IL-12 is a potent inducer of IFN-γ production and promotes a protective cell-mediated immune response after Mycobacterium tuberculosis infection. Recently, the IL-12-related cytokine IL-27 was discovered, and WSX-1 was identified as one component of the IL-27R complex. To determine the functional significance of IL-27/WSX-1 during tuberculosis, we analyzed the course of infection and the immune response in WSX-1-KO mice after aerosol infection with M. tuberculosis. In the absence of WSX-1, an increased production of the proinflammatory cytokines TNF and IL-12p40 resulted in elevated CD4+ T cell activation and IFN-γ production, which enhanced macrophage effector functions and reduced bacterial loads. This is the first occasion of a selectively gene-deficient mouse strain showing higher levels of protective immunity against M. tuberculosis infection than wild-type mice. However, a concomitantly increased chronic inflammatory response also accelerated death of infected WSX-1-KO mice. In vitro, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation. In conclusion, IL-27 both prevents optimal antimycobacterial protection and limits the pathological sequelae of chronic inflammation.
Tuberculosis is one of the most prevalent bacterial infections worldwide and constitutes a leading global health threat (1, 2). Human tuberculosis caused by Mycobacterium tuberculosis is responsible for eight million new cases and nearly two million deaths annually. Incomplete definition of the balance between protective and inflammatory immune responses to M. tuberculosis has hampered the development of more effective vaccines and therapies.
The cell-mediated immune response is known to be critical in the host defense against infection with intracellular pathogens such as mycobacteria. T lymphocytes, particularly CD4+ T cells, play an important role in granuloma formation by secreting type 1 cytokines, primarily IFN-γ and TNF (1, 3, 4, 5). These cytokines stimulate the antimicrobial activity of infected macrophages, allowing intracellular bacterial killing through reactive oxygen and nitrogen intermediates (6, 7). IL-12 indirectly promotes an effective cell-mediated immune response by inducing IFN-γ production from T and NK cells.
IL-12 is a heterodimeric cytokine composed of a p35 and a p40 subunit and is produced primarily by APCs. It promotes the differentiation of Th1 cells (8). A large number of studies in genetically deficient animals has shown that IL-12 is required for resistance to intracellular bacteria and parasites (9, 10, 11, 12, 13, 14). However, it is not absolutely essential for Th1 differentiation and the generation of protective immunity. Patients with a complete deficiency in IL-12p40 or the IL-12R β1 chain have only a limited susceptibility to mycobacteria and Salmonella infections and are not completely immunocompromised (15). Moreover, in the absence of IL-12, mice were able to develop a residual suboptimal immune response to intracellular pathogens (13, 16, 17). Importantly, phenotypic differences between IL-12p35- and IL-12p40-deficient (knockout (KO) 3) mice after infection with intracellular pathogens (10, 18, 19, 20) indicated that additional factors must be involved in promoting protective Th1 immune responses.
Recently, IL-12-related cytokines were discovered, which share some functions and receptor components of IL-12 that may contribute to the fine-tuning of T cell responses after infection with intracellular pathogens such as M. tuberculosis. IL-12p40 associates not only with the IL-12p35 subunit but also with a p19 chain to form a novel heterodimeric cytokine, IL-23 (21). Similar to IL-12, IL-23 is involved in promoting IFN-γ production and Th1 differentiation. Unlike IL-12, IL-23 selectively induces proliferation of memory T cells (21). More recently, a novel IL-12p35-related long chain four-helix bundle cytokine p28 was described, which forms, together with the EBV-induced protein 3 (EBI3), a receptor-like soluble chain homologous to IL-12p40 (22), another noncovalently linked heterodimeric cytokine, termed IL-27 (23). Recent studies identified gp130 and WSX-1 as components of the IL-27R complex (23, 24). WSX-1, a thus-far orphan class I cytokine receptor that is homologous to the IL-12Rβ2 chain of the IL-12R, is highly expressed on resting/naive CD4+ and CD8+ T cells (25, 26, 27) but also on myeloid cells (24).
Some functional parallels between IL-12/IL-12R and IL-27/WSX-1 have emerged. For example, WSX-1-deficient T cells are impaired in IFN-γ production (26, 27). Moreover, rIL-27, like IL-12, is capable of enhancing production of T-bet, IFN-γ, and IL-12Rβ2 expression in naive T cells through STAT1-dependent mechanisms (28, 29). In addition, IL-27 was shown to be involved in immune protection from infection with intracellular pathogens (26, 27) and from tumor growth (30). On the basis of these early data on the biology of IL-12-related cytokines, a tentative sequential model has emerged: IL-27, by inducing T-bet expression, IL-12Rβ2 chain expression, and proliferation of naive T cells, is an early inducer of Th1 commitment and differentiation; IL-12 serves to expand and stabilize the Th1 response; and IL-23 helps maintain Th1-commited memory cells (31, 32, 33). However, very recent findings seriously question such a simplified and possibly premature model. In fact, after infection with the intracellular parasites Toxoplasma gondii and Trypanosoma cruzi, WSX-1-KO mice showed a much higher lethality due to T cell hyperreactivity and excessive production of proinflammatory cytokines (34, 35), suggesting that IL-27 has an additional regulatory role during infection-induced immune responses. However, no regulatory mechanism has been ascribed to IL-27 so far.
To determine the functional significance of WSX-1 during tuberculosis, we analyzed the course and outcome of infection in WSX-1-KO mice after aerosol inoculation with M. tuberculosis. Together, our data suggest that IL-27/WSX-1 is a key regulator of immune-mediated protection and immunopathology in mycobacterial disease.
Materials and Methods
Breeding pairs of WSX-1-deficient (WSX-1-KO) mice (26) on a C57BL/6 genetic background (ninth generation backcrossed to C57BL/6) were provided by Amgen, Inc., and offspring were generated under specific pathogen-free conditions at the Research Center Borstel. As wild-type controls, C57BL/6 mice were purchased from Charles River. All mice used were between 8 and 16 wk old. In any given experiment, mice were matched for age and sex. For infection experiments, mice were maintained under barrier conditions in the BSL 3 facility at the Research Center Borstel in individually ventilated cages. All experiments performed were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment (Kiel, Germany).
