Role of interleukin-1beta in activating the CD11c(high) CD45RB- dendritic cell subset and priming Leishmania amazonensis-specific CD4+ T cells in vitro and in vivo

Cutaneous leishmaniasis associated with Leishmania amazonensis infection is characterized by uncontrolled parasite replication and profound immunosuppression; however, the underlying mechanisms remain largely unclear. One possibility is that the L. amazonensis parasite modulates antigen-presenting cells, favoring the generation of pathogenic Th cells that are capable of recruiting leukocytes but insufficient to fully activate their microbicidal activities. To test this possibility, we infected bone marrow-derived dendritic cells (DCs) of C57BL/6 mice with L. amazonensis or Leishmania major promastigotes and assessed the activation of DC subsets and their capacity in priming CD4(+) T cells in vitro. In comparison to L. major controls, L. amazonensis-infected DCs secreted lower levels of interleukin-1alpha (IL-1alpha) and IL-1beta, were less potent in activating the IL-12p40-producing CD11c(high) CD45RB(-) CD83(+) CD40(+) DC subset, and preferentially activated CD4(+) T cells with a IFN-gamma(low) IL-10(high) IL-17(high) phenotype. Although the addition of IL-1beta at the time of infection markedly enhanced DC activation and T-cell priming, it did not skew the cytokine profile of DCs and pathogenic Th cells, as local injection of IL-1beta following L. amazonensis infection accelerated Th cell activation and disease progression. This study suggests that intrinsic defects at the level of DC activation are responsible for the susceptible phenotype in L. amazonensis-infected hosts and that this parasite may have evolved unique mechanisms to interfere with innate and adaptive immunity.     

Impaired expression of inflammatory cytokines and chemokines at early stages of infection with Leishmania amazonensis

Infection of mice with Leishmania major results in disease progression or resolution, largely depending on the genetic backgrounds of the mouse strains. Infection with Leishmania amazonensis, on the other hand, causes progressive cutaneous lesions in most inbred strains of mice. We hypothesized that deficient activation of early immune responses contributes to the pathogenesis in L. amazonensis-infected mice. To distinguish early molecular events that determine the outcome of Leishmania infections, we examined cytokine gene expression in C57BL/6 mice infected with either L. amazonensis or L. major (a healing model). After 2 to 4 weeks, L. amazonensis-infected mice had significantly delayed and depressed expression of inflammatory cytokines (interleukin-12 [IL-12], gamma interferon, IL-1 alpha, IL-1 beta), CC chemokines (CC chemokine ligand 3 [CCL3]/macrophage inflammatory protein 1 alpha [MIP-1 alpha], CCL4/MIP-1 beta, CCL5/RANTES, MIP-2), and chemokine receptors (CCR1, CCR2, CCR5) in foot tissues and draining lymph nodes compared to the expression in L. major-infected controls. These findings correlated with defective T-cell responsiveness to parasite stimulation in vivo and in vitro. Adoptive transfer of L. amazonensis-specific Th1 cells prior to infection overcame the immune defects of the animals, leading to complete control of the disease. Studies with gene knockout mice suggested that IL-10, but not IL-4, contributed partially to compromised immunity in L. amazonensis-infected hosts. The data suggest that there is impairment in multiple immune functions at early stages of infection with L. amazonensis parasites and provide a compelling rationale to explore immune augmentation as an intervention in American cutaneous leishmaniasis.     

CXCL10/gamma interferon-inducible protein 10-mediated protection against Leishmania amazonensis infection in mice

Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4(+) T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 following L. amazonensis infection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.     

Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection

C3H and C57BL/6 mice are resistant to Leishmania major but develop chronic lesions with persistent parasite loads when they are infected with Leishmania amazonensis. These lesions develop in the absence of interleukin-4 (IL-4), indicating that susceptibility to this parasite is not a result of development of a Th2 response. Expression of the cytokine IL-10 during infection could account for the lack of IL-12 expression and poor cell-mediated immunity towards the parasite. Therefore, we tested the hypothesis that IL-10 plays a central role in downmodulating the Th1 response after L. amazonensis infection. Infection of C57BL/6 IL-10-deficient mice indicated that in the absence of IL-10 there was early enhancement of a Th1 response, which was downregulated during the more chronic stage of infection. In addition, although there were 1- to 2-log reductions in the parasite loads within the lesions, the parasites continued to persist, and they were associated with chronic lesions whose size was similar to that of the control lesions. These experiments indicated that L. amazonensis resistance to killing in vivo is only partially dependent on expression of host IL-10. However, IL-10-deficient mice had an enhanced delayed-type hypersensitivity response during the chronic phase of infection, indicating that there were Th1 type effector cells in vivo at this late stage of infection. These results indicate that although IL-10 plays a role in limiting the Th1 response during the acute infection phase, other immunomodulatory factors are responsible for limiting the Th1 response during the chronic phase.     

Vaccine-induced protection against Leishmania amazonensis is obtained in the absence of IL-12/23p40

Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.     

The TGF-beta response to Leishmania chagasi in the absence of IL-12

Cure of leishmaniasis requires a type 1 immune response characterized by IFN-gamma production. Leishmania major infection leads to a type 2 response suppressing cure of susceptible BALB/c mice, and L. major causes an exacerbated type 2 response in mouse strains with a gene knockout (KO) such that they lack IL-12p40 (IL-12KO mice). In contrast, type 1 responses are inhibited by TGF-beta without Th2 cell expansion in BALB/c mice infected with L. chagasi. We questioned whether the type 2 or the TGF-beta response would dominate during L. chagasi infection of IL-12KO mice. C57BL/6 mice developed self-resolving L. chagasi infection with abundant IFN-gamma. In contrast, L. chagasi disease was exacerbated and IFN-gamma was low in IL-12KO mice. Total TGF-beta was significantly higher in IL-12KO than control C57BL/6 mice, but IL-4 and IL-10 levels were similar. TGF-beta was further augmented in IL-12/IFN-gamma double-KO mice. Thus, in contrast to L. major, the TGF-beta response was exacerbated whereas type 2 cells were not expanded during L. chagasi infection of IL-12KO mice. We conclude that L. chagasi has an inherent propensity to elicit a prominent TGF-beta response that either suppresses, or is suppressed by, a type 1 response. We propose this be termed a "type 3" immune response, which can antagonize a type 1 response.     

Enhancement of experimental cutaneous leishmaniasis by Leishmania molecules is dependent on interleukin-4, serine protease/esterase activity, and parasite and host genetic backgrounds

Most inbred strains of mice, like the BALB/c strain, are susceptible to Leishmania amazonensis infections and resistant to Leishmania braziliensis infections. This parasite-related difference could result from the activity of an L. amazonensis-specific virulence factor. In agreement with this hypothesis, it is shown here that the intravenous injection of BALB/c mice with L. amazonensis amastigote extract (LaE) but not the L. braziliensis extract confers susceptibility to L. braziliensis infection. This effect was associated with high circulating levels of IgG1 anti-L. amazonensis antibodies and with an increase in interleukin-4 (IL-4) production and a decrease in gamma interferon production by draining lymph node cells. Moreover, the effect was absent in IL-4-knockout mice. The biological activity in the LaE was not mediated by amphiphilic molecules and was inhibited by pretreatment of the extract with irreversible serine protease inhibitors. These findings indicate that the LaE contains a virulence-related factor that (i) enhances the Leishmania infection by promoting Th2-type immune responses, (ii) is not one of the immunomodulatory Leishmania molecules described so far, and (iii) is either a serine protease or has an effect that depends on that protease activity. In addition to being Leishmania species specific, the infection-enhancing activity was also shown to depend on the host genetic makeup, as LaE injections did not affect the susceptibility of C57BL/6 mice to L. braziliensis infection. The identification of Leishmania molecules with infection-enhancing activity could be important for the development of a vaccine, since the up- or downmodulation of the immune response against a virulence factor could well contribute to controlling the infection.     

