A live Leishmania major vaccine containing CpG motifs induces the de novo generation of Th17 cells in C57BL/6 mice

Cutaneous leishmaniasis produces open sores that lead to scarring and disfiguration. We have reported that vaccination of C57BL/6 mice with live Leishmania major plus CpG DNA (Lm/CpG) prevents lesion development and provides long‐term immunity. Our current study aims to characterize the components of the adaptive immune response that are unique to Lm/CpG. We find that this vaccine enhances the proliferation of CD4⁺ Th17 cells, which contrasts with the highly polarized Th1 response caused by L. major alone; the Th17 response is dependent upon release of vaccine‐induced IL‐6. Neutralization of IFN‐γ and, in particular, IL‐17 caused increased parasite burdens in Lm/CpG‐vaccinated mice. IL‐17R‐deficient Lm/CpG‐vaccinated mice develop lesions, and display decreased IL‐17 and IFN‐γ, despite normal IL‐12, production. Neutrophil accumulation is also decreased in the IL‐17R‐deficient Lm/CpG‐vaccinated mice but Treg numbers are augmented. Our data demonstrate that activation of immune cells through CpG DNA, in the presence of live L. major, causes the specific induction of Th17 cells, which enhances the development of a protective cellular immunity against the parasite. Our study also demonstrates that vaccines combining live pathogens with immunomodulatory molecules may strikingly modify the natural immune response to infection in an alternative manner to that induced by killed or subunit vaccines.     

The MyD88 protein 88 pathway is differently involved in immune responses induced by distinct substrains of Leishmania major

Host resistance to Leishmania major is highly dependent on the development of a Th1 immune response. The TLR adaptator myeloid differentiation protein 88 (MyD88) has been implicated in the Th1 immune response associated with the resistant phenotype observed in C57BL/6 mice after infection with L. major. To investigate whether the MyD88 pathway is differentially used by distinct substrains of parasites, MyD88^−/− C57BL/6 mice were infected with two substrains of L. major, namely L. major LV39 and L. major IR75. MyD88^−/− mice were susceptible to both substrains of L. major, although with different kinetics of infection. The mechanisms involved during the immune response associated with susceptibility of MyD88^−/− mice to L. major is however, parasite substrain‐dependent. Susceptibility of MyD88^−/− mice infected with L. major IR75 is a consequence of Th2 immune‐deviation, whereas susceptibility of MyD88^−/− mice to infection with L. major LV39 resulted from an impaired Th1 response. Depletion of regulatory T cells (Treg) partially restored IFN‐γ secretion and the Th1 immune response in MyD88^−/− mice infected with L. major LV39, demonstrating a role of Treg activity in the development of an impaired Th1 response in these mice.     

c-Rel promotes type 1 and type 17 immune responses during Leishmania major infection

Recent studies demonstrated the crucial role of c‐Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell‐mediated autoimmune inflammation. We thus wondered whether these opposite functions of c‐Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T‐cell subsets on disease outcome. Here we show that c‐Rel‐deficient (rel^?/?) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN‐γ and IL‐17 production in response to L. major antigens was severely impaired in rel^?/? mice. Reconstitution of Rag1^?/? T‐cell deficient mice with rel^?/? CD4⁺ T cells followed by L. major infection demonstrated that c‐Rel‐deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4⁺ cells in vitro was normal. Notably, a selective defect in IL‐12 and IL‐23 production was observed in rel^?/? DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c‐Rel in myeloid cells is essential for clearance of L. major and that this c‐Rel‐mediated effect is dominant over the lack of Tregs.     

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major

Dendritic cell (DC)‐mediated vaccination against Leishmania major induces a parasite‐specific T helper 1 (Th1) response and long‐lasting protective immunity in susceptible mice. As the cytokine interleukin‐12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross‐presentation of transferred DC via resident DC of the host and cross‐activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC‐mediated vaccination. Co‐culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG‐containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross‐presented to a significant extent in vivo. In contrast, co‐culture of DC with NK cells led to cross‐activation of both cell populations with induction of interferon‐γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major‐susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross‐presentation of conditioned DC after DC‐based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross‐activation with NK cells of the host and NK‐cell‐derived interferon‐γ.     