Bacteria and infection
M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 broth (Difco) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase enrichment medium (Invitrogen Life Technologies), 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. All experiments were performed in the BSL 3 laboratories at the Research Center Borstel.
Before infection of experimental animals, stock solutions of M. tuberculosis were diluted in sterile distilled water, and pulmonary infection was performed using an inhalation exposure system (Glas-Col). To infect mice with a low dose of 100 CFU/lung, animals 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 undiluted homogenates of the entire lung of infected mice. Mice were regularly weighed before and after infection. In accordance with the Animal Research Ethics Board of the Ministry of Environment, mice that lost 25% of their original weight during the course of infection had to be sacrificed.
Colony enumeration assay and histology
Bacterial loads in lung, liver, and spleen were evaluated at different time points after infection with M. tuberculosis to follow the course of infection. Organs from sacrificed animals were removed aseptically, weighed, and homogenized in PBS containing a proteinase inhibitor mixture (Roche Diagnostics) for subsequent quantification of cytokines prepared according to the manufacturer’s instructions. Ten-fold serial dilutions of organ homogenates were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% oleic acid-albumin-dextrose-catalase and incubated at 37°C for 19–21 days. Colonies on plates were enumerated, and results were expressed as log10 CFU per organ. One lung lobe, a piece of liver, and spleen per mouse were fixed in 4% formalin-PBS, set in paraffin blocks, and sectioned (2–3 μm). Histology was performed using standard protocols for trichrome and H&E staining. Acid-fast bacilli were detected using a modified Ziehl-Neelson protocol (10).
Preparation of serum and single-cell suspensions
To determine TNF, IL-12p40, and IFN-γ production ex vivo, blood was collected from M. tuberculosis-infected mice at different time points, and serum was prepared using serum separator tubes (BD Biosciences).
For Ag-specific restimulation and flow-cytometric analysis, single-cell suspensions of mediastinal lymph nodes and lungs were prepared from M. tuberculosis-infected mice at different time points. Lymph node cells were isolated by straining through a metal sieve. After depletion of erythrocytes, cells were resuspended in complete IMDM (Invitrogen Life Technologies) supplemented with 10% FCS (Invitrogen Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich), and penicillin and streptomycin (100 U/ml and 100 μg/ml; Invitrogen Life Technologies), counted, and used for additional experiments. For preparation of single-cell suspensions from lungs, mice were anesthetized and injected i.p. with 150 U of heparin (Ratiopharm). Lungs were perfused through the right ventricle with warm PBS. Once lungs appeared white, they were removed and sectioned. Dissected lung tissue was then incubated in collagenase A (0.7 mg/ml; Roche Diagnostics) and DNase (30 μg/ml; Sigma-Aldrich) at 37°C for 2 h. Digested lung tissue was gently disrupted by subsequent passage through a 100-μm pore size nylon cell strainer. Recovered lung cells were counted, diluted in IMDM, and used for additional experiments.
Flow-cytometric analysis of surface markers, intracellular cytokines, and proliferation
For flow-cytometric analysis of surface markers, cells were washed and incubated with a mixture containing anti-FcγRIII/II mAb (clone 2.4G2), mouse and rat serum to block nonspecific binding to FcRs. Cells were then incubated in consecutive steps for 20 min with optimal concentrations of the following Abs: CD4-allophycocyanin, CD8-allophycocyanin, CD3-PerCP, DX5-FITC, CD44-FITC, CD62L-PE, CD4-FITC, CD69-biotin, streptavidin-CyChrome (all from BD Biosciences).
For detection of intracellular IFN-γ, an intracellular cytokine staining kit was used (BD Biosciences). Briefly, single-cell suspensions were prepared at 21 and 42 days after infection, and 2 × 106 cells were stimulated with plate-bound anti-CD3/CD28 mAb (clone 2C11 and clone 37/51 at 10 μg/ml, respectively) for 4 h in the presence of GolgiPlug (BD Biosciences). Nonspecific Ab binding was blocked by incubation with a mixture containing anti-FcγRIII/II mAb (clone 2.4G2), mouse and rat serum. Cells were washed and incubated with optimal concentrations of anti-CD4-FITC (BD Biosciences). After staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and intracellularly accumulated IFN-γ was stained with a PE-labeled anti-IFN-γ mAb (BD Biosciences).
To analyze proliferation of CD4+ T cells in lungs from infected mice, 1 mg of BrdU (BD Biosciences) was i.p. injected 3 days before analysis. After 21 and 42 days of infection, single-cell suspensions of lungs were prepared, and CD4+ T cells were stained with anti-CD4-allophycocyanin (BD Biosciences). After washing, cells were fixed and permeabilized for 30 min in Cytofix/Cytoperm and 10 min in Cytoperm Plus (both from BD Biosciences) buffer. Following treatment with 30 μg of DNase (BD Biosciences), incorporated BrdU was detected by staining with anti-BrdU-FITC (BD Biosciences).
Fluorescence intensity was analyzed on a FACSCalibur (BD Biosciences) gating on lymphocytes identified by forward-scatter/side-scatter profile.
Delayed-type hypersensitivity (DTH)
At 28 days after infection, mice were challenged with a s.c. injection of 10 μg of purified protein derivative (PPD; in 50 μl of PBS; Statens Serum Institute) in the right and 50 μl of PBS in the left footpad. Swelling in both footpads was measured after 24 h using a Mitutoyo micrometer caliper (Brütsch), and the difference was taken as the amount of Ag-specific DTH. The PPD preparation did not induce swelling in noninfected animals.
