Endogenous interleukin-18 is involved in immunity to Leishmania donovani but its absence does not adversely influence the therapeutic activity of sodium stibogluconate

Immunity to Leishmania donovani is associated with an interleukin (IL)-12 driven T helper 1 (Th1) response. In addition, the ability to respond to chemotherapy with sodium stibogluconate (SSG) requires a fully competent immune response and both Th1 and Th2 responses have been shown to positively influence the outcome of drug treatment. In the present study, the influence of IL-18, which can modulate both interferon (IFN)-gamma and IL-4 production, on the outcome of primary L. donovani infection and SSG therapy following infection was assessed using BALB/c IL-18-deficient and wild type mice. IL-18 deficiency was associated with an increased susceptibility to L. donovani infection, evident by day 40 post infection, resulting in higher parasite burdens in the spleen, liver, and bone marrow compared with wild type control animals. Infected IL-18-deficient mice had significantly lower splenocyte concanavalin A (ConA) induced IFN-gamma production as well as lower serum IL-12 and IFN-gamma levels, indicating a reduced Th1 response. However, drug treatment was equally effective in both mouse strains and restored serum IL-12 and IFN-gamma levels, and IFN-gamma production by ConA stimulated splenocytes of IL-18-deficient mice, to levels equivalent to similarly treated wild type mice.     

Up-regulation of IL-18 and predominance of a Th1 immune response is a hallmark of lupus nephritis

There is evidence that nephritis is dominated by a Th1 immune response in systemic lupus erythematosus. Since IL-18 promotes polarization of the immune response toward Th1, we investigated the role of this cytokine in lupus nephritis (LN). A total of 133 lupus patients and 44 healthy subjects were enrolled. Demographic and clinical characteristics with renal biopsy data were recorded. IL-18 along with IFN-gamma and IL-4, two prototypical of Th1 and Th2 cytokines, were measured in serum by ELISA. Peripheral blood lymphocytes were analysed by flow cytometry for IFN-gamma and IL-4. IL-18 expression was determined by immunohistochemistry in 13 renal biopsy specimens from patients with LN and 2 controls. Serum IL-18 was higher in lupus patients than in controls. Levels of IL-18 correlated with urinary microalbumin and were increased in patients with LN when compared to those without LN. IL-18 expression was also increased within the glomeruli of nephritic patients and was primarily detected within the mesangial matrix and in infiltrating mononuclear cells. Measurement of IFN-gamma and IL-4 in either sera or peripheral blood lymphocytes showed high IFN-gamma along with low IL-4 expression in LN patients compared to patients without nephritis. A positive correlation between serum IL-18 and IFN-gamma levels was found. IL-18 may play a prominent role in the pathogenesis of LN by promoting a cytokine imbalance towards a Th1 immune response. Measurement of IL-18 may be helpful for the early identification of lupus patients with LN and may help gauge the response to treatment in patients with active LN undergoing treatment.     

Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

Interleukin-18 is an essential element in host resistance to experimental group B streptococcal disease in neonates

Previous studies demonstrated that interleukin-12 (IL-12)-dependent gamma interferon (IFN-gamma) responses have a major role in restricting in vivo bacterial growth during infection of mice with group B streptococci (GBS), important human pathogens. Like IL-12, IL-18 is a potent IFN-gamma inducer. The role of IL-18 in experimental GBS infection was investigated here. Significant elevations of IL-18 levels over baseline values were detected in plasma samples from neonatal mice rendered septic with GBS. Neutralization of IL-18 significantly increased mortality and bacterial burden (P < 0.05). In contrast, administration of recombinant IL-18 (rIL-18) before or after GBS challenge remarkably improved survival and decreased blood colony counts, in association with increased IFN-gamma production by spleen cells. The beneficial effects of rIL-18 were counteracted by administration of neutralizing anti-IFN-gamma monoclonal antibodies, indicating that the effects of IL-18 were mediated by IFN-gamma. Finally, low rIL-18 doses that had no effect of their own on bacterial burden could act in synergy with rIL-12 to protect neonatal mice during GBS infection. Collectively, our data indicate that IL-18 responses have an important role in host defenses against GBS and that rIL-18 may be useful in alternative strategies to treat neonatal GBS disease.     