Selective down-regulation of Th2 immune responses following treatment with antigen-coupled splenocytes

Intravenous injection of antigen-coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen-specific immune responses. Using both well-characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen-coupled splenocytes strongly down-regulated antigen-specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite-specific Th2-like cells. Because administration of antigen-coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co-stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co-stimulatory signals than the priming of Th2 cells.     

The role of IL-12 in the maintenance of an established Th1 immune response in experimental leishmaniasis

IL-12 initiates the development of cell-mediated immunity by promoting the differentiation of naive T cells into the Th1 phenotype, and is essential in the development of a Th1 immune response to the intracellular protozoan parasite, Leishmania major. The present study investigated whether IL-12 is also required for the maintenance and effector function of an established Th1 immune response in L. major -infected mice. While neutralization of IL-12 com promised the ability of a leishmanial antigen-reactive Th1 cell clone to produce IFN-γ in vitro, lymphnode cells taken from 2-week L. major -infected mice were able to secrete IFN-γ in an IL-12-independent manner. However, when a short-term T cell line was established in vitro from lymph node cells, the production of IFN-γ again became IL-12 dependent. These results suggest that other factors may compensate for IL-12 in vivo in promoting IFN-γ production during L. major infection. To directly assess if IL-12 was required in vivo for resistance to L. major, we studied the effect of IL-12 neutralization on both a primary and secondary L. major infection in C3H mice. L. major infection in C3H mice is characterized by the development of a small lesion that heals by 8 weeks, and these animals are resistant to reinfection. As previously reported, administration of anti-IL-12 monoclonal antibody (mAb) during a primary infection led to severe disease. However, mice that had healed from a primary infection with L. major and were treated with anti-IL-12 mAb were as resistant as control animals. These findings suggest that once Th1 cells have developed, their effector function in vivo is independent of IL-12, and that this independence is not due to an intrinsic property of the T cell, but to the microenvironment created by the infection.     

IL‐1‐dependent,IL‐1R1‐independent resistance to gastrointestinal nematodes

IL‐1 null mice are unable to expel the intestinal nematode Trichuris muris; whereas WT littermates exhibit sterile immunity. Intriguingly the essential signalling components IL‐1R1 and IL‐1R accessory protein (AcP) are dispensable for expulsion of this parasite. IL‐1 is thus critical for CD4⁺ Th2‐mediated immunity to T. muris; however, this action is independent of the established IL‐1 signalling receptor. We also present data demonstrating that both IL‐1α and IL‐1β induce measurable effects on T. muris primed cells isolated from IL‐1R1 or IL‐1R AcP null mice. MLN cells from these mice restimulated with parasite antigen proliferated at a greater rate and produced more cytokines in response to exogenous IL‐1. This ability to respond to IL‐1 was restricted to these parasite‐primed cells and importantly was not evident in cells from naïve gene null mice. These in vitro data are consistent with the observed ability of mice with compromised IL‐1 signalling to expel the parasite, bolstering the premise that an alternative IL‐1 signalling mechanism is accessible in the context of an intestinal helminth‐driven Th2 immune response.     

