma) for S. aureus-infected mice (24). Proliferation assays were performed in triplicate in 96-well culture plates (Nunc, Roskilde, Denmark), with addition of 1 μCi of [³H]thymidine (Amersham, Aylesbury, U.K.) in 25 μl during the final 6 h of culture before harvesting onto a glass-fiber filter (Packard, Wallac, Milton Keynes, U.K.) using a Micromate 196 Harvester (Packard). [³H]Thymidine incorporation was measured using a Matrix 96, direct beta counter (Packard). In some experiments duplicate 500-μl cultures were performed for up to 96 h in 48-well plates (Costar, Cambridge, MA), and supernatants were stored at −70°C until estimation of cytokine content by ELISA.

FACS phenotyping

Splenic single cell suspensions were washed in serum-free RPMI, and then 10⁵ cells were incubated with 5 μl of conjugated specific anti-CD3(PE), anti-CD4(FITC), anti-CD8(FITC), anti-ThB (FITC), and anti-Pan NK(FITC) Abs (all from Sigma) or normal IgG control (PharMingen, San Diego, CA) for 20 min at room temperature in the dark and then processed as previously described (24).

Statistical analysis

Statistical analysis was performed using Minitab software (Minitab, State College, PA). Analysis was performed using log-rank, χ², Mann-Whitney, Student’s t, or two-tailed log-rank test.

Generation of IL-18^−/− mice

The IL-18 gene was disrupted by homologous recombination in E14 ES cells with a replacement targeting construct (Fig. 1⇓A). Seven targeted clones were identified among 278 G418- and gancyclovir-resistant clones by Southern blot using 3′ external probe, and confirmed by 5′ external probe. Southern blot using the neomycin gene as probe demonstrated that only one copy was hybridized at the expected size in all seven clones (data not shown). Three targeted ES cell clones were microinjected into blastocysts; all successfully transmitted the disrupted IL-18 gene through the germline. A replacement gene allele was carried by homozygous mutant and heterozygous mice, which was confirmed by Southern blot using 3′ and 5′ end external probes (Fig. 1⇓, B and C). IL-18^−/− mice were bred from one of these clones. They were born at the expected ratio (+/+:+/−:−/− = 32:59:30). The homozygous IL-18-deficient mice grew normally and showed no obvious abnormalities up to 40 wk. IL-18 mRNA was not detectable by RT-PCR in activated macrophages from the mutant mice, but was strongly present in the cells from control mice (Fig. 1⇓D). Naive IL-18^−/− or wild-type mice have similar percentages of CD4⁺, CD8⁺ T cells, B cells, and NK cell by flow cytometric analysis (data not shown). Splenic T cells also produced similar levels of proliferative response and cytokine (IFN-γ and IL-4) when activated with Con A (mitogen) or anti-CD3 Ab (polyclonal T cell activation; data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Disruption of the IL-18 gene. A, The structure of the IL-18 locus, the targeting vector, and the predicted homologous recombination events are shown. A targeting vector was designed to replace about 5 kb of the IL-18 gene, which contains exons 4, 5, and 6 and part of exons 3 and 7. The start codon for IL-18 was also deleted by the replacement with bacterial β-galactosidase- and neomycin-resistant genes. Southern blot analysis of tail DNA of offspring from the heterozygous intercross with primer P1 (B) or primer P2 (C), as indicated in A, was performed. D, IL-18 mRNA was not detected in the peritoneal macrophages from homozygous mice by RT-PCR. The RT-PCR product was 397 bp. R, EcoRI restriction sites.

