A Protective and Agonistic Function of IL-12p40 in Mycobacterial Infection

Mice

IL-12p35^−/− (23) and IL-12p40^−/− mice (24) on a 129Sv/Ev background, were backcrossed five times to C57BL/6. Mutant and C57BL/6 wild-type mice were maintained under specific pathogen-free conditions. For generation of homozygous double-deficient mice, IL-12p35^−/− and IL-12p40^−/− mice were intercrossed and genotyped by PCR. For experiments, animals were kept in autoclaved cages with autoclaved bedding, food, water, and microfilter lids. All experiments performed were in accordance with the guidelines of the Animal Research Ethics Board of the University of Cape Town (Cape Town, South Africa).

Bacteria

Mycobacteria were grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook oleic acid, dextrose, and catalase enrichment medium (Life Technologies, Gaithersburg, MD), 0.002% glycerol, and 0.05% Tween 80. Midlog phase cultures were harvested, aliquoted, and frozen at −80°C. After thawing, viable cell counts were determined by plating serial dilutions of the cultures on Middlebrook 7H10 agar plates followed by incubation at 37°C. Mycobacterial strains M. bovis BCG (Pasteur) and M. tuberculosis (H37Rv) were used and experiments were performed in level II and level III biohazard facilities. Before infection with M. bovis BCG or M. tuberculosis, stock solutions of mycobacteria were diluted in PBS, and the preparation was sonicated to ensure proper dispersion of mycobacteria. For i.v. infection with M. bovis BCG, mice were infected with 2 × 10⁶ CFU mycobacteria via a lateral tail vein. Pulmonary infection with M. tuberculosis was performed in an inhalation exposure system (Glas-Col, Terre-Haute, IN). Mice were exposed for 40 min to an aerosol generated by nebulizing ∼5.5 ml of a suspension containing 10⁷ live bacteria. Inoculum size was checked 24 h after infection by determining the bacterial load in the lung of infected mice.

Colony enumeration assay

Bacterial loads in the lung, liver, and spleen of infected mice were evaluated at different time points after infection with M. bovis BCG or M. tuberculosis. Organs were weighed and defined aliquots were homogenized in 0.04% Tween 80 saline. Tenfold serial dilutions of organ homogenates were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% oleic acid, dextrose, and catalase and incubated at 37°C for 19–21 days. Colonies on plates were enumerated and results are expressed as log₁₀ CFU per organ.

Histopathological analysis

For analysis mice were killed by cervical dislocation. Organs were fixed in 4% buffered formalin and paraffin-embedded. Two- to 3-μm sections were stained with H&E and a modified Ziehl-Nielsen method. The latter involved staining in a prewarmed (60°C) carbol-fuchsin solution for 30 min followed by 30 min at room temperature; destaining was performed for 1 min in 0.5% HCl-ethanol before counterstaining with hematoxylin for 4 min.

Immunohistochemistry

Frozen tissue sections were prepared using a cryostat (Leica CM 1850) and 3-aminopropyltriethoxysilane-coated slides. Seven-micrometer sections were air-dried and fixed in 75% acetone and 25% ethanol for 5 min before storage at −80°C. Slides were washed in PBS and incubated with a primary Ab against CD4 (clone GK 1.5) for 90 min at room temperature. After incubation with appropriately diluted rabbit anti-rat mAb coupled with Texas Red (Jackson Immuno-Research Laboratories, West Grove, PA) for 30 min at room temperature, sections were mounted in Fluoromount (DAKO, Carpinteria, CA) and assessed by immunofluorescence microscopy (Nikon, Randburg, South Africa).

Delayed-type hypersensitivity

Infected mice were challenged in the right footpad with 10 μg of purified protein derivative (PPD; State Vaccine Institute, Pinelands, South Africa) in 50 μl of PBS and in the left footpad with 50 μl of PBS alone. Swelling in each footpad was measured after 24, 48, and 72 h using a Mitutoyo micrometer caliper (Brütsch, Zürich, Switzerland), and the difference was taken as the amount of Ag-specific delayed-type hypersensitivity (DTH). The PPD preparation did not induce swelling in noninfected animals.