Ag-specific restimulation of CD4+ T cells
For measuring Ag-specific production of IFN-γ by CD4+ T cells in lymph nodes, single-cell suspension of mediastinal lymph nodes were prepared in IMDM from mice 19 days after infection with M. tuberculosis. To enrich CD4+ T cells, single-cell suspensions from lymph nodes were incubated with magnetic CD4 microbeads (Miltenyi Biotec) and separated from other cells on a MACS separation unit (Miltenyi Biotec). Separated CD4+ T cells were collected in IMDM, counted, and diluted. Purity of enriched CD4+ T cells was >95% as determined by flow cytometry. For Ag-specific restimulation, 4 × 105 enriched CD4+ lymph node cells were incubated with 2 × 105 peritoneal macrophages that were pulsed with 25 μg/ml short-term culture filtrate from M. tuberculosis (a kind gift of P. Andersen, Statens Serum Institute, Copenhagen, Denmark) in IMDM. Resident peritoneal macrophages were obtained 1 day before the experiment by peritoneal lavage of uninfected C57BL/6 mice and incubated overnight in 96-well flat-bottom microplates (Nunc) in complete IMDM. After 72 h of restimulation, supernatants were collected and frozen at −80°C until production of IFN-γ was quantified by ELISA.
Before and at different time points after aerosol infection with M. tuberculosis, weighed lung samples were homogenized in 5 ml of 4 M guanidinium-isothiocyanate buffer, and total RNA was extracted by acid phenol extraction. To quantitatively compare WSX-1 gene expression in CD4+ T cells and macrophages, RNA was extracted from purified lymph node CD4+ T cells and elicited peritoneal macrophages using a total RNA isolation kit (Promega). cDNA was obtained using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) and oligo(dT) (12- to 18-mer; Sigma-Aldrich) as a primer. To assess expression of IL-27p28, EBI3, and WSX-1, PCR was performed on a thermal cycler (Dyad; MJ Research). Amplified DNA was separated on agarose gels and visualized under UV light after ethidium bromide incorporation. Quantitative PCR was performed on a Light Cycler (Roche Diagnostics) as previously described (36). Data were analyzed using the “fit points” and “standard curve method” using β2-microglubulin (β2m) as housekeeping gene to calculate the level of gene expression normalized for β2m expression. The following primer sets were used: β2m, sense, 5′-TCA CCG GCT TGT ATG CTA TC-3′, and antisense, 5′-CAG TGT GAG CCA GGA TAT AG-3′; IL-27p28, sense, 5′-GGC CAT GAG GCT GGA TCT C-3′, and antisense, 5′-AAC ATT TGA ATC CTG CAG CCA-3′; EBI3, sense, 5′-ACC CAT TGA AGC CAC GAC TT-3′, and antisense, 5′-AGT ATT GCA TCC AGG TGT CAG CT-3′; WSX-1, sense, 5′-CAA GAA GAG GTC CCG TGC TG-3′, and antisense, 5′-TTG AGC CCA GTC CAC CAC AT-3′; TNF, sense, 5′-TCT CAT CAG TTC TAT GGC CC-3′, and antisense, 5′-GGG AGT AGA CAA GGT ACA AC-3′; IL-12p40, sense, 5′-CTG GCC AGT ACA CCT GCC AC-3′, and antisense, 5′-GTG CTT CCA ACG CCA GTT CA-3′; NOS2, sense, 5′-AGC TCC TCC CAG GAC CAC AC-3′, and antisense, 5′-ACG CTG AGT ACC TCA TTG GC-3′; LRG-47, sense, 5′-AGC CGC GAA GAC AAT AAC TG-3′, and antisense, 5′-CAT TTC CGA TAA GGC TTG G-3′.
Quantification of TNF, IL-12p40, and IFN-γ by ELISA
To determine cytokine production after pulmonary infection of experimental animals, lung homogenates, serum, and supernatants were analyzed in 3-fold serial dilutions using the sandwich ELISA system OptEia (BD Biosciences) using a modified protocol. Before adding biotinylated Abs, endogenous biotin was blocked by incubating samples with an avidin/biotin block reagent (Vector Laboratories). After incubation with HRP coupled to avidin and developing with TMB substrate reagent, the absorbance was read on a microplate reader (Sunrise; Tecan). Using a test wavelength of 450 nm and a reference wavelength of 630 nm, samples were compared with appropriate recombinant cytokine standards. The detection limits for all cytokines were 5 pg/ml.
Stimulation of peritoneal macrophages
Mice were injected i.p. with 1 ml 3% thioglycolate (Invitrogen Life Technologies) and elicited peritoneal exudate cells were harvested after 5 days, and incubated for 4 h at 37°C and 5% CO2 in complete IMDM. To analyze STAT3 phosphorylation, adherent macrophages were incubated with medium, recombinant murine IL-27 (10 ng/ml) or IL-10 (10 ng/ml; BD Pharmingen) for 30 min. Cell lysates were subjected to Western blot analysis with anti-STAT3 and anti-phospho-STAT3 Abs (Cell Signaling Technology). Detection was conducted using IRDye800-conjugated goat anti-rabbit Abs (Rockland) and visualized with the LI-COR detection system (LI-COR Biosciences) according to the manufacturer’s instructions. For induction of proinflammatory cytokine production, triplicate cultures of adherent macrophages were stimulated with IFN-γ (100 U/ml; BD Pharmingen) and LPS (15 ng/ml; Sigma-Aldrich) or live M. tuberculosis (MOI, 3:1) (37) in the presence of medium, recombinant murine IL-27 (10 ng/ml) or IL-10 (10 ng/ml; BD Pharmingen) for 24 h. Recombinant murine IL-27 used in the present study was produced as described (30) and induced proliferation of WSX-1-expressing and gp130-transfected Ba/F3 cells (J. Scheller and C. Hölscher, unpublished observation). Supernatants were harvested and assayed for TNF and IL-12p40.
Quantifiable data are expressed as the means of individual determinations and SDs. Statistical analysis was performed using the unpaired Student’s t test defining different error probabilities: ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001.
WSX-1 regulates protection against M. tuberculosis infection
To assess the role of WSX-1 during the immune response to M. tuberculosis, infection-induced expression levels of IL-27 and WSX-1 were determined. Wild-type C57BL/6 mice were infected with 100 CFU of M. tuberculosis via the aerosol route, and RT-PCR was used to measure the levels of mRNA for IL-27p28, EBI3, and WSX-1 in the lungs. Before infection, EBI3 and WSX-1 were constitutively expressed, whereas IL-27p28 was not detectable (Fig. 1 a). After 21 days of infection, IL-27p28, EBI3, and WSX-1 were up-regulated.