Mast cells have no impact on cutaneous leishmaniasis severity and related Th2 differentiation in resistant and susceptible mice

The genus leishmania comprises different protozoan parasites which are causative agents of muco‐cutaneous and systemic, potentially lethal diseases. After infection with the species Leishmania major, resistant mice expand Th1 cells which stimulate macrophages for Leishmania destruction. In contrast, susceptible mice generate Th2 cells which deactivate macrophages, leading to systemic spread of the pathogens. Th‐cell differentiation is determined within the first days, and Th2 cell differentiation requires IL‐4, whereby the initial IL‐4 source is often unknown. Mast cells are potential sources of IL‐4, and hence their role in murine leishmaniasis has previously been studied in mast cell‐deficient Kit mutant mice, although these mice display immunological phenotypes beyond mast cell deficiency. We therefore readdressed this question by infecting Kit‐independent mast cell‐deficient mice that are Th1 (C57BL/6 Cpa^Cre) or Th2 (BALB/c Cpa^Cre) prone with L. major. Using different parasite doses and intra‐ or subcutaneous infection routes, the results demonstrate no role of mast cells on lesion size development, parasite load, immune cell phenotypes expanding in draining lymph nodes, and cytokine production during murine cutaneous leishmaniasis. Thus, other cell types such as ILCs or T cells have to be considered as primary source of Th2‐driving IL‐4.     

Developmental maturation of innate immune cell function correlates with susceptibility to central nervous system autoimmunity

MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two‐week‐old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid‐derived suppressor cells and plasma‐cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti‐inflammatory IL‐10. When used as APCs, splenocytes from 2‐week‐old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T‐cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2‐week‐old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T‐cell‐mediated CNS autoimmune disease.     

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions

 The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L. major infection is the paradigm for polarized T helper cell (Th) 1 and Th2 differentiation. In this model system a Th1 response characterized by interleukin (IL)-2 and interferon (IFN)-γ secretion leads to self-curing disease, whereas a Th2 response (IL-4, IL-10) leads to nonhealing disease. Numerous manipulations, including the injection of cytokines and of neutralizing anti-cytokine antibodies, cytokine transgene expression, and more recently cytokine and cytokine receptor gene knockout studies, have all provided intriguing new pieces to the still incomplete mosaic of our understanding of the immune response. Some of these findings were clearly unexpected and are still incompletely understood. For instance, based on earlier neutralizing anti-IL-4 monoclonal antibody injection studies, IL-4 gene-disrupted BALB/c mice were expected to be unable to mount the biased Th2 response typical of the IL-4^+/+ wild-type mice and to be able to control their lesions; quite unexpectedly, the BALB/c IL-4 knockout mice remain unable to heal their L. major infection. Based on these unexpected findings, we reexamine the literature in an attempt to resolve this apparent paradox and to relate the large body of experimental findings in the mouse system to that which is known about natural and experimental infections in the human. Received: 18 February 1997 / Accepted: 31 July 1997     

Dietary nucleotides can up-regulate antigen-specific Th1 immune responses and suppress antigen-specific IgE responses in mice

BACKGROUND: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. METHODS: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. RESULTS: The level of production of antigen-specific interferon-gamma by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet. CONCLUSION: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.     

Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires the development of a resistance-promoting CD4⁺ -mediated Th1 response. Epidermal Langerhans cells (LC) are critically involved in the induction of the primary immune response to Leishmania infection. They are able to ingest the parasites, to express MHC class II molecules with extraordinarily long half-life and to activate naive L. major -specific Th cells. Considering these unique properties, we studied the capacity of LC to mediate resistance to L. major in vivo. A single i.v. application of LC that had been pulsed with L. major antigen in vitro induced the protection in susceptible BALB/c mice against subsequent challenges with L. major parasites. Resistance could neither be induced by unpulsed LC, nor by L. major antigen alone or by L. major -pulsed macrophages. Development of resistance was paralleled by a reduced parasite burden and by a shift of the cytokine expression towards a Th1-like pattern. In contrast, control mice developed a Th2 response. In vitro exposure of LC to L. major antigen induced the expression of IL-12 (p40) mRNA. In conclusion, our data demonstrate that LC are able to serve as a natural adjuvant and to induce a protective immune response to L. major infection. This effect is based on the initiation of a Th1-like response that is likely to be mediated by IL-12.     