Ag-specific IFN-γ production

For measuring Ag-specific production of IFN-γ, single cell suspension of peripheral lymph nodes were prepared from mice 8 wk after infection with M. bovis BCG. Cells were resuspended in IMDM (Life Technologies, Paisley, U.K.) supplemented with 10% FBS (Life Technologies), 0.05 mM 2-ME (Sigma, Cape Town, South Africa), and penicillin and streptomycin (100 U/ml and 100 μg/ml; Life Technologies). Lymph node cells (2 × 10⁶) were incubated with 2 × 10⁴ peritoneal macrophages that were pulsed for 4 h with 2 × 10⁵ M. bovis BCG and 1 μg of PPD in antibiotic-free IMDM. Resident peritoneal macrophages were obtained 1 day before the experiment by peritoneal lavage of uninfected C57BL/6 mice and incubated in 96-well flat-bottom microplates (Nunc, Naperville, IL) in complete IMDM.

To determine mycobacterial Ag-specific IFN-γ production, 100 μl of supernatants from cultures were taken and kept frozen at −80°C. IFN-γ levels were analyzed in 3-fold serial dilutions using a sandwich ELISA with coating and biotinylated anti-IFN-γ mAbs (BD PharMingen, San Diego, CA), as previously described (25). The detection limit was 200 pg/ml.

CD4⁺ and CD8⁺ T cell enrichment

Peripheral lymph nodes were obtained 8 wk after infection with M. bovis BCG and single cell suspensions were prepared in IMDM. Enrichment of CD4⁺ T cells was performed by positive selection with magnetic mouse CD4 Dynabeads and mouse CD4 DETACHaBEAD (Dynal, Lake Success, NY). CD8⁺ cells were enriched by incubation of the CD4⁺-depleted cell suspension with anti-B220-specific Dynabeads. Positively selected CD4⁺ T cells from lymph nodes contained >90% CD4⁺ cells, and negatively enriched CD8⁺ T cell suspensions contained >90% CD8⁺ cells, as determined by flow cytometry analysis (26).

Functional CD4⁺ T cell assays

In vitro responses to mycobacterial Ags were measured as described previously (27), with slight modifications: 4 × 10⁵ CD4⁺ enriched lymph node cells obtained from infected mice were cultured with 2 × 10⁴ peritoneal macrophages pulsed with 2 × 10⁵ M. bovis BCG and 1 μg of PPD for 4 h in antibiotic-free IMDM. Resident peritoneal macrophages were obtained 1 day before the experiment from peritoneal lavages of uninfected C57BL/6 mice and incubated in 96-well flat-bottom microplates (Nunc) in complete IMDM. CD4⁺ T cells and pulsed macrophages were incubated for 72 h at 37°C and 5% CO₂. For the proliferation assay, 1 μCi of [³H]thymidine was added for the last 10 h of culture. After harvesting, thymidine incorporation was measured in a liquid scintillation counter.

Cytotoxic CD8⁺ T cell assay

The cytotoxic activity of CD8⁺ T cell-enriched lymph node cells was performed as previously described in a 4-h ⁵¹Cr release assay (28). Effector cells were harvested and cultured in complete IMDM with 10 μg/ml PPD at 37°C and 5% CO₂. For a source of target cells, macrophages were obtained from peritoneal lavages of uninfected C57BL/6 mice, seeded at 1 × 10⁴ cells/well in 96-well round-bottom microplates (Nunc) in antibiotic-free IMDM. Cells were then pulsed with 1 × 10⁵ M. bovis BCG, 1 μg of PPD, and 2 μCi of ⁵¹Cr (Amersham Pharmacia Biotech, Piscataway, NJ). After washing of target cells, effector cells were added at different E:T ratios and plates were incubated for 4 h at 37°C. A 100-μl volume of supernatant was then collected from each well, and radioactivity release was measured in a Packard gamma counter (Cobra III; Packard Intrument, Downers Grove, IL). The percentage of specific lysis was calculated taking into account spontaneous and maximum release of target cells incubated with medium alone or 100% Triton X-100 as follows: [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100.