To determine the functional significance of this infection-induced increase in IL-27 expression, the course of infection with 100 CFU of M. tuberculosis via the aerosol route was monitored in WSX-1-KO mice. On day 7 after infection, bacterial loads in lungs from M. tuberculosis-infected WSX-1-KO mice were slightly increased (4.1 ± 0.3 × 104) compared with infected C57BL/6 mice (2.1 ± 0.4 × 104) (Fig. 1,b). Because of this initially increased CFU in lungs from WSX-1-KO mice, the transiently higher bacterial loads in livers (Fig. 1,c) and spleens (d) at day 21 of infection are likely due to differential dissemination from the lungs rather than increased bacterial replication in these organs. Later during infection, WSX-1-KO mice coped better with M. tuberculosis infection than C57BL/6 mice showing a significantly reduced (∼0.5–1 log10 lower) bacterial load in the lungs (Fig. 1 b), liver (c), and spleen (d) from day 42 onwards. These data suggest that IL-27/WSX-1 inhibits cell-mediated immune responses and eventually suppresses protection from M. tuberculosis infection.
Histopathological changes in lung tissue from M. tuberculosis-infected WSX-1-KO mice
Effector mechanisms against mycobacteria conducted by macrophages and lymphocytes take effect in granulomas at the site of infection. To assess the formation and function of granulomas in lungs after aerosol infection with M. tuberculosis, tissue sections from C57BL/6 and WSX-1-KO mice were histologically examined 42 and 98 days after infection (Fig. 2). Compared with C57BL/6 mice, WSX-1-KO mice showed a slightly enhanced cellular infiltration into lung tissue 42 days after aerosol infection (Fig. 2, a–d) with more but smaller inflammatory foci in C57BL/6 mice. The cellular composition of granulomas did not differ between wild-type and WSX-1-KO mice, and both groups of mice showed epithelioid cell differentiation, lymphoid follicle formation, and highly structured, compact granuloma development. Whereas granulomas in lungs from C57BL/6 mice contained many acid-fast bacilli 98 days after infection (Fig. 2 e), bacteria were hardly detectable in lung granulomas from WSX-1-KO mice at this time point (f) indicating a more effective anti-mycobacterial response in the absence of WSX-1.
Enhanced expression of TNF and IL-12p40 in the absence of WSX-1
To evaluate the impact of WSX-1-mediated signaling on inflammatory responses during tuberculosis at the molecular level, the expression of proinflammatory cytokines in the lung were determined in C57BL/6 and WSX-1-KO mice after aerosol infection with M. tuberculosis (Fig. 3). Uninfected mice expressed negligible mRNA levels of TNF and IL-12p40 as determined by quantitative RT-PCR (Fig. 3,a). Gene expression of these proinflammatory cytokines were induced in lung homogenates 21 and 42 days after aerosol infection with M. tuberculosis (Fig. 3,a). WSX-1-KO mice expressed significantly higher amounts of TNF and IL-12p40 transcripts than C57BL/6 mice, and TNF and IL-12p40 protein levels were elevated in lungs from WSX-1-KO mice as determined by ELISA (Fig. 3 b). In contrast to the elevated expression of proinflammatory cytokines in the absence of WSX-1, RT-PCR of RNA isolated from lungs of uninfected and infected mice revealed that anti-inflammatory cytokines such as IL-4, IL-13, and IL-10 were not induced in either strain during the first 6 wk of infection with M. tuberculosis (data not shown). These results indicate that, after infection with M. tuberculosis, WSX-1-associated signaling events prevent a more pronounced proinflammatory cytokine response.
Increased recruitment, proliferation, and activation of T cells in WSX-1-KO mice
Effective recruitment and activation of macrophages, dendritic cells, and lymphocytes in the lungs of infected mice is essential for protective immunity against M. tuberculosis. Although macrophages are important cells for the induction of proinflammatory immune responses and the development of antibacterial effector functions, dendritic cells are indispensable for Ag presentation and the induction of adaptive immune responses executed by lymphocytes. In the lung, CD4+ T cells are the most significant lymphocyte population responsible for an efficient protective effector responses to M. tuberculosis infection (38). In addition to CD4+ T cells, CD8+ T cells are also believed to contribute to the host protective immune response against mycobacteria by IFN-γ production and cytotoxic effector functions (39), whereas NK cells appear to play a limited role in protection (40). To assess the contribution of these leukocyte populations to the increased level of protection against tuberculosis apparent in WSX-1-KO mice, quantitative analysis and phenotypical analysis of the recruited leukocyte populations was performed in the lungs of M. tuberculosis-infected C57BL/6 and WSX-1-KO mice (Fig. 4). The total cell number recruited to the lungs of WSX-1-KO mice was increased as early as day 21 of aerosol infection with M. tuberculosis (Fig. 4,a). Whereas the accumulation of MAC3+ macrophages and CD11c+ dendritic cells was not significantly different in lungs from either mouse strain (data not shown), WSX-1-KO mice showed a more efficient recruitment of lymphocytes into the lungs (Fig. 4, b–d). The amount of DX5+ NK (Fig. 4 b) and CD8+ T cells (c) was consistently higher in the lungs of infected WSX-1-KO than in organs of C57BL/6 mice. CD4+ T cell numbers were similar in lungs of both groups of mice until day 14 of infection (d). From day 21 on, the amount of infiltrating CD4+ T cells was increased in WSX-1-KO compared with C57BL/6 mice with a significant difference.
The higher numbers of CD4+ and CD8+ T cells also showed an increased expression of activation markers, as revealed by flow-cytometric analysis of CD3-expressing CD4+ T cells from infected WSX-1-KO mice (Fig. 4, e and f). Compared with M. tuberculosis-infected C57BL/6 mice, an increased population of CD4+ T cells in lungs from infected WSX-1-KO mice was found to be CD44high and CD62Llow from day 21 on with a significant difference on day 42 (Fig. 4,e). This increased activation was also observed for CD8+ T cells (data not shown). More strikingly, CD4+ T cells in lungs from WSX-1-KO mice constitutively expressed significantly elevated levels of the early activation marker CD69 at all time points examined after infection with M. tuberculosis (Fig. 4 f).