Spontaneous arthritis in MRL/lpr mice is aggravated by Staphylococcus aureus and ameliorated by Nippostrongylus brasiliensis infections

Rheumatoid arthritis is an autoimmune disease that affects human beings worldwide. Infections have been associated to autoimmune diseases because their ability to induce a dominant cytokine response. Joint inflammation has been related to Th1 response because they induce high expression of proinflammatory cytokines TNF-alpha, IL-1, IFN-gamma. MRL/lpr mice spontaneously develop an autoimmune disease affecting joints, kidneys, etc. We compared incidence and severity of arthritis, antibody response, cytokine production, in mice infected with bacteria or helminthes in the Murphy Roths Large (MRL)lpr mice. Infections with helminthes Heligmosomoides polygyrus, Nippostrongylus brasiliensis or bacteria Nocardia brasiliensis and Staphylococcus aureus were studied. IL-4, IFN-gamma and IgG1, IgG2a antibody productions were determined. IFN-gamma was increased in all groups, the highest production was observed after bacterial infection; IL-4 production was higher after helminthes infection. IgG1 sera levels were increased in the helminthes infected group. IgG2a sera concentration was stimulated by bacterial infection. The histopathology showed that 100% of bacterial infected mice developed arthritis and severe tissue damage such as cartilage erosion and bone destruction. Animals infected with parasites showed a decreased incidence and severity of arthritis. Severity of tissue damage in joints is correlated with increased numbers of lymphocytes and macrophages immunoreactive to proinflammatory cytokines.     

IL-12 and IFN-gamma production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections

Resistance to acute Trypanosoma cruzi infection is mainly associated with a Th1 immune response, characterized by gamma-interferon (IFN-gamma) production and activation of macrophages. The outcome of the Th1 response in the spleen and serum of BALB/c and C3H mice infected with T. cruzi, Tulahuén strain was studied. The levels of interleukin-12 p40 (IL-12 p40) and IFN-gamma, as well as natural killer (NK) cell cytotoxicity were determined at different time-points during the acute phase, and the production of cytokines was also studied in the chronic infection. At 2 days post-infection (pi), spleen cells from C3H mice increased their NK cell activity and the ex vivo spontaneous release of both IL-12 p40 and IFN-gamma. On the other hand, BALB/c mice reached low levels of NK cell cytotoxicity and no IFN-gamma production was detected at this time pi, but the cytokine was released at high amounts in the second week of the infection. Seric IL-12 p40 concentrations showed a 3-fold increase in both mouse strains on the second day pi and remained high throughout the acute phase. However, seric IFN-gamma levels increased during the late acute infection and were higher in BALB/c than in C3H mice. In chronically infected mice IL-12 p40 was as high as in the acute phase in the serum of both strains, but only BALB/c mice still produced IFN-gamma. To the authors' knowledge this is the first report showing the protein levels of IL-12 p40 determined in vivo in acute and chronic T. cruzi infections. The results reveal differences between both mouse strains in the mechanisms controlling the onset and fate of the Th1 response triggered by the parasite and a long lasting pro-inflammatory stimuli.     

Serum interleukin-18 and soluble tumour necrosis factor receptor 2 are associated with disease severity in patients with paracoccidioidomycosis

Interleukin (IL)-18 is a proinflammatory cytokine of the IL-1 superfamily that exhibits broad functional effects in innate and acquired immune responses and which has been found in high levels in several chronic inflammatory and autoimmune diseases. Over-expression of IL-18 may promote early resolution of infection or could promote a detrimental exaggerated immune response. The aim of this study was to determine serum levels of IL-18 and other inflammatory mediators [IL-12, soluble intercellular adhesion molecule 1 (sICAM-1), soluble tumour necrosis factor receptor 1 (TNF-RI), sTNF-RII, CXC chemokine ligand 9 (CXCL9), CXCL10] at baseline and after anti-fungal therapy in serum from patients with juvenile (JF) and adult (AF) forms of paracoccidioidomycosis (PCM), as well as in healthy controls (C), and to assess their possible relationships to the severity of disease. IL-18 and sTNF-RII levels in patients with the JF of PCM were significantly higher than those in the AF and controls. In relation to sICAM-1, no difference was observed between JF and AF patients but both presented higher levels than controls. sTNF-RI levels were higher in patients with PCM than in controls, and significantly higher concentrations were detected in AF patients compared to JF patients. Moreover, IL-12 and chemokines CXCL9 and CXCL10 were also higher in patients than in controls. In JF patients IL-18 levels correlated significantly with sICAM-1 (r=0 x 62, P<0 x 0001), sTNF-RI (r=0 x 63, P<0 x 0001), sTNF-RII (r=0 x 51, P=0 x 02), as well as with clinical severity. The results suggest the value of serum IL-18 and sTNF-Rs levels as a parameter of PCM severity and may support a possible role for them in the pathogenesis of the disease.     

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.     