IL-12p(40)₂ administration and immunoblotting

Homodimeric IL-12p40 was produced by Chinese hamster ovary cells stably transfected with mouse IL-12p40 cDNA and purified as described previously (29). Purified IL-12p(40)₂ was >95% pure, as assessed by SDS-PAGE. Briefly, samples were boiled in SDS-PAGE buffer for 2 min and separated on a 5–15% polyacrylamide gradient gel (Life Technologies). Gels were stained in Coomassie brilliant blue (Sigma-Aldrich, St. Louis, MO) for 6 h and destained overnight before visualization of protein bands. For immunoblotting, procedures were conducted at room temperature and Abs were diluted in blocking buffer (PBS/5% skim milk powder). Proteins were transferred (semidry in-house blotting apparatus) onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked for 4 h and incubated in a 1/1500 dilution of rat anti-mouse IL-12p40 mAb (C15.6, 2 mg/ml; BD PharMingen) for 3 h. After three washes in PBS/0.05% Tween 20, membranes were incubated for 1 h in a 1/1000 dilution of rabbit anti-rat IgG (H and L chains) alkaline phosphatase-conjugated Ab (1 mg/ml; Southern Biotechnology Associates, Birmingham, AL). Washing was repeated and membranes were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate tablets (Roche Diagnostic Systems, Indianapolis, IN) for 15 min. SDS-PAGE and results were verified using IL-12 anti-sera. Endotoxin contamination was <10 IU/mg IL-12p(40)₂, as measured by the Limulus amebocyte lysate assay (M. Gately, unpublished observation). M. bovis BCG-infected mice received either 60 μg of rIL-12p(40)₂ i.p. or PBS as control daily for 28 days.

Quantification of IL-12p40, IL-23p19, and IL-18 by real-time PCR

Total RNA was extracted from homogenized mouse spleen, liver, and lung using TriReagent (Molecular Research Center, Cincinnati, OH), and DNA contamination was digested by molecular grade RNase-free DNaseI (Promega, Madison, WI) as per the manufacturer’s instructions. The purity and integrity of the RNA was controlled by absorbance at A_260/280 and gel electrophoresis. Absence of contaminating genomic DNA was verified by real-time PCR amplification of the RNA using the β-actin primers. Reverse transcription was performed in 30-μl total volume, including 10 ng of total RNA in 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM MgCl₂, 110 mM DTT, 0.5 mM each dNTPs, 0.7 U/μl RNasin (Promega), and 16 U/μl Moloney murine leukemia virus reverse transcriptase (Life Technologies), and incubated at 40°C for 2.5 h before inactivation of enzyme activity at 75°C for 15 min.

The levels of β-actin, IL-12p40, p19, and IL-18 mRNA in the cDNA were quantified by real-time PCR on a Lightcycler (Roche Diagnostic Systems). Hot start PCR was performed in 20 μl containing 50 mM Tris-HCL (pH 8.3), 4 mM MgCl2, 500 μg/ml BSA, 200 μM dNTPs, 0.125 U/μl Taq, 0.175 μM Taqstart Abs, 0.25 μM primers, and 2 μl of cDNA template and monitored in real time with the fluorescent DNA binding dye SYBR green (Roche Diagnostic Systems). The following primer pairs were used: β-actin (forward 5′-ACCCACACTGTGCCCATCTA-3′, reverse 5′-ATCGGAACCGCTCGTTGC; 321-bp product), IL-12p40 (forward 5′-CAGCTCGCAGCAAAGCAA-3′, reverse 5′-GACGCCATTCCACATGTCACT3′; 530-bp product), IL-18 (forward 5′-TGGTTCCATGCTTTCTGG-3′, reverse 5′-TCCGTATTACTGCGGTTGT-3′; 279-bp product), and p19 (forward 5′-TGCTGGATTGCAGAGCAGTAA-3′, reverse 5′-GCATG CAGAGATTCCGAGAGA-3′; 121-bp product). Standard curves for β-actin, IL-12p40, IL-18, and p19 were generated by performing 2-fold dilutions of positive samples. The mRNA level for each sample was normalized by dividing the calculated value by the housekeeping β-actin value. The various mRNA levels in the mutant mice were then relatively quantified in comparison to the respective levels in wild-type mice using the relative standard curve method (Applied Biosystems, Foster City, CA).