Leishmania major-infected murine langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis

Leishmania major-infected C57BL / 6 skin-dendritic cells (DC) are activated and release cytokines (including IL-12 p70), and likely initiate protective Th1 immunity in vivo (von Stebut, E. et al., J. Exp. Med. 188: 1547 - 1552). To characterize differences in DC function in mice that are genetically susceptible (BALB / c) and resistant (C57BL / 6) to cutaneous leishmaniasis, we analyzed the effects of L. major on Langerhans cell-like, fetal skin-derived DC (FSDDC) from both strains. BALB / c- and C57BL / 6-FSDDC ingested similar numbers of amastigotes, but did not ingest metacyclic promastigotes. Like C57BL / 6-FSDDC, infection of BALB / c-FSDDC led to up-regulation of MHC class I and II antigens, CD40, CD54, and CD86 within 18 h. L. major-induced BALB / c DC activation also led to the release of TNF-alpha, IL-6 and IL-12 p40 into 18-h supernatants. Infected BALB / c- and C57BL / 6-DC both released small amounts of IL-12 p70 within 72 h. Additional stimulation with IFN-gamma and / or anti-CD40 induced the release of more IL-12 p70 from infected BALB / c-DC than C57BL / 6-DC. Co-culture of control or infected BALB / c- and C57BL / 6-DC with naive syngeneic CD4(+) T cells and soluble anti-CD3 resulted in mixed, IFN-gamma-predominant responses after restimulation with immobilized anti-CD3. Finally, syngeneic L. major-infected DC effectively vaccinated BALB / c mice against cutaneous leishmaniasis. Genetic susceptibility to L. major that results from induction of Th2 predominant immune responses after infection does not appear to reflect failure of skin DC to internalize or respond to parasites, or the inability of BALB / c T cells to mount a Th1 response to DC-associated Leishmania antigens.     

The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice.

The immune response against Leishmania donovani infection has been investigated in one resistant mouse strain (C3H/HeJ) and three susceptible mouse strains (C57BL/6, BALB/c, and B10D2/n). In order to correlate the strain-specific course of infection with the individual T cell response phenotype, the ex vivo cytokine secretion patterns of splenic lymphocytes were assessed by ELISA (interferon-y [IFN-gamma], interleukin-4 [IL-4], IL-10) or by bioassay (IL-2). The strain-dependent differences in the course of infection correlated closely with the potency of T cells to produce IFN-gamma. C3H/HeJ mice produced high amounts of IFN-gamma before and during infection, whereas susceptible mice produced low amounts of IFN-gamma early during L. donovani infection. However, C57BL/6 mice, which recovered from the infection rapidly after the acute stage, developed marked IFN-gamma response within the first 30 days of infection. In contrast, in BALB/c and B10D2/n mice, the IFN-gamma production diminished during the acute stage, and this was associated with a delay in recovery and with subsequent switching into the chronic stage. Interestingly, CD8+ T cells contributed significantly to IFN-gamma production during this phase. In contrast to IFN-y, the levels of IL-4 in response to antigen or mitogen ex vivo were always very low. Moreover, neutralization of endogenous IL-4 in vivo by treatment with soluble murine IL-4 receptor did not result in significant decreases in the parasite burdens in spleen and liver but did cause a decrease in the serum IgE level of L. donovani-infected BALB/c mice. These results confirm that in visceral leishmaniasis a Thl-dominated immune response is protective against the L. donovani parasites and, furthermore, that the capacity to produce IFN-gamma rather than the presence of IL-4 determines the efficacy of the immune response in susceptible mice. The data show that CD8+ T cells represent an important source of IFN-gamma during L. donovani infection in susceptible mice, implying a role for this cell type in healing and development of protective immunity.     

Non-major histocompatibility complex control of antibody isotype and Th1 versus Th2 cytokines during experimental infection of mice with Mycobacterium avium