After infection with M. tuberculosis, enhanced recruitment and/or proliferation of CD4+ T cells could have been responsible for the observed expansion of these cells in lungs from WSX-1-KO mice. To assess the proliferative capacity of T cells in vivo, mice received an i.p. injection of BrdU 3 days before flow-cytometric analysis. Compared with wild-type mice, BrdU incorporation in CD4+ (Fig. 5 a) and CD8+ (data not shown) T cells from WSX-1-KO mice was increased 21 and 42 days after infection by 50%, indicating that an enhanced proliferation contributed to the profound expansion of CD4+ T cells in lungs from M. tuberculosis-infected WSX-1-KO mice.
Production of IFN-γ is the key effector mechanism against M. tuberculosis mediated by T cells at the site of infection. IFN-γ leads to efficient macrophage activation and subsequent containment of intracellular mycobacterial growth. To determine the capability of CD4+ effector T cells to produce IFN-γ in the absence of WSX-1, intracellular cytokine staining and subsequent flow-cytometric analysis of single-cell suspensions of lungs from C57BL/6 and WSX-1-KO mice was performed. Lymphocytes isolated from the lungs were stimulated with plate-bound anti-CD3/CD28 21 and 42 days after aerosol infection with M. tuberculosis. Whereas unstimulated suspensions did not contain appreciable amounts of IFN-γ-producing CD4+ T cells (data not shown), an increased proportion of CD4+ T cells were found to be positive for IFN-γ after stimulation with anti-CD3/CD28 (Fig. 5 b). Compared with stimulated cell suspensions from C57BL/6 mice, significantly more CD4+ T cells isolated from infected WSX-1-KO mice produced intracellular IFN-γ. Similarly, CD8+ T cells from lungs of infected WSX-1-KO mice produced elevated levels of IFN-γ in response to anti-CD3/CD28 stimulation (data not shown).
Recall responses of Ag-specific CD4+ T cells are optimal during the third and fourth week of infection, after which these responses decline. Enriched CD4+ T cell suspensions were prepared from mediastinal lymph nodes on day 19 after infection with M. tuberculosis by magnetic cell sorting. Enriched CD4+ T cells with a purity of >95% were restimulated in vitro with Ag-pulsed peritoneal macrophages, and IFN-γ production was measured (Fig. 5,c). After incubation with short-term culture filtrate-pulsed macrophages, CD4+ T cells from uninfected animals produce negligible amounts of IFN-γ (data not shown). Whereas unstimulated CD4+ T cells did not produce appreciable levels of IFN-γ, Ag-restimulated CD4+ T cells from lymph nodes of infected WSX-1-KO mice secreted significantly increased levels of IFN-γ when compared with CD4+ T cells of infected C57BL/6 mice. To monitor the development of Ag-specific immune responses, the footpad rechallenge model was used (DTH; Fig. 5,d). Mice received a single s.c. injection of PPD into one hind footpad at 4 wk of infection, and the relative footpad swelling was measured 24 h later. In contrast to C57BL/6 mice, WSX-1-KO mice exhibited a significantly increased DTH, suggesting an enhanced Ag-specific cell-mediated immune response to M. tuberculosis in the absence of WSX-1 (Fig. 5 d).
Taken together, the presented data demonstrate that the expansion of NK and T cells into lungs from M. tuberculosis-infected mice is regulated by WSX-1. Moreover, in the absence of WSX-1, CD4+ T cells were sufficiently primed, highly activated, and capable of efficient proliferation and IFN-γ production. Thus, WSX-1 modulates protection against tuberculosis by inhibiting activation, proliferation, and effector functions of DTH-conferring CD4+ T cells.
Increased IFN-γ production and macrophage activation in WSX-1-KO mice
Next, IFN-γ was quantified in lung homogenates and sera from M. tuberculosis-infected C57BL/6 and WSX-1-KO mice by ELISA (Fig. 6 a). Whereas uninfected mice in both groups produced negligible amounts of IFN-γ, the IFN-γ concentration 21 days after aerosol infection with M. tuberculosis was significantly increased in homogenates and sera from WSX-1-KO mice when compared with the levels of IFN-γ found in C57BL/6 mice. By day 42, IFN-γ production in WSX-1-KO mice declined to levels found in wild-type mice.
IFN-γ-induced effector functions in macrophages are key mechanisms for intracellular elimination of mycobacteria (41). NOS2-dependent reactive nitrogen intermediates produced by IFN-γ-activated macrophages are essential effector molecules against M. tuberculosis (42). In addition, the IFN-γ-dependent GTPase LRG-47 efficiently contributes to the intracellular elimination of mycobacteria by activated macrophages (43). To determine IFN-γ-dependent macrophage effector functions in M. tuberculosis-infected WSX-1-KO mice, RT-PCR of NOS2 and LRG-47 expression in lungs from infected mice was performed (Fig. 6 b). In relation to uninfected mice, mRNA for both enzymes were considerably induced in C57BL/6 mice 21 and 42 days after aerosol infection with M. tuberculosis. Compared with this, NOS2 as well as LRG-47 expression was significantly enhanced in lungs from infected WSX-1-KO mice after 21 and 42 days.
In line with the heightened resistance of WSX-1-KO mice to M. tuberculosis infection, IFN-γ production and the expression of IFN-γ-induced effector molecules was elevated in the absence of WSX-1-mediated signaling. These results indicate that WSX-1, rather than promoting IFN-γ production, effectively inhibits it. As a consequence, WSX-1 is involved in suppressing macrophage effector functions and thus protection from tuberculosis by regulating IFN-γ production.
Chronic hyperinflammatory responses and accelerated death of WSX-1-KO mice
Recently, it was reported that WSX-1-KO mice rapidly die after infection with T. gondii or T. cruzi due to an uncontrolled systemic hyperinflammation leading to severe immunopathology and organ failure (34, 35). We therefore also evaluated inflammatory responses and survival of WSX-1-KO mice after a protracted course of M. tuberculosis infection.