Statistical analysis

Data are expressed as the means of individual determinations and SDs. Statistical analysis was performed using the Student’s t test or the Mann-Whitney rank sum test.

IL-12p40 mediates protective immune responses to infection with M. bovis BCG

To determine the role of IL-12p40 during infection with M. bovis BCG, we compared IL-12p35^−/−p40^−/−, IL-12p35^−/−, and C57BL/6 wild-type mice. IL-12p35^−/− mice produce normal levels of endogenous IL-12p40 (23). Wild-type as well as IL-12p35^−/− mice were able to clear bacterial burden in infected organs, shown by decreased bacterial loads in spleen, liver, and lung during the monitored 17 wk of infection (Fig. 1⇓a). In contrast, IL-12p35^−/−p40^−/− mice showed 3- to 4-log higher CFUs relative to IL-12p35^−/− and wild-type mice, resulting in an inability to control infection (10⁷–10⁸ CFU/organ). Of importance, all IL-12p35^−/−p40^−/− mice survived infection but were chronically infected, with multiple bacterial and inflammatory infiltrates resulting in severe pathology in spleen (splenomegaly), liver, and lung (data not shown), 7 mo postinfection. Four weeks postinfection, T cell-driven Ag-specific immune responses were present in IL-12p35^−/− mice, which also exhibited a similar DTH reaction to wild types, using the Ag-specific footpad rechallenge model by injection of PPD of M. tuberculosis, measured 24, 48, and 72 h postinjection. In striking contrast, IL-12p35^−/−p40^−/− mice were unable to mount a DTH response 4 (Fig. 1⇓b) or 6 wk (data not shown) postinfection. These results suggest a role of IL-12p40 in mediating specific immune responses during infection with M. bovis BCG, which is independent of bioactive IL-12p70.

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FIGURE 1.

IL-12p35^−/− mice clear i.v. infection with M. bovis BCG with an Ag-specific immune response. Mice were infected i.v. with 2 × 10⁶ CFU of M. bovis BCG. a, For mycobacterial colony enumeration, spleen, liver, and lungs were taken from five C57BL/6 (•), IL-12p35^−/− (⋄), and IL-12p35^−/−p40^−/− mice (○) at 5, 9, and 17 wk postinfection. Differences between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were statistically significant (∗, p < 0.01; ∗∗, p < 0.001). b, For measuring DTH, C57BL/6 (filled bar), IL-12p35^−/− (shaded bar), and IL-12p35^−/−p40^−/− mice (open bar) were challenged in the left hind footpad with PBS and the right footpad with 10 μg of PPD of M. tuberculosis at wk 4 after infection. Differences in footpad swelling between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were highly significant (p < 0.0001). These results are representative of four experiments.

A role for IL-12p40 in protective granuloma formation and function

Protective immunity during mycobacterial infection is dependent on granuloma formation leading to control and containment of the infection, which is closely associated with T cell responses. In agreement with the bacterial burden, histological examination of acid fast bacilli in hepatic granulomas 9 wk postinfection revealed only few bacilli in wild-type (Fig. 2⇓, a and d) or IL-12p35^−/− mice (Fig. 2⇓, b and d) but abundant bacilli in IL-12p35^−/−p40^−/− mice (Fig. 2⇓, c and d). Increased numbers of multibacillary granulomas were also observed in IL-12p35^−/−p40^−/− mice at 5 wk postinfection when CFUs between mutant strains were still comparable (Table I⇓). This indicates an impairment of granuloma function in double-deficient mice. Subsequent cellular analysis by immunohistochemistry showed sparsely infiltrated CD8⁺ T cells within the granulomas of all strains of mice (data not shown). In IL-12p35^−/− mice (Fig. 2⇓f) similar numbers of infiltrating CD4⁺ T cells were found compared with C57BL/6 controls (Fig. 2⇓, e and h). In contrast, IL-12p35^−/−p40^−/− mice showed significant reduced CD4⁺ T cell infiltrates (Fig. 2⇓, g and h) with diffuse formation within the granuloma comprising large areas with foamy macrophages (Fig. 2⇓g) due to the presence of many bacilli (Fig. 2⇓c). Together, these data show that granuloma formation and function is present in IL-12p35^−/− mice but impaired in IL-12p35^−/−p40^−/− mice.

