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.     

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.     

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.     

Induction of murine T-helper-cell responses to the filarial nematode Brugia malayi.

The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens.     

Interleukin-12 is indispensable for protective immunity against Leishmania major.

Interleukin-12 (IL-12)-deficient mice derived from a strain genetically resistant to infection with Leishmania major were recently shown to be susceptible toward this parasite, developing a strong Th2 response after injection of a large number of parasites. We further investigated the role of IL-12 in L. major infection by studying the responses of mutant mice against smaller numbers of parasites. IL-12-deficient mice infected with only small numbers of parasites showed the progressive lesion development and high parasite burden associated with a polarized Th2 response. Our data show that IL-12 is indispensable for protective immunity against L. major. Even at low inocula, no salvage pathway appears to compensate for the lack of IL-12. However, genetically susceptible BALB/c mice infected with small numbers of parasites were able to resolve lesions and restrict the parasite burden to levels which were 10(5)-fold lower than those in IL-12-deficient mice. In contrast to mutant mice, BALB/c mice mounted a type 1 response against low inocula of L. major. IL-12-deficient BALB/c mice, however, developed a type 2 response. These data emphasize the essential role of IL-12 in resistance against L. major. In addition, this study suggests that in the absence of IL-12, susceptibility to L. major is determined by the inability to induce a Th1 response rather than the development of a Th2 response. Our results are relevant for potential vaccination strategies that use low inocula of infective microorganisms which fail to induce a protective type 1 response at higher inocula.     

CXCR3-/- mice mount an efficient Th1 response but fail to control Leishmania major infection

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using CXCR3-/- C57BL/6 mice. We found that Leishmania major-infected CXCR3-/- mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1-associated IgG2a and significant production of IFN-gamma and IL-12 by the draining lymph node cells, restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non-healing lesions. Furthermore, the inability of CXCR3-/- mice to control cutaneous L. major growth was associated with fewer CD4+ and CD8+ T cells and significantly lower levels of IFN-gamma in their lesions as compared to CXCR3+/+ mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major. Furthermore, they also suggest that the susceptibility of CXCR3-/- mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFN-gamma at the site of infection rather than to their inability to mount a parasite-specific Th1 response.     

T-helper-cell cytokines in the early evolution of murine Lyme arthritis.

Genetic susceptibility to murine Lyme arthritis has been correlated with the dominance of T-helper (Th1)- or Th2-cell-associated cytokines. To determine when commitment of the Th cell phenotype occurs, we examined the kinetics of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production by lymph node T cells of disease-susceptible C3H/HeN and disease-resistant BALB/c mice from days 2 through 30 of infection, a period encompassing the evolution of disease and early regression. BALB/c mice produced more IFN-gamma on day 2 of infection than did C3H/HeN mice, whereas IL-4 was first detected on day 14. In contrast, only IFN-gamma could be detected in C3H/HeN mice, and the levels steadily increased from day 2 to surpass those seen in BALB/c mice by day 14 of infection. Despite the difference in cytokine profiles, both BALB/c and C3H/HeN mice developed comparable arthritis assessed at 14 days of infection. Arthritis regressed by day 30 in BALB/c mice but persisted in C3H/HeN mice. These studies are the first to demonstrate that the Th2 response to Borrelia burgdorferi infection of BALB/c mice is preceded by a Th1 cytokine response. Moreover, the timing of the appearance of IL-4 suggests that its primary effect is not in preventing disease, as suggested by others, but, rather, in hastening the resolution of inflammation. The implications of these findings for the orchestration of host defense against B. burgdorferi infection are discussed.     

Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis.

Leishmaniae are protozoans which, depending upon both the host and parasite species, can cause either a healing or nonhealing infection. While C57BL/10 mice are able to heal following infection with Leishmania major, they fail to heal following infection with Leishmania amazonensis. In order to address the role of Th1 and Th2 cell responses in the outcome of these infections in C57BL/10 mice, gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production was assessed. While cells from L. major-infected C57BL/10 mice produced high levels of IFN-gamma, cells from L. amazonensis-infected animals produced little or no IFN-gamma. On the other hand, IL-4 was produced only by cells from L. amazonensis-infected C57BL/10 mice, but this production was restricted to the first few weeks of infection. Later in infection, when lesions were evident, no IL-4 was detected. Treatment of BALB/c mice with a monoclonal antibody (11B11) directed against IL-4 induced a dramatic reduction in L. amazonensis lesions. This reduction was associated with a decrease in IL-4 levels and an increase in IFN-gamma production. However, only a slight reduction in lesion sizes and parasite numbers was observed when anti-IL-4-treated C57BL/10 mice were infected with L. amazonensis. These results suggest that IL-4 may have an important role in mediating susceptibility to L. amazonensis in BALB/c mice, as previously demonstrated for L. major. More importantly, however, the data suggest that susceptibility to L. amazonensis in C57BL/10 mice is due to the absence of a Th1 cell response, rather than to the presence of a Th2 cell response.     

Transgenic Expression of CXCR3 on T Cells Enhances Susceptibility to Cutaneous Leishmania major Infection by Inhibiting Monocyte Maturation and Promoting a Th2 Response

Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3^Tg) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3^Tg mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3^Tg mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.     

Stimulation of B-cell lymphopoiesis by interleukin-7 leads to aggravation of murine leishmaniasis.