Histological examination of lungs taken from moribund WSX-1-KO mice showed advanced inflammatory cell infiltration, interstitial fibrosis, and deposition of cholesterol crystals, similar to the pathology observed in moribund C57BL/6 mice at a significantly later time point (Fig. 7,a). This accelerated tissue pathology in the lungs of WSX-1-KO mice was also reflected by a profound splenomegaly (the spleen body weight ratio of spleens from infected WSX-1-KO mice amounted to 21.3 ± 2.5, the ratio of spleens from C57BL/6 mice was 8.1 ± 0.5) and destruction of the splenic architecture when examined on day 254 of infection (Fig. 7 b). Whereas white and red pulpae as well as germinal centers were clearly distinguishable in spleens from wild-type mice, this structure was largely obliterated in spleens from WSX-1-KO mice.
TNF and IL-12p40 levels steadily increased in the lungs (Fig. 8,a) and sera (b) of C57BL/6 mice until day 254. However, the amount of both proinflammatory cytokines were significantly higher in lung homogenates and sera from infected WSX-1-KO mice throughout the period of infection, reaching levels almost twice as high as those in wild-type control infected mice. Moreover, during chronic infection, weight loss in WSX-1-KO mice amounted to 25% of the original weight on day 254 postinfection, whereas the body weight of C57BL/6 mice had slightly increased to 105% of the original weight before infection (Fig. 8,c). In line with this observed cachexia, WSX-1-KO mice succumbed to M. tuberculosis infection significantly earlier than wild-type mice (Fig. 8 d). Although C57BL/6 mice started to die after day 300 of aerosol infection with 100 M. tuberculosis, death of WSX-1-KO mice was observed as early as 200 days of infection. Although bacterial loads were not increased in WSX-1-KO mice immediately before death (data not shown), all mutant mice died before day 300 of infection. This accelerated mortality of WSX-1-KO mice was strictly dependent on M. tuberculosis infection because the lifespan of uninfected mutant mice was not different from naive C57BL/6 mice.
Although the absence of WSX-1 was accompanied by a significant reduction of M. tuberculosis replication in infected organs, a concomitant hyperinflammatory response accelerated the death of WSX-1 KO mice. This indicates that yet-unknown anti-inflammatory effects of IL-27/WSX-1 may regulate immunopathology during chronic infection.
IL-27 induces STAT3 phosphorylation and suppresses secretion of proinflammatory cytokines by activated peritoneal macrophages
Because WSX-1 is not only expressed on CD4+ T cells but also on myeloid cells (26, 27), we speculated that IL-27 may directly affect activated macrophages. To analyze the possible impact of IL-27 on inflammatory macrophages after infection with M. tuberculosis, we induced inflammatory peritoneal macrophages by i.p. injection of 3% thioglycolate. To confirm that WSX-1 is present on macrophages, we analyzed the expression of WSX-1 on elicited adherent peritoneal cells (Fig. 9, a and b). Peritoneal macrophages from C57BL/6 mice expressed WSX-1 (Fig. 9a). Quantitative real-time PCR revealed that WSX-1 expression in peritoneal macrophages was comparable to the expression level found in CD4+ T cells that had been purified from the lymph nodes of C57BL/6 mice (Fig. 9 b).
On a functional level, we determined whether peritoneal macrophages responded to IL-27. After incubation with medium, IL-27, or IL-10, we measured STAT3 phosphorylation in cell lysates (Fig. 9,c). Although medium did not induce any phosphorylation, STAT3 was phosphorylated after stimulation with IL-27 albeit to a lesser extent when compared with an incubation with IL-10. To confirm our hypothesis that IL-27 directly affects macrophage activation, elicited adherent peritoneal cells from C57BL/6 and WSX-1-KO mice were coincubated with IFN-γ/LPS and medium, IL-27, or IL-10. After 24 h, supernatants were assayed for the production of TNF and IL-12p40 by ELISA (Fig. 9,d). Adherent peritoneal cells from either mouse strain responded to stimulation with IFN-γ/LPS with a marked production of TNF and IL-12p40, which was reduced by coincubation with IL-10. Whereas the addition of IL-27 to IFN-γ/LPS-stimulated wild-type peritoneal macrophages also resulted in a decreased release of TNF and IL-12p40, coincubation of IL-27 had no effect on TNF and IL-12p40 production by activated peritoneal macrophages from WSX-1-KO mice. Although the suppressive activity of IL-27 on the secretion of proinflammatory cytokines by wild-type peritoneal cells was lower than that of IL-10 (mean suppression by IL-27: TNF, 44.5% ±14.6; IL-12p40, 44.5% ±21.4), the suppression of proinflammatory mediator release by IL-27 was reproducible and significant (n = 7) (Fig. 9,d). Because we were not able to detect any IL-10 in the supernatants of IFN-γ/LPS/IL-27-stimulated peritoneal macrophages, this suppression of inflammatory macrophages appeared to be mediated directly by IL-27 and not as a secondary effect through the induction of endogenous IL-10. Finally, we checked whether IL-27 also suppresses the production of TNF in M. tuberculosis-infected macrophages and coincubated elicited adherent peritoneal cells from C57BL/6 and WSX-1-KO mice with live M. tuberculosis and medium, IL-27, or IL-10. After 24 h, adherent peritoneal cells from either mouse strain responded to infection with TNF production, which was reduced in wild-type cells by coincubation with either IL-27 or IL-10 (Fig. 9 e).
Our results suggest that IL-27 may control pathological Th1 immune responses by regulating TNF and IL-12 production at the level of the activated inflammatory macrophage.
This study demonstrates that the IL-27R chain WSX-1 is not required for the development of a Th1-dominated cell-mediated immune response after infection with M. tuberculosis. Rather, WSX-1 was found to prevent optimal immunity, because WSX-1-KO mice had significantly lower bacterial loads in infected organs and higher tissue levels of Ag-specific IFN-γ. That WSX-1 was also important for the ultimate outcome of chronic infection is evident from the accelerated death rates in KO mice. Thus, IL-27/WSX-1 seems to play a dual role in protective immunity to M. tuberculosis: it prevents maximal antibacterial containment, but protects from an excessive chronic inflammatory response. IL-27/WSX-1 therefore emerges as a key mediator regulating the balance between protection and pathology in tuberculosis infection.