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FIGURE 2.

Efficient granuloma formation and function in IL-12p35^−/− mice. Nine weeks follow an i.v. infection with 2 × 10⁶ CFU of M. bovis BCG, livers were taken from C57BL/6, IL-12p35^−/−, and IL-12p35^−/−p40^−/− mice (five mice per group) for histological analysis. a–c, Sections were stained with Ziehl-Nielsen and counterstained with hematoxylin. Bacteria (red/pink staining) were essentially absent in hepatic granulomas from C57BL/6 (a) and IL-12p35^−/− (b) mice but abundant in IL-12p35^−/−p40^−/− (c) mice (bar = 50 μm). d, Bacteria containing granuloma. Results are shown as means and SDs of five mice per group. e–g, Immunofluorescence of CD4⁺ T cells within hepatic granulomas. CD4⁺ T cells were prominent in tissue from C57BL/6 (e) and IL-12p35^−/− (f) mice (five mice per group) but dispersed in tissue from IL-12p35^−/− p40^−/− mice (g) (bar = 50 μm). h, CD4⁺ cells were counted from 25 granulomas of five mice per group. Differences between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were highly significant (p < 0.0001).

In vivo supplementation with IL-12p40 leads to a more resistant phenotype in IL-12p35^−/−p40^−/− mice

To determine whether the observed differences between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were directly mediated by IL-12p40, M. bovis BCG-infected IL-12p35^−/−p40^−/− mice were supplemented in vivo by administering rIL-12p(40)₂, produced by a transfected Chinese hamster ovary cell line (kindly provided by M. Gately, Hoffmann-LaRoche, Nutley, NJ). Immunoblotting of purified IL-12p(40)₂ with anti-IL-12p40 mAbs revealed a purity of 99% IL-12p40 homodimer and ∼1% IL-12p40 monomer (Fig. 3⇓a). After 9 wk postinfection, increased resistance in p40-supplemented IL-12p35^−/−p40^−/− mice was observed, resulting in a 10- to 15-fold reduction of bacterial burden in infected organs compared with nonsupplemented but infected IL-12p35^−/−p40^−/− mice. Similar CFUs in liver and lung were found in IL-12p35^−/− mice as compared with wild-type mice (Fig. 3⇓b). Importantly, supplementation with IL-12p40 was able to fully restore the Ag-specific recall response in infected IL-12p35^−/−p40^−/− mice, demonstrated by a comparable DTH response to infected IL-12p35^−/− and C57BL/6 mice (Fig. 3⇓c). In summary, these results demonstrate that exogenously administrated p(40)₂ is able to restore some of the defective functions observed in IL-12p35^−/−p40^−/− mice, which suggests an agonistic role for IL-12p40 in the absence of bioactive IL-12p70.

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FIGURE 3.

Supplementation with IL-12p(40)₂ reduces bacterial burden and restores Ag-specific DTH of BCG-infected IL-12p35^−/−p40^−/− mice. a, SDS-PAGE and Western blot of purified IL-12p(40)₂; first lane, 20 μg of Coomassie brilliant blue-stained IL-12p(40)₂; other lanes, identification of homodimeric p(40)₂ or monomeric p40 in a Western blot using rat anti-IL-12p40 mAb and rabbit anti-rat IgG (H and L chains) alkaline phosphatase-conjugated Ab under nonreduced and reduced conditions as a standard (20–0.02 μg loaded), showing ∼1% monomeric p40. b and c, C57BL/6, IL-12p35^−/−, and IL-12p35^−/−p40^−/− mice that were infected i.v. with 2 × 10⁶ CFU of M. bovis BCG received i.p. 200 μl of PBS daily as control. Three IL-12p35^−/−p40^−/− mice received a daily i.p. injection of 60 μg/day of rIL-12p(40)₂ for 28 days, the remaining PBS only. b, Mycobacterial CFUs from spleen, liver, and lungs at 9 wk postinfection. Data are shown from individual mice and the means are indicated. ∗, p < 0.05 compared with PBS-treated IL-12p35^−/−p40^−/− mice (Mann-Whitney rank sum test). c, Four weeks postinfection DTH was measured using 10 μg of PPD of M. tuberculosis. Differences in footpad swelling between IL-12p35^−/−p40^−/− plus PBS and IL-12p35^−/− p40^−/− plus p(40)₂ mice, shown at 48 h, were significant (p < 0.01). These results are representative of two experiments.