Infection of different strains of mice with Mycobacterium avium has revealed genetic control of the immunoglobulin isotype induced and of the balance between Th1 and Th2 cytokines. Female BALB/c or C57BL/10 mice were infected intranasally with 10(5) M. avium organisms. The antibody response was measured over 18 weeks by enzyme-linked immunosorbent assay and Western blotting, while numbers of cytokine-producing cells were assessed at 12 to 15 weeks by ELISPOT assay. Upon infection, C57BL/10 mice produced a clear Th1 response with strong gamma interferon (IFN-gamma) production, no interleukin-4 (IL-4), and almost entirely immunoglobulin G2a (IgG2a) antibody. In contrast, BALB/c mice developed T cells producing IL-4, as well as those producing IFN-gamma, while the antibody response was a mixture of IgG1 and IgG2a. Antibodies from BALB/c mice were also able to recognize a greater range of antigens than were C56BL/10 mice. B10D2 mice, which carry the BALB/c major histocompatibility complex haplotype on a C57BL/10 background, followed the C57BL/10 cytokine pattern. Mice infected with Listeria monocytogenes did not show a similar response dichotomy.     

Interleukin-4-independent acceleration of cutaneous leishmaniasis in susceptible BALB/c mice following treatment with anti-CTLA4 antibody

BALB/c mice are susceptible to progressive infection with Leishmania major due to the preferential development of CD4(+) T cells that secrete Th2 cytokines. Although Th2 cell development and susceptibility are disrupted by blockade of CD86 function early in infection, CD28-deficient BALB/c mice remain susceptible to leishmaniasis. We therefore examined whether the alternative CD86 ligand, CTLA4, contributes to the expression of susceptibility. BALB/c mice treated for 2 weeks of infection with anti-CTLA4 monoclonal antibody developed more rapidly progressive disease than sham-treated mice, whereas normally resistant C57BL/6 mice were unaffected. The draining lymph node cells of anti-CTLA4-treated BALB/c mice produced up to sixfold more interleukin-4 (IL-4) and IL-13 than control mice in the first 2 weeks of infection, but IFN-gamma synthesis was reciprocally decreased. Anti-CTLA4 treatment of BALB/c mice pretreated with neutralizing anti-IL-4 antibody or genetically deficient in IL-4 also caused significant worsening of leishmaniasis. Exacerbation in IL-4 KO mice was associated with increased IL-13 and decreased gamma interferon (IFN-gamma) and inducible nitric oxide synthase (iNOS) mRNA expression in vivo. These data indicate that anti-CTLA4 antibody induced earlier and more-polarized Th2 responses in susceptible BALB/c mice infected with L. major. The mechanism of disease worsening was partially IL-4 independent, indicating that increased IL-13 and/or decreased IFN-gamma production may have disrupted nitric oxide-based microbicidal responses. We conclude that CTLA4 significantly modulates Th2 development in murine leishmaniasis and that the Th2-polarizing effects of anti-CTLA4 treatment result in IL-4-independent exacerbation of disease.     

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.     

Cytokine regulation of murine leishmaniasis: interleukin 4 is not sufficient to mediate progressive disease in resistant C57BL/6 mice.

Neutralization of interleukin 4 (IL-4) at the time of infection with Leishmania major allowed susceptible BALB/c mice to heal. Recombinant IL-4, however, had little effect on the course of L. major infection in resistant C57BL/6 mice, nor did coinfection with Nippostrongylus brasiliensis, despite marked elevation of endogenous IL-4 levels.     

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.     

Control of New World cutaneous leishmaniasis is IL-12 independent but STAT4 dependent

Leishmania mexicana, a New World protozoan parasite, induces small, chronic, but non-progressive lesions in C57BL/6 (B6) mice. In this study we investigated the role of IL-12, and subsequent Th1 factors, in controlling cutaneous L. mexicana infection. IL-12 treatment failed to promote disease resolution, suggesting that the inability of mice to heal is not related to a deficiency of endogenous IL-12 production. Surprisingly, L. mexicana-induced cutaneous lesions in wild-type and IL-12p40-deficient mice were indistinguishable, with similar parasite burdens, immune responses, and lesion histopathology. In contrast, iNOS, IFN-gamma, and STAT4-deficient mice developed progressive disease and uncontrolled parasite growth. These results differ dramatically from L. major infection, in which IL-12p40-deficient mice are highly susceptible, with very rapid lesion growth, very large parasite burdens, and the development of a strong Th2 response. These data uncover the existence of an alternate IFN-gamma and iNOS pathway for control of Leishmania lesions, which is IL-12 independent, but which unexpectedly requires STAT4.