IL-12 is required to maintain a Th1 response during Leishmania major infection

IL-12 initiates Th1 cell development and cell-mediated immunity, but whether IL-12 contributes to the maintenance of a Th1 response is unclear. To address this question, we infected IL-12 p40-/- C57BL/6 mice with Leishmania major, an intracellular protozoan parasite controlled by a cell-mediated immune response, and simultaneously administered IL-12. Whereas untreated p40-/- mice developed an uncontrolled infection, p40-/- mice treated with IL-12 for the first 2 or 4 wk of infection developed a Th1 response and resolved their lesions. However, the induction of this protective Th1 cell response by IL-12 treatment was not associated with long term immunity. We observed that on rechallenge in the absence of IL-12, the mice exhibited a susceptible phenotype. In addition, without rechallenge, lesions in the IL-12-treated p40-/- mice developed several weeks after cessation of IL-12 treatment. In both cases, disease was associated with the loss of a Th1 response and the development of a Th2 response. Our observations are not limited to the C57BL/6 strain, because IL-12 treatment was also unable to provide lasting protection to p40-/- BALB/c mice. Finally, we found that although Th1 cells from healed wild-type C57BL/6 mice adoptively transferred protection to L. major-infected RAG-/- mice, they were unable to protect p40-/- mice. In conclusion, these studies provide the first demonstration that IL-12 is required not only to initiate Th1 cell development but also throughout infection to maintain a Th1 cell response and resistance to L. major.     

The immunology of susceptibility and resistance to Leishmania major in mice

Established models of T-helper-2-cell dominance in BALB/c mice infected with Leishmania major -- involving the early production of interleukin-4 by a small subset of Leishmania-specific CD4+ T cells -- have been refined by accumulating evidence that this response is not sufficient and, under some circumstances, not required to promote susceptibility. In addition, more recent studies in L. major-resistant mice have revealed complexities in the mechanisms responsible for acquired immunity, which necessitate the redesign of vaccines against Leishmania and other pathogens that require sustained cell-mediated immune responses.     

The central memory CD4+ T cell population generated during Leishmania major infection requires IL-12 to produce IFN-gamma

Central memory CD4(+) T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4(+) T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4(+) T cells require IL-12 to produce IFN-gamma, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-gamma in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4(+) T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4(+) T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4(+) T cells.     

The role of IL-12, IL-23 and IFN-gamma in immunity to viruses

IL-12, IL-23 and IFN-gamma form a loop and have been thought to play a crucial role against infectious viruses, which are the prototype of "intracellular" pathogens. In the last 10 years, the generation of knock-out (KO) mice for genes that control IL-12/IL-23-dependent IFN-gamma-dependent mediated immunity (STAT1, IFN-gammaR1, IFNgammaR2, IL-12p40 and IL-12Rbeta1) and the identification of patients with spontaneous germline mutations in these genes has led to a re-examination of the role of these cytokines in anti-viral immunity. We here review viral infections in mice and humans with genetic defects in the IL-12/IL-23-IFN-gamma axis. A comparison of the phenotypes observed in KO mice and deficient patients suggests that the human IL-12/IL-23-IFN-gamma axis plays a redundant role in immunity to most viruses, whereas its mouse counterparts play a more important role against several viruses.     

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.     

LIGHT Is critical for IL-12 production by dendritic cells, optimal CD4+ Th1 cell response, and resistance to Leishmania major

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.     

IL-12 induction of resistance to pulmonary blastomycosis

BACKGROUND: Why severity varies in blastomycosis outbreaks remains unresolved. In experimental pulmonary blastomycosis, susceptibility varied in mouse strains. In susceptible BALB/c the response is Th-2, in immunized, resistance is associated with Th-1. Can susceptibility be redirected by IL-12? Methods, results: BALB/c bronchoalveolar and peritoneal macrophages (PM) were shown deficient in IL-12 production in response to IFN-gamma+LPS. High dose IL-12 (1 microg, subcutaneously) treatment of BALB/c infected intranasally with Blastomyces resulted in enhanced survival (P<0.008). Since IL-12 was poorly tolerated, a new protocol for infected mice, IL-12 0.1 or 0.3 microg, every other day, resulted in minimal toxicity; almost all treated mice survived (P<0.002 vs. controls). When lungs of surviving mice were cultured, the 0.1 microg regimen resulted in fewer (P<0.02) cfu. For weeks after treatment, in vitro IFN-gamma treatment enabled PM Blastomyces killing. After infection spleen cells from IL-12 treated mice produced 4-fold more IFN-gamma and 3-fold less IL-10 in response to Blastomyces. IL-10 abrogated activation of macrophages by IFN-gamma for enhanced Blastomyces killing. Conclusions: A proper IL-12 treatment protocol induces resistance (survival and decreased growth in lungs), low toxicity, macrophage responsiveness to IFN-gamma for killing Blastomyces, up-regulation of IFN-gamma and down-regulation of IL-10 production.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