An agonistic role of IL-12p40 in Ag-specific T cell responses

IL-12p70 promotes Th1 differentiation leading to IFN-γ production and a type 1 cellular immune response. Therefore, we compared Ag-specific T cell functions in response to BCG infection. Lymph nodes from infected mice were isolated 9 wk postinfection and cells were restimulated with Ag-presenting peritoneal macrophages in vitro to measure IFN-γ responses by T cells. The ratio and percentage of CD4⁺ and CD8⁺ T cells were similar in all three strains as determined by FACS analysis (data not shown). Lymphocytes from wild-type as well as from IL-12p35^−/− mice showed a marked and comparable IFN-γ response (Fig. 4⇓a). In contrast, this response was abolished in lymphocytes from IL-12p35^−/−p40^−/− mice. Next, the Ag-specific proliferative response of CD4⁺ T cells was determined by coculturing enriched CD4⁺ T cells from infected mice with M. bovis BCG-infected peritoneal macrophages (Fig. 4⇓b). In comparison to the Ag-specific proliferative response of wild-type mice, proliferation of CD4⁺ T cells was reduced in IL-12p35^−/− mice. However, proliferation was completely abrogated in IL-12p35^−/−p40^−/− mice. These results demonstrate a defective Ag-specific proliferative response in the absence of endogenous IL-12p40. Finally, CD8⁺ T cells were analyzed for their Ag-specific cytotoxic activity. Syngenic peritoneal macrophages pulsed with M. bovis BCG and PPD were used as target cells for CD8⁺ T cells isolated from infected mice. Cytotoxic activity was present in IL-12p35^−/− mice, albeit clearly lower than that observed in CD8⁺ cells from wild-type mice. The cytotoxic activity of CD8⁺ T cells from IL-12p35^−/−p40^−/− mice was further reduced to 32% of the maximum killing observed in wild-type mice. Collectively, these data show striking evidence for an agonistic and protective role of IL-12p40 by promoting Ag-specific Th1 and CTL effector functions.

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FIGURE 4.

M. bovis BCG-infected IL-12p35^−/− but not IL-12p35^−/−p40^−/− mice are able to mount Ag-specific T cell responses in vitro. Lymph nodes from C57BL/6, IL-12p35^−/−, and IL-12p35^−/−p40^−/− mice were obtained 9 wk after i.v. infection with 2 × 10⁶ M. bovis BCG. a, Lymph node cells were stimulated in vitro with M. bovis BCG-pulsed peritoneal macrophages and IFN-γ levels weredetermined in the supernatant 72 h later by ELISA. b, CD4⁺ T cell proliferation of triplicate cultures of pooled cells from five mice per group was measured after stimulation in vitro with M. bovis BCG-infected peritoneal macrophages. Differences between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were significant (p < 0.001 and p < 0.01). c, Cytotoxicity of enriched CD8⁺ T cells from C57BL/6 (•), IL-12p35^−/− (⋄), and IL-12p35^−/−p40^−/− (○) mice was assessed on M. bovis BCG/PPD-pulsed peritoneal macrophages from quadruplicate cultures as described in Materials and Methods. Results in a–c are expressed as the means with SD of three independent experiments.