Early studies in WSX-1-KO mice supported the notion that IL-27 primarily promotes the differentiation of naive T cells by inducing the expression of T-bet, IL-12Rβ2, and IFN-γ, and thereby directs protective immune responses early after infection (28, 31). In line with this perception, WSX-1-KO mice were more susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) (26) and Listeria monocytogenes (27). In both studies, analyses were performed not later than day 14 or day 9 after infection, respectively. In the case of M. bovis BCG, bacterial loads in liver tissue from WSX-1-KO were increased 2 and 4 days after infection. However, later during infection, bacterial loads in WSX-1-KO mice appeared to be reduced compared with wild-type mice. Our own data corroborate these findings: bacterial loads in WSX-1-KO mice were always slightly increased early after infection (<day 14 in the lungs, up to day 21 in livers and spleens). During later stages, however, mutant mice consistently displayed a significantly increased resistance to M. tuberculosis infection (>day 21; see Fig. 1, b–d). IL-27/WSX-1 therefore promotes distinct mechanisms during the acute and chronic phases of intracellular infections.
Over time, however, T cell recruitment and proliferation were significantly increased in the lungs of M. tuberculosis-infected WSX-1-KO mice. This was accompanied by a profound production of proinflammatory cytokines and the development of highly activated CD4+ T cells capable of secreting elevated amounts of IFN-γ. To our knowledge, this is the first study in experimental tuberculosis demonstrating increased levels of antimycobacterial protection in a KO mouse strain. In contrast to M. tuberculosis-infected WSX-1-KO mice, mutant mice were not able to control parasite growth after infection with T. cruzi despite unimpaired IFN-γ production (35), indicating that WSX-1-mediated events support macrophage trypanocidal effector functions (6). However, after T. cruzi infection, the anti-inflammatory cytokines IL-4 and IL-13, capable of suppressing IFN-γ-induced NOS2-mediated effector mechanisms in macrophages (44), were up-regulated in the absence of WSX-1. Because neutralization of IL-4 significantly decreased parasitemia in T. cruzi-infected WSX-1-KO mice, WSX-1-mediated signals are capable of promoting parasite elimination by suppressing IL-4 production rather than by enhancing macrophage effector functions directly (35). Susceptibility to Leishmania major infection is also strongly dependent on the production of IL-4 (45), the IL-4-related cytokine IL-13 (46), and the presence of the IL-4Rα (44). Again, a decreased resistance of WSX-1-KO mice to infection with L. major was accompanied by a normal IFN-γ response, whereas the expression of IL-4 was elevated 6 wk after infection (26). Neutralization of IL-4 abolished the requirement for WSX-1 to promote early IFN-γ production and control of L. major (47), suggesting that WSX-1 signaling is dispensable for Th1 T cell differentiation in the absence of IL-4. In contrast to L. major infection, resolving infection with gastrointestinal nematodes is dependent on the genetically determined expression of IL-4 and IL-13 (48). Accordingly, WSX-1-KO mice developed a profound resistance to infection with Trichuris muris, because production of IL-4 was increased in these mice (49). In M. tuberculosis infection, however, expression of IL-4 or IL-13 was hardly detectable in the lungs of either C57BL/6 or WSX-1-KO mice (data not shown). This might explain why the absence of WSX-1 also did not affect the generation of effective cell-mediated immunity in this infection model.
The enhanced antimycobacterial immunity in WSX-1-KO mice could be attributed to their unfettered capacity to accumulate activated CD4+ and CD8+ T cells and to elaborate large amounts of IFN-γ at the site of infection, effectively inducing antimicrobially active effector molecules such as NOS2 and LRG-47. However, it should be noted that increasing the expression of endogenous IFN-γ does not necessarily lead to enhanced protection from mycobacterial infection (50). Quite to the contrary, IFN-γ and NOS2 have also been implicated in modulating the local cellular response to mycobacterial infection by down-regulating lymphocyte activation and by driving T cells into apoptosis, which may ultimately lead to a deteriorated protective immune response (41, 51, 52). Thus, the immunological mechanisms resulting in an increased resistance to M. tuberculosis infection in the absence of WSX-1 may be more complex.
In the absence of WSX-1, hyperactivation of T cells after infection with T. gondii and T. cruzi (34, 35) or in Con A-induced hepatitis (53) was reported. More specifically, after T. cruzi infection of WSX-1-KO mice (35), Ag-specific IFN-γ production by CD4+ T cells was only slightly increased, whereas the Th2 cytokines IL-4 and IL-5 were found to be significantly enhanced in lymphoid tissue. In T. cruzi-infected liver tissue, a generalized enhanced response of Th1 and Th2 cytokines was also observed, but the profound proinflammatory cytokine response with elevated levels of TNF, IL-6, GM-CSF, and IFN-γ was taken to be responsible for the pathological hyperactivated immune status in WSX-1-KO mice. After T. gondii infection, a similarly dysregulated cytokine response was found in spleen cells from WSX-1-KO mice, which produced increased amounts of IL-12p40, IFN-γ, IL-2, and IL-10. CD4+ T cells from T. gondii-infected WSX-1-KO mice were highly activated and capable of secreting elevated amounts of IFN-γ. Therefore, in toxoplasmosis and experimental Chagas’ disease, WSX-1-mediated signals down-regulate a pathological cell-mediated immune response that may otherwise lead to liver necrosis, organ failure, and premature death. In murine tuberculosis, no pathological changes in lung and liver such as tissue destruction or necrosis were observed in WSX-1-KO mice up to at least day 100 of infection. However, during later phases of murine tuberculosis, the absence of WSX-1 was associated with an unmitigated systemic production of proinflammatory cytokines, caused extensive inflammation, splenomegaly, precipitated cachexia, and accelerated death of infected mice. IL-27/WSX-1 therefore appears to be an essential component of a regulatory system required to prevent the pathological consequences of an unrestrained antimicrobial response.