IL-17 Mediates Immunopathology in the Absence of IL-10 Following Leishmania major Infection

Leishmaniasis, resulting from infection with the protozoan parasite Leishmania, consists of a wide spectrum of clinical manifestations, from healing cutaneous lesions to fatal visceral infections. A particularly severe form of cutaneous leishmaniasis, termed mucosal leishmaniasis, exhibits decreased IL-10 levels and an exaggerated inflammatory response that perpetuates the disease. Using a mouse model of leishmaniasis, we investigated what cytokines contribute to increased pathology when IL-10-mediated regulation is absent. Leishmania major infected C57BL/6 mice lacking IL-10 regulation developed larger lesions than controls, but fewer parasites. Both IFN-γ and IL-17 levels were substantially elevated in mice lacking the capacity to respond to IL-10. IFN-γ promoted an increased infiltration of monocytes, while IL-17 contributed to an increase in neutrophils. Surprisingly, however, we found that IFN-γ did not contribute to increased pathology, but instead regulated the IL-17 response. Thus, blocking IFN-γ led to a significant increase in IL-17, neutrophils and disease. Similarly, the production of IL-17 by cells from leishmaniasis patients was also regulated by IL-10 and IFN-γ. Additional studies found that the IL-1 receptor was required for both the IL-17 response and increased pathology. Therefore, we propose that regulating IL-17, possibly by downregulating IL-1β, may be a useful approach for controlling immunopathology in leishmaniasis.     

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling

Background

Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines.

Methodology/Principal findings

We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T_H1 CD4⁺ and CD8⁺ T cells and a systemic LACK-specific T_H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T_H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T_H1 response.

Conclusions/Significance

This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.     

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44^hiCD4⁺ T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44^hiCD4⁺ T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4⁺ T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T_reg cells.     

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 involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.     

NKT cells mediate organ-specific resistance against Leishmania major infection

Whereas the acquired T cell-mediated protection against intracellular pathogens such as Leishmania major has been well studied in the past, the cells and mechanisms involved in their innate control are still poorly understood. Here, we investigated the role of natural killer T (NKT) cells in a high dose L. major mouse infection model. In vitro, L. major only weakly stimulated NKT cells and antagonized their response to the prototypic NKT cell ligand alpha-galactosylceramide, indicating that L. major partially escapes the activation of NKT cells. NKT cell deficiency as analyzed by subcutaneous infection of Jalpha281-/- mice (lacking invariant CD1d-restricted NKT cells) and CD1-/- mice (lacking all CD1d-restricted NKT cells) led to a transient increase in skin lesions, but did not impair the clinical cure of the infection, NK cell cytotoxicity, the production of IFN-gamma, the expression of inducible nitric oxide synthase, and the control of the parasites in the lymph node. In the spleen, however, NKT cells were required for NK cell cytotoxicity and early IFN-gamma production, they lowered the parasite burden, and exerted bystander effects on Leishmania antigen-specific T cell responses, most notably after systemic infection. Thus, NKT cells fulfill organ-specific protective functions during infection with L. major, but are not essential for parasite control.     

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids—the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes—the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.     

IL-4- and IL-4 receptor-deficient BALB/c mice reveal differences in susceptibility to Leishmania major parasite substrains

Using genetically pure BALB/c mice deficient in IL-4 (IL-4-/-) or IL-4 receptor alpha-chain (IL-4Ralpha-/-), we have observed different disease outcomes to Leishmania major infection depending on the parasite substrain. Infection with L. major LV39 caused progressive, nonhealing ulcers and uncontrolled parasite growth in both IL-4-/- and IL-4Ralpha-/- mice. In contrast, infection with L. major IR173 was partially controlled in IL-4-/- mice but efficiently controlled in IL-4Ralpha-/- mice. Both IL-4-/- and IL-4Ralpha-/- mice infected with either substrain displayed reduced Th2 responses. Surprisingly, IFN-gamma secretion was not up-regulated in the mutant mice, even in the IL-4Ralpha-/- mice, which were resistant to L. major IR173. The lack of increased IFN-gamma production suggests that cytokine cross-regulation may not be operating in this model and that the effective ratios of Th1/Th2 cytokines become more indicative of disease outcome. The partial vs complete resistance to IR173 in IL-4-/- or IL-4Ralpha-/- mice implies that, in addition to IL-4, IL-13 may be involved in disease progression during L. major infection. The results with LV39 infection indicate that yet another unidentified factor is capable of causing susceptibility to L. major in the absence of IL-4 or IL-4 signaling.     

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.     

Folylpolyglutamates in Leishmania major

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.