IL-18-deficient mice are highly susceptible to L. major infection

Groups of IL-18^−/− mice and heterozygous littermates were infected with L. major in the right hind footpad. The lesion was visible at 2 wk after infection and was indistinguishable in both strains of mice. However, disease development began to divert from 3 wk after infection. While lesions in the heterozygous mice were contained, those in the IL-18^−/− mice continued to progress (Fig. 2⇓A). By 40 days, lesions in the mutant mice began to ulcerate, and the experiments were terminated as required by the guidelines of animal experimentation of the Home Office, U.K. At this stage, the parasite loads in the footpads of the IL-18^−/− mice were 2.5 log₁₀ higher than those in the heterozygous mice. Experiments were repeated twice by comparing the phenotypes of the mutant and wild-type mice. Again, the IL-18^−/− mice were highly susceptible to L. major infection compared with the IL-18^+/+ mice (Fig. 3⇓A). These results show directly that IL-18 plays a critical role in host resistance against the intracellular protozoa parasite, L. major.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Mice lacking IL-18 failed to control L. major infection. A, Groups of mice were infected in the footpads with 1 × 10⁶ parasites, and lesion development was followed at regular intervals with a dial caliper and expressed as the difference in the thickness of the infected and the uninfected contralateral footpads. Homozygous mice developed progressive and uncontrolled lesions and have significantly higher parasite load at the end of the experiment (day 40) compared with heterozygous control mice. Data are the mean ± SEM (n = 5). ∗∗, p < 0.01 vs heterozygous controls (experiments were terminated on day 40 when the lesion began to ulcerate, as required by Home Office U.K. guidelines). B, Sera were collected from the sacrificed mice and assayed for IL-12 (p40 plus p70), IFN-γ, and IL-4 by ELISA. C, Spleen cells were cultured with soluble L. major Ags, and T cell proliferation (3 days culture), IFN-γ, and IL-4 in the supernatants (2-day culture) were determined. Data are the mean ± SEM (n = 5). ∗, p < 0.05; ∗∗, p < 0.01 (compared with heterozygous control). Similar results were obtained with draining lymph node cells.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Further analysis of Leishmania infection in IL-18^−/− mice. Groups of IL-18^−/− and IL-18^+/+ mice were infected as described in Fig. 2⇑. A, Mutant mice were significantly more susceptible to L. major infection than +/+ mice. B, Draining lymph node cells from the IL-18^−/− mice produced significantly less IFN-γ and TNF-α, but markedly more IL-4 and IL-10 (2-day culture), than those from infected IL-18^+/+ mice when cultured with killed L. major. Data are the mean ± SEM (n = 8). ∗, p < 0.05; ∗∗, p < 0.01. Data are representative of two experiments.

Immunological analysis of L. major-infected mice

Serum IL-12 and IFN-γ were measured on day 40 after infection. Serum from the mutant mice contained markedly higher IL-12 (p40 plus p70), but significantly less IFN-γ, than serum from the heterozygous littermates. IL-4 concentrations for both groups were similar (Fig. 2⇑B). Spleen cells from both groups of mice proliferated to a similar level against L. major Ags and had similar percentages of CD3⁺, CD4⁺, CD8⁺, CD19⁺, and NK cells (data not shown). However, cells from the IL-18^−/− mice produced significantly less IFN-γ than cells from the IL-18^+/− mice. In contrast, cells from the mutant mice produced significantly more IL-4 than those from the IL-18^+/− mice (Fig. 2⇑C). The lymph node cells from the infected mutant mice also produced markedly less IFN-γ and TNF-α, but more IL-4 and IL-10, compared with similarly infected IF-18^+/+ mice following stimulation with killed parasites in vitro (Fig. 3⇑B). Similar results were obtained with spleen cells from these mice (data not shown).

To determine the kinetics of induction of Th1 response, IL-18^−/− and wild-type mice were infected in the footpad with 1 × 10⁶ L. major promastigotes. Mice were sacrificed on days 4, 7, 10, and 14 postinfection, and serum was harvested. Spleen cells were cultured with killed parasites, and culture supernatants were analyzed for a panel of cytokines. The wild-type mice had significantly larger spleens than the infected mutant mice (138 ± 16 vs 83 ± 7 mg; n = 10; p < 0.05; cumulative data for mice 4–14 days after infection). Their spleen cells also produced stronger proliferative responses than those of the mutant mice (Fig. 4⇓A). Spleen cells from the IL-18^−/− mice produced significantly less IFN-γ and TNF-α, but more IL-4, IL-5, and IL-10, compared with those from the +/+ mice when cultured with killed parasite (Fig. 4⇓). IL-12 was not detectable in the culture supernatants. Serum cytokine levels were generally below detection levels, except for IL-12 which was comparable in the two groups of mice on day 7 after infection (+/+, 95.22 ± 48.37; −/−, 104.88 ± 48.87 pg/ml). Thus, the absence of IL-18 predisposes the animals to develop a Th2 phenotype following immunological activation, as illustrated in vivo by the prototypic Th1/Th2 polarization model, murine cutaneous leishmaniasis.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Production of cytokines by spleen cells from −/− and +/+ mice during the first 2 wk of L. major infection. Spleen cells were harvested on days 4, 7, 10, and 14 after infection and cultured with graded numbers of killed L. major in vitro. Supernatants were collected at 48 h, and cytokine concentrations were determined by ELISA. Cytokines were not detectable in cultures of cells from mice 4 days after infection. Data from day 10 were similar to those from day 14. IL-12 was not detected. Data are the mean ± SEM (n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

IL-18^−/− mice developed less septicemia but more severe arthritis than intact mice after S. aureus infection