Expression level of IL-23p19 in M. bovis-infected IL-12p35^−/−p40^−/− mice

Because the agonistic IL-12p40-dependent activities described above leave open whether homodimeric IL-12p40 or the recently discovered p40p19 heterodimer (designated as IL-23) are responsible for protection we quantified expression levels by real-time RT-PCR in spleen from M. bovis BCG-infected mice 2 mo postinfection. In comparison to wild-type controls, IL-12p35^−/− and IL-12p35^−/−p40^−/− mice showed a 3- and 8-fold increase in p19 mRNA levels (Fig. 5⇓ and Table II⇓). Neither IL-12p40 nor IL-18 was significantly increased in mutant mice compared to infected controls at this time point of infection (Table II⇓).

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FIGURE 5.

Real-time PCR analysis of IL-23p19 mRNA expression in splenocytes of M. bovis-infected mice. Mice were infected i.v. with 2 × 10⁶ CFU of M. bovis BCG (see Fig. 1⇑) and cDNA prepared from splenocytes of five mice per group. RNA from splenocytes from five mice per group was combined and PCR was monitored in real time with the fluorescent DNA binding dye SYBR green. The housekeeping mRNA β-actin was normalized before p19-specific PCR, and fold differences were calculated using the relative standard curve method (see Materials and Methods). Low abundance of p19-specific mRNA shown by relative high cycle numbers with 3-fold increase in splenocytes from IL-12p35^−/− mice (circled line) and 8-fold increase in splenocytes from IL-12p35p40^−/− mice (crossed line) compared with C57BL/6 (solid line). For comparison, see Table II⇓.

IL-12p40 is protective in pulmonary infection with M. tuberculosis

Airborne infection with M. tuberculosis is the most prevalent infectious disease for humans worldwide. Consequently, we addressed whether the protective and agonistic role of IL-12p40, which we observed after infection with M. bovis BCG, has relevance for infection with virulent M. tuberculosis. To mimic the natural route of infection in humans, mice were infected with 100 CFU of the virulent M. tuberculosis strain H37Rv in an inhalation exposure system to induce pulmonary tuberculosis. In the absence of bioactive IL-12p70 all mutant mice died, whereas wild-type mice survived infection, confirming an essential protective role for IL-12p70 in experimental tuberculosis (Fig. 6⇓a). IL-12p35^−/− mice survived up to 20 wk postinfection, whereas the life span of infected IL-12p35^−/−p40^−/− mice did not exceed the first 10 wk postinfection. IL-12p35^−/− mice were also able to mount a DTH response similar to wild-type controls (Fig. 6⇓b) and similar IL-12p40 serum levels as wild-type controls (5 and 5.5 ng/ml, respectively) 16 wk postinfection. Infected IL-12p35^−/−p40^−/− mice were completely defective in their DTH response, suggesting impaired cell-mediated responses as demonstrated in the BCG model. Indeed, lymphocyte infiltration to the site of infection during early pulmonary tuberculosis was comparable to that observed in wild-type (Fig. 6⇓c) and IL-12p35^−/− mice (Fig. 6⇓d), but appeared to be reduced in granulomas of IL-12p35^−/−p40^−/− mice (Fig. 6⇓e). Together, these results demonstrate residual resistance to M. tuberculosis in IL-12p35^−/− mice which is dependent on the presence of endogenous IL-12p40.

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FIGURE 6.

A protective role for IL-12p40 in M. tuberculosis infection. Ten mice per group were infected by the pulmonary route with 100 CFU of M. tuberculosis. a, Mortality of C57BL/6 (•), IL-12p35^−/− (⋄), and IL-12p35^−/−p40^−/− (○) mice. Differences in survival time between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were highly significant (p < 0.0001). Similar results were found in an independent mortality study using 13 mice per group. b, DTH response of C57BL/6 (filled bar), IL-12p35^−/− (shaded bar), and IL-12p35^−/−p40^−/− (open bar) mice 28 days postinfection (five mice per group). Differences between IL-12p35^−/− and IL-12p35^−/−p40^−/− mice were highly significant (p < 0.0001). c–e, Histological analysis of lungs 35 days postinfection. At sites of mononuclear infiltration lymphocytes were more prominent in C57BL/6 (c) or IL-12p35^−/− (d) compared with IL-12p35^−/−p40^−/− mice (e) (bar = 50 μm).