The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis

Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.     

IL-4 induces a Th2 response in Leishmania major-infected mice.

The infection of mice with Leishmania major can cause either a fatal disseminated disease or a localized healing disease, depending on the genetic background of the mice. A strong correlation has been shown between disease outcome and the nature of the T cell response, with healer strains developing a Th1-like response and nonhealer strains a Th2-like response. The treatment of nonhealer BALB/c mice with a single dose of an anti-IL-4 antibody, given at the time of infection with L. major, allowed these mice to develop healing Th1-like responses, suggesting that IL-4 is required in BALB/c mice for the differentiation of Th cells into Th2 cells. Anti-IL-4 had to be present during the first 2 wk of infection to have this effect. Anti-IL-4 caused a marked shift from a Th2 to a Th1 pattern of cytokine expression within 4 days, in vivo, and injections of IL-4 had the opposite effect on the early response in healer C3H/HeN mice. These findings demonstrate that IL-4 can induce the development of Th2 response to L. major infection in vivo.     

BALB/c mice bearing a transgenic IL-12 receptor beta 2 gene exhibit a nonhealing phenotype to Leishmania major infection despite intact IL-12 signaling

In BALB/c mice infected with Leishmania major, early secretion of IL-4 leads to a Th2-type response and nonhealing. We explored the role of IL-4-induced down-regulation of the IL-12Rbeta2 chain in the establishment of this Th2 response. First, we showed that the draining lymph nodes of resistant C57BL/6 mice infected with L. major were enriched in CD4+/IL-12Rbeta2 chain+ cells producing IFN-gamma. Next, we demonstrated that BALB/c background mice bearing an IL-12Rbeta2-chain transgene manifested a nonhealing phenotype similar to wild-type littermates despite the persistence of their ability to undergo STAT4 activation. Finally, we found that such transgenic mice display more severe infection than wild-type littermates when treated with IL-12 7 days after infection, and under this condition, the mice display increased Leishmania Ag-induced IL-4 secretion. These studies indicate that although CD4+/IL-12Rbeta2 chain+ T cells are important components of the Th1 response, maintenance of IL-12Rbeta2 chain expression is not sufficient to change a Th2 response to a Th1 response in vivo and thus to allow BALB/c mice to heal L. major infection.     

The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R alpha-chain (IL-4R alpha), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4R alpha(-/-) mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4R alpha(-/-) mice were crossed with BALB/c IL-10(-/-) mice. In contrast to the lack of resistance conferred by IL-4R alpha gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4R alpha(-/-) mice and IL-4R alpha x IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4R alpha(-/-) mice infected with L. major IR173. Production of IFN-gamma was only increased relative to wild-type or littermate controls in IL-4R alpha(-/-) mice with complementary defects in IL-10. Comparisons of IFN-gamma-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-gamma than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-gamma mediated killing.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.     

IL-13 is a susceptibility factor for Leishmania major infection

Leishmania major infection is useful as an experimental model to define factors responsible for the development and maintenance of Th cell immune responses. Studies using inbred mouse strains have identified that the Th1 response characteristic of C57BL/6 mice results in healing, whereas BALB/c mice fail to control the infection due to the generation of an inappropriate Th2 response. We now demonstrate that IL-13 is a key factor in determining susceptibility to L. major infection. Overexpression of IL-13 in transgenic mice makes the normally resistant C57BL/6 mouse strain susceptible to L. major infection even in the absence of IL-4 expression. This susceptibility correlates with a suppression of IL-12 and IFN-gamma expression. Furthermore, using BALB/c mice deficient in the expression of IL-4, IL-13, or both IL-13 and IL-4, we demonstrate that IL-13-deficient mice are resistant to infection and that there is an additive effect of deleting both IL-4 and IL-13.     

The role of IL-12 in maintaining resistance to Leishmania major

IL-12p40 is required for the maintenance of resistance during Leishmania major infection. In this study, we addressed how IL-12 mediates this function. First, we demonstrated that both subunits of IL-12, p40 and p35, were required for continued resistance to L. major. Second, using IL-12, IL-4 doubly deficient mice, we investigated the possibility that IL-12 inhibits IL-4-induced outgrowth of Th2 cells that might compete with Th1 cells. We found that even in the absence of a Th2 response, IL-12 was still required to maintain resistance. Next, using adoptive transfer of Thy-1 disparate CD4(+) T cells from L. major-healed mice, we were able to show that the loss of a protective response in L. major-infected IL-12-deficient mice is linked with the loss of Th1 cells. In contrast, there was an equal recovery of CD4(+) Th1 cells from wild-type and IL-12-deficient mice when transferred into mice that were not challenged with L. major. The ability of Th1 cells to survive regardless of IL-12 levels in the absence of Ag stimulation was confirmed by adoptive transfer studies of CD4(+) Th1 cells from DO11.10 TCR transgenic mice. Taken together, these results indicate that, rather than modulating Th2 responses or optimizing IFN-gamma production, the critical role for IL-12 in maintaining cell-mediated immunity may be to prevent the loss of Th1 cells during a challenge infection.     

Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response

Host resistance to the intracellular protozoan Leishmania major is highly dependent on IL-12 production by APCs. Genetically resistant C57BL/6 mice develop IL-12-mediated Th1 immune response dominated by IFN-gamma and exhibit only small cutaneous lesions that resolve spontaneously. In contrast, because of several genetic differences, BALB/c mice develop an IL-4-mediated Th2 immune response and a chronic mutilating disease. Myeloid differentiation marker 88 (MyD88) is an adaptator protein that links the IL-1/Toll-like receptor family to IL-1R-associated protein kinase. Toll-like receptors recognize pathogen associated molecular patterns and are crucially implicated in the induction of IL-12 secretion by APC. The role of MyD88 protein in the development of protective immune response against parasites is largely unknown. Following inoculation of L. major, MyD88(-/-) C57BL/6 mice presented large footpad lesions containing numerous infected cells and frequent mutilations. In response to soluble Leishmania Ag, cells from lesion-draining lymph node showed a typical Th2 profile, similar to infected BALB/c mice. IL-12p40 plasma level collapses in infected MyD88(-/-) mice compared with infected wild-type C57BL/6 mice. Importantly, administration of exogenous IL-12 rescues L. major-infected MyD88(-/-) mice, demonstrating that the susceptibility of these mice is a direct consequence of IL-12 deficiency. In conclusion, MyD88-dependent pathways appear essential for the development of the protective IL-12-mediated Th1 response against the Leishmania major parasite. In absence of MyD88 protein, infected mice develop a nonprotective Th2 response.     

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.     

Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection

Effector responses induced by polarized CD4+ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha). IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(cre)IL-4Ralpha(-/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Ralpha(-/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Ralpha(-/lox) mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4+ T cells induce protective responses.     

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.     

Reconstitution of Leishmania immunity in severe combined immunodeficient mice using Th1- and Th2-like cell lines.

Leishmania major disseminates in genetically susceptible BALB/c mice to cause fatal disease. Progressive infection has been linked to the failure of parasite-specific Th1, IFN-gamma-producing, CD4+ T lymphocytes to expand and direct macrophage activation and control of intracellular parasitism. In contrast, Th2 CD4+ cell expansion accompanies disease progression. Immunomodulation using CD4 cell depletion at the time of infection results in control of infection and Th1 CD4+ cell expansion. A Th1-like cell line, H1A, was established from the draining lymph nodes of an anti-CD4-pretreated BALB/c mouse infected with L. major, H1A was CD4, TCR(+)-alpha/beta, and released IL-2 and IFN-gamma in response to parasite Ag. A Th2-like cell line, U1A, was established from the lymph node cells of an infected BALB/c mouse that was also CD4, TCR(+)-alpha/beta but released IL-4 and IL-5 after stimulation. Mice with severe combined immunodeficiency were reconstituted with H1A and U1A before infection with L. major. Non-reconstituted mice were unable to restrict parasite growth. Mice reconstituted with H1A healed infection, whereas mice reconstituted with U1A suffered exacerbation of disease. Analysis of spleen cells by flow cytometry confirmed the reconstitution of CD4+ cells in both instances, and stimulation with mitogen established that the lymphokine profile of the donor cells had been maintained during 6 to 8 wk of infection. Histologic analysis of the lesions confirmed migration of donated cells to sites of infection. Neutralization of IFN-gamma in H1A-reconstituted mice and IL-4 in U1A-reconstituted mice reversed the disease phenotype mediated by the two cell lines. These data demonstrate the capacity of CD4+ T cells alone to modulate both positively and negatively the course of leishmaniasis in a lymphokine-dependent manner.     

Genetically resistant mice lacking IL-18 gene develop Th1 response and control cutaneous Leishmania major infection

IL-18 has been shown to play a critical role in the development of a Th1 response and immunity against intracellular pathogens. To determine the role of IL-18 in the development of protective immunity against Leishmania major, we have analyzed the course of cutaneous L. major in IL-18-deficient C57BL/6 mice (IL-18-/-) compared with similarly infected wild-type mice (IL-18+/+). After L. major infection, IL-18-/- mice may develop larger lesions during early phase of infection but eventually will resolve them as efficiently as IL-18+/+ mice. By 2 wk after infection, although Ag-stimulated lymph node cells from L. major-infected IL-18+/+ and IL-18-/- mice produced similar levels of IFN-gamma, those from IL-18-/- mice produced significantly more IL-12 and IL-4. By 10 wk after infection, both IL-18+/+ and IL-18-/- mice had resolved L. major infection. At this time, lymph node cells from both IL-18+/+ and IL-18-/- mice produced IL-12 and IFN-gamma but no IL-4. Furthermore, administration of anti-IFN-gamma Abs to IL-18-/- mice rendered them susceptible to L. major. These results indicate that despite the role IL-18 may play in early control of cutaneous L. major lesion growth, this cytokine is not critical for development of protective Th1 response and resolution of L. major infection.     

Leishmania amazonensis-dendritic cell interactions in vitro and the priming of parasite-specific CD4(+) T cells in vivo

The progressive disease following Leishmania amazonensis infection in mice requires functional CD4(+) T cells, which are primed to a disease-promoting phenotype during the infection. To understand how these pathogenic T cells are generated and the role of dendritic cells (DCs) in this process, we use DCs of susceptible BALB/c and resistant C3H/HeJ mice to examine parasite-DC interactions in vitro as well as the effector phenotype of T cells primed by parasite-exposed DCs in vivo. Our results demonstrate that amastigotes and metacyclics efficiently enter and activate DCs of both genetic backgrounds. Infection with amastigotes fails to induce CD40-dependent IL-12 production, but rather potentiates IL-4 production in BALB/c DCs. Upon transfer into syngeneic recipients, amastigote-exposed BALB/c DCs prime parasite-specific Th cells to produce significantly higher levels of IL-4 and IL-10 than their C3H/HeJ counterparts. Transfer studies with IL-4(-/-) DCs indicate that this enhanced Th2 priming seen in BALB/c mice is partially due to the IL-4 production by amastigote-carrying DCs. These results suggest that L. amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4(+) T cells, and thereby provide a new perspective on the pathogenesis of cutaneous leishmaniasis and protozoan parasite-host interactions in general.