Within 3 days after i.v. S. aureus infection, all the wild-type mice showed clinical evidence of septicemia, which progressed up to 7 days. In contrast, only 28% of the IL-18^−/− mice developed septicemia by day 5 after infection (Fig. 5⇓A). Interestingly, despite the lower levels of septicemia, the IL-18^−/− mice developed markedly more severe septic arthritis than the control wild-type mice. Clinical signs of arthritis were observed within 2 days of infection. After 7 days, whereas articular inflammation was evident in only 50% of wild-type mice, up to 75% of IL-18^−/− mice developed arthritis (Fig. 5⇓B). Moreover, the mean arthritic score was significantly higher in arthritic homozygous mice than in arthritic control mice from day 2 (Fig. 5⇓C).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

IL-18^−/− mice developed reduced septicemia (severity score; A), but a higher incidence of arthritis (B), from day 2 after iv S. aureus infection compared with similarly infected wild-type mice (n = 15). The arthritic IL-18^−/− mice also exhibited significantly more severe arthritic score than controls (C). Data pooled from two experiments are the mean ± SEM. ∗, p < 0.05; ∗∗, p < 0.01.

To determine whether these observations were reflected in altered bacterial distribution and survival in vivo, cultures of blood and tissues were performed. Viable staphylococci were detected in blood on day 3 after infection, but not in paws or kidneys of the wild-type mice. In contrast, bacteria were present in blood and, more strikingly, in paws and kidneys of the mutant mice on day 3 (Fig. 6⇓A). By day 7 the levels of bacteria in the paws and kidneys were comparable in the two strains of mice. The mutant mice, however, showed lower bacterial count in blood than the control mice (Fig. 6⇓B).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Viable bacterial counts in mice 3 days (A) and 7 days (B) after infection with S. aureus. On day 3 samples from three mice per group were pooled. On day 7 samples were analyzed from five mice per group. A large number of viable bacteria were present in kidneys and paws of the IL-18^−/− mice 3 days after infection; this was not detectable (ND) in samples from the corresponding wild-type mice.

Immunoregulatory effect of IL-18 deficiency

We next investigated whether the altered clinical phenotype in the IL-18^−/− mice was reflected in the immune response following staphylococcal infection. Seven days after infection, spleen cells from the IL-18^−/− mice displayed significantly lower proliferative responses to TSST-1 (an exotoxin secreted by the S. aureus LS-1) and SEA than cells from the control wild-type mice (Fig. 7⇓A). Culture supernatant of cells from the IL-18^−/− mice also produced markedly lower concentrations of IFN-γ, but larger amounts of IL-4, compared with cells from wild-type mice (Fig. 7⇓, B and C). Interestingly, spleen cells from the infected mutant mice also produced significantly less TNF-α compared with cells from the control mice when stimulated with the superantigens TSST-1 and SEA (Fig. 7⇓D).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Spleen cells from IL-18^−/− mice (A) proliferated more than those from wild-type controls in response to SEA or TSST-1 after 48 h culture in vitro. IFN-γ (B) and TNF-α (D) syntheses were enhanced, but IL-4 production (C) was decreased, in parallel cultures of spleen cells compared with those in cells from the control wild-type mice. Data are the mean ± SEM (n = 3) and are representative of two experiments. ∗, p < 0.05; ∗∗, p < 0.01.

We then compared the kinetics of cytokine production in the mutant and wild-type mice following S. aureus infection. Groups of mice were sacrificed on days 1, 2, 3, and 7 after infection, and their production of cytokines was analyzed by ELISA. Sera from the −/− mice contained significantly lower concentrations of IFN-γ and TNF-α than those from the +/+ mice. IL-12 concentrations were comparable in the two groups of mice (Fig. 8⇓). IL-4 and IL-10 were below detectable levels. Spleen cells were cultured with graded numbers of heat-killed bacteria. Cells from the mutant mice consistently produced less IFN-γ, TNF-α, and IL-6 than those from the +/+ mice (Fig. 9⇓). IL-4, IL-10, and IL-12 were not detected in the culture supernatants of both groups of mice.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

Cytokine concentrations in the serum of IL-18^−/− or wild-type mice infected with S. aureus. IL-4 and IL-10 were not detectable. Data are the mean ± SEM (n = 3). ∗, p < 0.05; ∗∗, p < 0.01.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 9.

Cytokine production by spleen cells from IL-18^−/− or IL-18^+/+ mice infected with S. aureus and cultured with heat-killed bacteria in vitro. IL-4, IL-10, and IL-12 were not detectable. Data are the mean ± SEM (n = 3). ∗, p < 0.05; ∗∗, p < 0.01.