Elevated cytokine expression in the absence of WSX-1 may be a secondary phenomenon reflecting the lower levels of mycobacteria found in different organs, because it was shown that M. tuberculosis can actively suppress IL-12p40 and TNF production (54). However, in light of recent findings, it has also been speculated that IL-27 may modulate proinflammatory macrophage responses (55). In search for a mechanism explaining the anti-inflammatory effect of IL-27, we found that WSX-1 was expressed on murine macrophages and that IL-27 was able to suppress the release of proinflammatory cytokines by IFN-γ/LPS-stimulated or M. tuberculosis-infected elicited adherent peritoneal macrophages. It was reported that IL-27 can activate STAT3 in purified human monocytes (24). In this study, we show that IL-27 also induces STAT3 phosphorylation in inflammatory macrophages. Because cell-specific deletion of STAT3 in macrophages results in elevated production of proinflammatory cytokines leading to hyperinflammation and accelerated death during endotoxemia (56), it is possible that a lack of IL-27/WSX-1-induced STAT3 phosphorylation contributes to the enhanced secretion of inflammatory cytokines observed in M. tuberculosis-challenged WSX-1-KO mice.
At this stage, we cannot exclude that the elevated proinflammatory immune response in M. tuberculosis-infected WSX-1-KO mice is mediated by the absence of WSX-1-mediated signaling in CD4+ T cells rather than in macrophages. In this scenario, increased IL-12p40 and TNF secretion by macrophages would have been secondary to unsuppressed CD4+ T cell responses. Recently, it has been shown that, after infection with T. muris, IL-27 could directly affect recall responses in differentiated Th2 cells (47). However, incubation with IL-27 had no effect on differentiated CD4+ T cells from M. tuberculosis-infected mice during polyclonal restimulation (data not shown). To unequivocally define the molecular mechanism underlying WSX-1-mediated anti-inflammatory mechanisms in vivo, it will be important to manipulate the expression of WSX-1 on leukocyte subsets. As we have recently described for the IL-4Rα (57), a cell-specific deletion of the IL-27R component WSX-1 in either macrophages or T cells should unravel the mechanisms responsible for the elevated but uncontrolled immune response in M. tuberculosis-infected WSX-1-KO mice.
The anti-inflammatory function of IL-27 appears to be similar to other classical anti-inflammatory cytokines such as IL-4, IL-13, and IL-10. To date, however, there has been no evidence in murine tuberculosis that endogenous levels of IL-10, IL-4, or the presence of IL-4Rα play any role during the natural course of infection (58) (C. Hölscher, unpublished data), although overproduction of IL-10 leads to compromised immunity to M. tuberculosis (59) (T. Schreiber, S. Ehlers, A. Hölscher, R. Lang, and C. Hölscher, unpublished data). In contradistinction, the presence of the IL-27R component WSX-1 is necessary for this pronounced immunoregulatory effect. WSX-1 exerts its anti-inflammatory effects likely by down-regulating expression of TNF and IL-12p40 in activated inflammatory macrophages. High levels of TNF have long been associated with cachexia and septic shock (60). Moreover, the toxic inflammatory side effects of high-dose IL-12 therapy are well documented (61). Tight regulation of these cytokines may therefore be necessary to prevent systemic organ failure during chronic infection-induced inflammation. Unfortunately, it is extremely difficult technically to only partially neutralize TNF and IL-12 function in the chronic phase of infection to provide definitive proof that it is the uncontrolled proinflammatory cytokine response in WSX-1 KO mice that is responsible for the accelerated death of these mice.
Taken together, IL-27 appears to be a dichotomous cytokine promoting early Th1 cell development and limiting chronic inflammatory immune responses, depending on the pathogen, the site of infection, as well as the cell types involved in the respective immune response (26, 27, 34, 35, 47). For example, WSX-1-KO mice show a transient, early defect in IFN-γ production when infected with L. major or M. bovis BCG, but infection with T. gondii or T. cruzi leads to elevated Th1 responses (26, 34, 35, 47). A key difference between these pathogens is their capacity to induce IL-12 (8). Whereas T. gondii and T. cruzi promote strong innate immune responses that lead to systemic IL-12 levels early during infection, acute leishmaniasis induces much less IL-12 production (62). M. tuberculosis appears to be a low IL-12 inducer, and may even inhibit LPS-induced IL-12 production (63). One might predict, therefore, that differences in the virulence of M. tuberculosis strains and the dose of infection would result in different kinetics of initially reduced Th1 development and increased inflammatory responses at later time points in the absence of IL-27-mediated signaling. Although, in this report, we have not directly addressed T-bet expression and IL-12Rβ2 induction in CD4+ T cells from WSX-1-KO mice early after infection, we clearly show that, during later stages of tuberculosis infection, WSX-1 is indispensable for limiting an otherwise-detrimental proinflammatory immune response. Moreover, our report indicates a possible feedback mechanism during Th cell development after infection. At the onset of infection, WSX-1 signaling induces T-bet in naive CD4+ T cells and subsequently the expression of a functional IL-12R. Because IL-27 production by macrophages and dendritic cells precedes that of IL-12, IL-27 appears to sensitize naive CD4+ T cells to the Th1-polarizing influences of IL-12. However, during the course of infection, IL-27 may suppress IL-12 production by inflammatory macrophages, eventually limiting excessive Th1 immune responses.
In conclusion, our studies in WSX-1-KO mice revealed IL-27/WSX-1 to be a dual regulator of the cell-mediated immune response against M. tuberculosis infection. On the one hand, IL-27/WSX-1 prevents the maximal expression of antimycobacterial immunity by limiting T cell expansion and IFN-γ production. In contrast, IL-27/WSX-1 prevents a pathological systemic hyperinflammatory response probably by suppression of TNF and IL-12 secretion by activated inflammatory macrophages. Thus, IL-27 appears to occupy a central regulatory role in mediating protective and pathological immune responses to intracellular infections. This proverbial function of a “double-edged sword” is a hitherto-underappreciated facet of IL-27.
The authors have no financial conflict of interest.
We thank Susanne Metken and Manfred Richter for excellent technical assistance; Ilka Monath, Sven Mohr, and Claus Möller for organizing the animal facility and taking care of the mice; and Gottfried Alber and Frank Brombacher for critical reading of the manuscript and fruitful discussions.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by German Research Foundation Research Grants HO 2145/2-1 and SFB367-C9.
Abbreviations used in this paper: KO, knockout; EBI3, EBV-induced protein 3; DTH, delayed-type hypersensitivity; PPD, purified protein derivative; β2m, β2-microglubulin; BCG, bacillus Calmette-Guérin.