Characterization of the in vitro and in vivo activity of monoclonal antibodies to human IL-18

IL-18 is a cytokine with potent IFN-gamma inducing activities as well as an important mediator of Th1 polarized immune responses. In this study we demonstrated that IL-18 induces the concentration-dependent production of the proinflammatory mediators IFN-gamma, IL-6, and GM-CSF, but not the anti-inflammatory cytokine, IL-10 from peripheral blood lymphocytes in the presence of mitogen. Three neutralizing IL-18 monoclonal antibodies (MAbs) were investigated, one of which (2C10) inhibited IL-18 bioactivity with an IC50 of 0.1 nM and had a K(D) of 3.9 x 10(-11) M. A NOD/SCID mouse model engrafted with human peripheral blood lymphocytes was developed to test the in vivo efficacy of this MAb. The IFN-gamma production induced by LPS administration was inhibited approximately 90% by prior dosing of MAb 2C10. The therapeutic utility of a high-affinity IL-18 MAb may be of benefit in Th1-driven autoimmune diseases such as rheumatoid arthritis and Crohn's Disease, where elevated levels of IL-18 have been observed.     

Th1 and Th1-inducing cytokines in Salmonella infection

Thl and Thl-inducing cytokines and T cell responses were investigated in human salmonellosis. Serum IFN-gamma, IL-12 and IL-18 levels were increased significantly in patients with salmonellosis. The increase in serum IL-15 and IL-18 levels was more significant and prolonged in patients with the systemic form of salmonellosis than in those with the gastroenteric form. The serum IFN-gamma level was correlated significantly with IL-12 and IL18 levels, and the IL-15 level was correlated significantly with IL-18. Upon stimulation with Salmonella in vitro, mononuclear cells from salmonellosis patients produced significantly higher amounts of IFN-gamma and IL-12 compared with those from healthy controls. Anti-IL-12 moAb or anti-IL18 MoAb significantly inhibited Salmonella-induced IFN-gamma production in vitro. gamma delta T cells expressed significantly higher levels of IFN-gamma mRNA in salmonellosis patients than in healthy controls. The results suggest that Th1-inducing cytokines appear to be involved in the in vivo response against Salmonella infection, promoting IFN-gamma production by alpha beta and gamma delta T cells which plays a protective role against Salmonella.     

Pentoxifylline inhibits the synthesis and IFN-gamma-inducing activity of IL-18.

The effect of phosphodiesterase-inhibiting anti-inflammatory drug pentoxifylline (PTX) on LPS-induced IL-18 synthesis and IL-18-mediated IFN-gamma-induction were investigated. In a dose-dependent manner PTX inhibited production of IL-18 in LPS-treated cultures of murine spleen cells and bone marrow-derived macrophages. Similarly, PTX treatment significantly reduced blood IL-18 levels and expression of spleen IL-18 mRNA in LPS-challenged mice. The inhibitory effect of PTX was specific for IL-18, since LPS-induced IL-12 p40 release was not suppressed either in splenocyte cultures or blood of LPS-injected animals. Synergistic induction of IFN-gamma by combined IL-12/IL-18 treatment was also inhibited by PTX in vitro and in vivo. Experiments with IL-12 pretreatment of splenocytes, followed by IL-18 stimulation, revealed that PTX suppressed both IL-12 and IL-18 signals responsible for IFN-gamma induction. These results suggest that interference with IL-18 synthesis and IFN-gamma-inducing activity might contribute to anti-inflammatory actions of PTX.     

Differential role of IL-18 and IL-12 in the host defense against disseminated Candida albicans infection

IFN-gamma plays a crucial role in the defense against infection with Candida albicans. Since IL-18 and IL-12 are strong stimuli of IFN-gamma production, we investigated whether endogenous IL-18 and IL-12 are involved in the host defense during disseminated candidiasis. IL-18 knockout (IL-18-/-) mice, but not IL-12-/- mice, displayed an increased mortality due to C. albicans infection, accompanied by a decreased clearance of the yeasts from the kidneys late during the course of infection. Histopathology of the organs, combined with phagocyte recruitment experiments, showed a decreased influx of monocytes at the sites of Candida infection, mainly in the IL-18-/- mice. Whereas production of the chemokine KC was decreased in both IL-18-/- and IL-12-/- mice, MIP-2 production was deficient only in IL-18-/- animals, which may explain the differences in phagocyte recruitment. In addition, although IFN-gamma production capacity, as a parameter of the Th1-protective immunity, was reduced by 65 to 80% in the IL-12-/- mice, this defect was even more pronounced in the IL-18-/- mice (85 to 95% down-modulation). In conclusion, the anticandidal effects of endogenous IL-18 are mediated late during the infection by assuring a proper IFN-gamma response and promoting the infiltration of the site of infection by monocytes.     

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.     

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C

DS-Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS-Nh mice with AD-like dermatitis and that the serum levels of anti-SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC-producing S. aureus to DS-Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL-4 and IFN-gamma from spleen mononuclear cells (MNC) from DS-Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL-4 responses and lower IFN-gamma responses to SEC showed correlation with TCRBV8S2-positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC-producing S. aureus was able to survive in DS-Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host-S. aureus relationships.     