The development of effector and memory T cells in cutaneous leishmaniasis: the implications for vaccine development

Summary: Leishmania major infections induce the development of a CD4⁺ T‐helper 1 (Th1) response that not only controls the primary infection but also results in life‐long immunity to reinfection. How that immunity is maintained is unknown, although because of the existence of infection‐induced immunity, there has been an assumption that the development of a vaccine against leishmaniasis would be relatively easy. This has turned out not to be the case. One problem has been the finding that a large part of the immunity induced by a primary infection depends upon the presence of persistent parasites. Nevertheless, there are ample situations where immunologic memory persists without the continued presence of antigen, providing the prospect that a non‐live vaccine for leishmaniasis can be developed. To do so will require an understanding of the events involved in the development of an effective protective T‐cell response and, more importantly, an understanding of how to maintain that response. Here, we review work from our laboratory, describing how Th1 cells develop in L. major‐infected mice, the nature of the memory T cells that provide protection to reinfection, and how that information may be utilized in the development of vaccines.     

Re-examination of the immunosuppressive mechanisms mediating non-cure of Leishmania infection in mice

Summary: The interleukin (IL)‐4 driven, polarized T‐helper 2 cell (Th2) response that controls non‐healing infection with Leishmania major in BALB/c mice has long been embraced as the underlying principle with which to consider the pathogenesis of non‐healing and systemic forms of leishmaniasis in humans. The inability, however, to reveal a Th2 polarity associated with non‐curing clinical disease has suggested that alternative cells and cytokines are involved in susceptibility. In this review, various mouse models of non‐curing infection with L. major and other Leishmania species are re‐examined in the context of the suppression mediated by IL‐10 and regulatory T (Treg) cells. These activities are revealed in L. major‐infected BALB/c IL‐4 knockout (KO) and IL‐4Rα KO mice and especially in non‐cure resistant mice that do not default to a Th2 pathway as a result of inherent defects in Th1 differentiation. In contrast to the extreme BALB/c susceptibility arising from an aberrant Th2 response, non‐cure in resistant mice arises from an imbalance in Treg cells that are activated in the context of an ongoing Th1 response and whose primary function may be to suppress the immunopathology associated with persistent antiparasite responses in infected tissues.     

Dietary fish oil impairs induction of γ‐interferon and delayed‐type hypersensitivity during a systemic Salmonella enteritidis infection in rats

Snel J, Born L, van der Meer R. Dietary fish oil impairs induction of γ‐interferon and delayed‐type hypersensitivity during a systemic Salmonella enteritidis infection in rats. APMIS 2010; 118: 578–84. Fish oil that is rich in n‐3 polyunsaturated fatty acids markedly modulates immunological responses. Literature data indicate that the fish oil reduces cellular immunity and therefore impairs resistance to infections. We have investigated how dietary fish oil affects the immune response against a facultative intracellular pathogen, Salmonella enteritidis. Wistar rats were fed a diet containing 16% (w/w) of either fish oil or corn oil. After a 4‐week adaptation period, rats were intraperitoneally challenged with 4 × 10⁵ cfu of S. enteritidis. During the 14‐day infection period, urine was collected on a daily basis. At days 2 and 14, eight rats per group were sacrificed. Urinary nitrate, used as a marker for NO production, was lower on a fish oil diet during days 3–8. At day 2, serum γ‐interferon was 48 ± 7 pg/mL in the fish oil‐fed rats compared with 162 ± 52 pg/mL in the corn oil‐fed rats. No effects were found on living salmonella in liver and spleen. At day 14, as markers of an impaired T‐helper 1 (Th‐1) response, a 38% lower delayed‐type hypersensitivity responses and a lower salmonella‐specific IgG2b were observed in the fish oil‐fed rats. Although here dietary fish oil has affected only immune parameters, this impairment of the innate and Th‐1‐mediated immune response may have implications for the host resistance against other intracellular pathogens.     

Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge

BALB/c mice are susceptible to a high-dose infection of the protozoan Leishmania major, which induces a parasite-specific antibody, Th2-like response, exclusive of a significant and protective cell-mediated Th1 component. We have shown, in contrast, that infection with a low number of parasites induces cell-mediated immunity exclusive of antibody production, and results in resistance to substantial subsequent high-dose infection. Low-dose exposure thus constitutes effective vaccination. In the present study, we analyze lymphokine production by parasite-specific T cells from these low-dose exposed, resistant mice and from normal, susceptible mice following high-dose infection. Two findings stand out. First, the parasite-specific T cells in mice rendered resistant appear not to be in an activated, effector state at the time of parasite challenge, as assessed by lack of lymphokine production on short-term stimulation with parasite antigens, but to be rather in a memory state. Second, the ratio of parasite antigen-dependent production of interferon-γ to that of interleukin-4 by spleen cells of low-dose exposed and normal mice upon high-dose challenge takes a dramatically different course. This ratio is similar in both groups of mice shortly after challenge, but increases dramatically in the resistant and declines dramatically in the control mice over a period of weeks, such that these ratios differ by about 60-fold 12 weeks after the high-dose challenge. In addition, we show that a similar state of resistance occurs following low-dose infection with a more virulent strain of L. major. In toto, our observations suggest that resistance may be generally achievable by low-dose exposure and may be associated with a memory state which, when activated by parasite challenge, results in the evolution of the response over weeks such that the protective, Th1 component becomes ever more dominant over the Th2 component.     

Deficiency of prolactin‐inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice

Although the strategic production of prolactin‐inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4⁺ T cells in peripheral lymphoid tissues and impaired CD4⁺ Th1‐cell differentiation in vitro. In vivo, CD4⁺ T cells from OVA‐immunized, PIP‐deficient mice showed significantly impaired proliferation and IFN‐γ production following in vitro restimulation. Furthermore, PIP‐deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN‐γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4⁺ Th1‐cell‐mediated immunity.     

Development and regulation of cell-mediated immunity in experimental leishmaniasis

The development of a Th1 response is critical for controlling many intracellular pathogens. Our laboratory has focused on the role IL-12 plays in initiating such a Th1 response following infection with the obligate intracellular protozoan, Leishmania. Infection of several mouse strains with L. major is associated with IL-12 production and the development of a Th1 response and resistance, although, interestingly, some species of Leishmania (L. mexicana and L. amazonenesis) fail to initiate a Th1 response in the same mouse strains. Consistent with these observations was our finding that IL-12 is an effective adjuvant for the induction of a Th1 response in leishmaniasis (1). Surprisingly, however, in spite of the fact that following resolution of a primary leishmanial infection there is substantial and long-lived resistance to reinfection, an effective prophylactic or therapeutic vaccine for human leishmaniasis does not exist. Our ability to induce a Th1 response in a primate Leishmania vaccine model, but not protection, suggests that long-term resistance to Leishmania requires more than simply initiating a Th1 response (2). Therefore, we recently expanded our studies to investigate how infection-induced resistance to Leishmania operates. We made the unexpected finding that IL-12 is required for L. major-infected mice to remain immune (3). We are now studying how IL-12 participates in maintaining cell-mediated immunity, and more broadly, how immunologic memory works in L. major-healed mice, as well as defining parasite factors that may block the development of cell-mediated immunity.     

Mast cells are crucial in the resistance against Toxoplasma gondii oral infection

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC‐deficient (W/W^v) mice. We show that in the absence of MCs the resistance of W/W^v mice to oral infection was considerably reduced. W/W^v mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/W^v mice correlated with a delayed Th1‐cell response, since IFN‐γ and IL‐12 levels peaked in the later phase of the infection. In vitro, BM‐derived MCs were able to recognize parasite lysate in a MyD88‐dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/W^v mice reconstituted with BM‐derived MCs from control mice retrieved an early strong Th1‐cell response and specially a significant IL‐12 production. In conclusion, MCs play an important role for the development of a protective immune response during oral infection with T. gondii.