The effect of recombinant interleukin-7 (IL-7) on the clinical course of murine leishmaniasis and the development of the accompanying immune response was investigated. Previously, IL-7 has been shown to possess stimulatory capacity for different cell types of the immune and haematopoietic system critically involved in the defence against Leishmania major (L. major), such as macrophages which are activated for the elimination of the parasite by IL-7. In contrast to these in vitro data, the present study indicates that treatment of genetically susceptible BALB/c mice with IL-7 at the onset of the infection leads to enhanced lesion development and a significantly accelerated death of the animals. This was correlated with a 40-fold increased parasite burden in spleens and lymph nodes. While the specific antibody response against L. major was not altered and lymphocytes of IL-7-treated mice produced comparable amounts of the T-helper type-2 (Th2) cytokines IL-4 and IL-10, less interferon-gamma (IFN-gamma) was measurable after antigenic stimulation of lymph node and spleen cells in vitro. One of the major changes appearing by the first week after infection in IL-7-treated mice was the increase of the total cell number in spleen and lymph nodes draining the local infection. Analysis of the cellular composition revealed that the enhanced cellularity was predominantly due to a rise in the B-cell compartment. Since antigen presentation by B cells has been implicated in the development of Th2 cells, the disease-aggravating activity of IL-7 is thought to be primarily due to augmentation of B lymphopoiesis.     

Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major

Protection against Leishmania major is dependent on IL-12 release from L. major-infected dendritic cells (DC) that induce IFN-gamma-producing Th1/Tc1 cells. IL-27, a novel member of the IL-12 family, is a heterodimer composed of p28 and IL-12p40-related Epstein-Barr virus-induced gene 3 (EBI3), and was shown to be produced by DC. In this study, we utilized EBI3-deficient mice to investigate the role of IL-27 in leishmaniasis using physiological low-dose infections that mimic natural transmissions. Lesions in EBI3(-/-) mice were significantly larger between weeks 3 and 10 post infection, reaching up to approximately threefold increased lesion volumes compared to wild types. In parallel, dermal lesions of EBI3(-/-) mice contained greater parasite numbers, reaching a peak load that was 2-log higher than in C57BL/6 mice. However, lesions in EBI3(-/-) and wild-type mice resolved after 12 weeks. At early time points, the antigen-specific cytokine response in EBI3(-/-) lymph nodes showed increased levels of IL-4, IL-10 and IL-13 and decreased IFN-gamma production. IL-27 production was restricted to the DC population, since the majority of EBI3 expression in lymph nodes of infected mice was found in CD11c(+) cells. In conclusion, our data show that DC-derived IL-27 is critical for the timely initiation of efficient anti-parasite Th1 immunity early in infections.     

Stimulation of hematopoiesis by the Fms-like tyrosine kinase 3 ligand restores bacterial induction of Th1 cytokines in thermally injured mice

Patients with large burn injuries are susceptible to opportunistic infections due to impaired functions of multiple effector cells of innate immunity and acquired immunity, including macrophages, dendritic cells (DC), natural killer (NK) cells, and T cells. The ability of a host to produce Th1 cytokines, such as gamma interferon (IFN-gamma) and interleukin-12 (IL-12), upon infectious challenge is also impaired after burn injury. Stimulation of hematopoiesis, to regenerate new immune cells, may be an effective strategy for improving resistance to infections after severe burn trauma. Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that stimulates the expansion and differentiation of NK cells and DC. Using a mouse model, we tested the hypothesis that Flt3L treatments after burn injury stimulate the production of functional effector cells of innate immunity and restore appropriate Th1 cytokine responses to Pseudomonas aeruginosa, a common source of pneumonia and wound infections in burn victims. Flt3L increased splenic cellularity in sham (uninjured) and burned mice and increased the numbers of NK cells (DX5(+)) and DC (CD11c(+)). In response to P. aeruginosa, significant increases in the serum IFN-gamma levels and the numbers of splenic IFN-gamma-producing DC, NK cells, and T cells were observed in Flt3L-treated burned mice compared to the values obtained for untreated burned mice. The splenic levels of IL-12 and IL-15 mRNAs and the IL-12 and IL-15 receptors were also increased. In addition, Flt3L treatment restored the ability of splenic cultures prepared from burned mice to produce IFN-gamma and IL-12 after in vitro challenge with P. aeruginosa. Flt3L may have potential for restoring NK cell and DC functions and improving immunity after burn injury.     

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.     

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.     

An in vitro model for infection with Leishmania major that mimics the immune response in mice.

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).     

Administration of plasmids expressing interleukin-4 and interleukin-10 causes BALB/c mice to induce a T helper 2-type response despite the expected T helper 1-type response with a low-dose infection of Leishmania major

BALB/c mice are susceptible to developing an infection with Leishmania major as a result of a fatal T helper 2 (Th2)-type response. However, mice infected with a low dose of parasites are reported to be able to overcome the lesions associated with a T helper 1 (Th1)-type response. To clarify why a difference in the dose of parasites induces a difference in the polarization of the Th phenotype, we first attempted to measure cytokine production. Soon after infection, the mice given high doses of parasites produced elevated levels of both Th1 [interferon-gamma (IFN-gamma)] and Th2 [interleukin (IL)-4 and IL-10] cytokines. However, when assessed at 1 and 2 weeks after infection, no significant difference in the balance of Th1 and Th2 cytokines could be detected between mice infected with low or high doses of L. major. These results support the notion that the Th2 cytokine levels at an early phase of infection could be a key factor for the induction of a Th2 response. In order to assess the efficacy of Th2 cytokines, the mice infected with low doses of L. major were co-administered IL-4 plasmid and IL-10 plasmid. Consequently, the mice (which originally exhibited a Th1 response) showed progressive disease and developed a Th2 response. However, administration of these plasmids at 7 days postinfection could not alter the Th polarization. Furthermore, production of IL-12 from the spleen cells stimulated by L. major was suppressed in the presence of IL-4 and IL-10. These results strongly suggest that the susceptibility to L. major in BALB/c mice depends on the persistence of Th2 cytokine levels at an early phase of infection.