A mutant of staphylococcal enterotoxin C devoid of bacterial superantigenic activity elicits a Th2 immune response for protection against Staphylococcus aureus infection

Staphylococcal enterotoxin C (SEC), a bacterial superantigenic exotoxin, is commonly produced by invasive Staphylococcus aureus isolates, especially methicillin-resistant strains and isolates from animal diseases. We constructed and expressed a nontoxic mutant SEC (mSEC) and investigated whether immunization with mSEC, which is devoid of superantigenic activity, can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. The bacterial counts in the organs of mSEC-immunized mice were significantly lower and the survival rate was higher than the corresponding values for the control group. Immunization with mSEC strongly induced the production of T-helper 2 type antibodies, immunoglobulin G1, and immunoglobulin G2b. The production of interleukin-10 (IL-10) and IL-4 was significantly greater in immunized mice challenged with S. aureus than in the control mice, whereas the production of gamma interferon (IFN-gamma) was significantly decreased in the immunized mice. The cytokine response in a spleen cell culture that was stimulated with heat-killed S. aureus or SEC showed that immunization with mSEC inhibited IFN-gamma production and up-regulated IL-10 production in vitro. Furthermore, IFN-gamma and tumor necrosis factor alpha production in vitro was significantly inhibited by sera from mSEC-immunized mice but not by sera from control mice. These results suggest that immunization with mSEC devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by SEC-specific neutralizing antibodies.     

Stress as an endogenous adjuvant: augmentation of the immunization phase of cell-mediated immunity

Stress is thought to be immunosuppressive but paradoxically exacerbates inflammatory and autoimmune diseases. We initially showed that acute stress enhances skin immunity. Such immunoenhancement could promote immunoprotection in case of wounding, infection or vaccination but could also exacerbate immunopathological diseases. Here we identify the molecular and cellular mediators of the immunoenhancing effects of acute stress. Compared with non-stressed mice, acutely stressed animals showed significantly greater pinna swelling and leukocyte infiltration, and up-regulated macrophage chemoattractant protein-1, macrophage inflammatory protein-3alpha, IL-1alpha, IL-1beta, IL-6, tumor necrosis factor-alpha and IFN-gamma, but not IL-4 gene expression at the site of primary antigen exposure. Stressed animals also showed enhanced maturation and trafficking of dendritic cells (DCs) from skin to lymph nodes (LNs), higher numbers of activated macrophages in skin and LNs, increased T cell activation in LNs, and enhanced recruitment of surveillance T cells to skin. These findings show that important interactive components of innate (DCs and macrophages) and adaptive (surveillance T cells) immunity are mediators of the stress-induced enhancement of a primary immune response. Such enhancement during primary immunization may induce a long-term increase in immunologic memory resulting in subsequent augmentation of the immune response during secondary antigen exposure. Thus, the evolutionarily adaptive fight-or-flight stress response may protectively prepare the immune system for impending danger (e.g. infection and wounding by a predator), but may also contribute to stress-induced exacerbation of inflammatory and autoimmune diseases.     

Increased levels of interferon-gamma primed by culture filtrate proteins antigen and CpG-ODN immunization do not confer significant protection against Mycobacterium tuberculosis infection

The results of various animal model studies of tuberculosis (TB) suggest that culture filtrate proteins (CFPs), which are antigens secreted by Mycobacterium tuberculosis, are largely responsible for improvements in TB vaccines. The great obstacle to developing protein subunit vaccines is that adjuvants are required in order to stimulate relevant protective immune responses. Acting as immune adjuvants, CpG-oligodeoxynucleotides (CpG-ODNs) promote the activation of Th1 cells and of pro-inflammatory cytokines. To evaluate the adjuvant role of CpG-ODNs in conferring enhanced immunogenic capacity and protection against M. tuberculosis, we immunized mice with CFP antigen combined with synthetic CpG-ODNs (CFP/CpG) or with incomplete Freund's adjuvant (IFA) (CFP/IFA). Immunization with CFP/CpG induced a T helper 1 (Th1)-biased response accompanied by a higher immunoglobulin G2a (IgG2a) antibody/IgG1 antibody ratio, elevated production of interferon-gamma (IFN-gamma) by spleen cells and in lungs. However, CFP/IFA-immunized mice presented higher levels of IgG1 antibodies, as well as increased production of IFN-gamma, interleukin (IL)-5, and IL-10 by spleen cells, together with lower levels of IFN-gamma in the lungs. Despite the stronger Th1 response seen in both groups, believed to be necessary for protection against TB, only mice immunized with CFP/IFA were protected after M. tuberculosis infection. Lung histology revealed that lung parenchyma were better preserved in CFP/IFA-immunized mice, which also presented intense lymphocyte recruitment to the lesion, whereas CFP/CpG-immunized mice presented severe pulmonary injury accompanied by necrosis. Based on the data presented, we discuss the widely accepted paradigm that high levels of IFN-gamma are directly correlated with protection against experimental TB.