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.     

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.     

Expression of T-cell-associated serine proteinase 1 during murine Leishmania major infection correlates with susceptibility to disease.

The expression of T-cell-associated serine proteinase 1 (MTSP-1) in vivo during Leishmania major infection was analyzed in genetically resistant C57BL/6 mice and in genetically susceptible BALB/c mice. Using a monoclonal antibody as well as an RNA probe specific for MTSP-1 to stain tissue sections, we found T cells expressing MTSP-1 in skin lesions and spleens of mice of both strains. In skin lesions, MTSP-1-positive T cells could be detected as early as 3 days after infection. Most importantly, the frequency of T cells expressing MTSP-1 was significantly higher in susceptible BALB/c mice than in resistant C57BL/6 mice. These findings suggest that MTSP-1 is associated with disease-promoting T cells and that it may be an effector molecule involved in the pathogenesis of cutaneous leishmaniasis.     

TH1 and TH2 T-cell subsets are differentially activated by macrophages and B cells in murine leishmaniasis.

The role of antigen-presenting cells in the differential expansion of TH1 and TH2 T cells in murine leishmaniasis was investigated. In general, macrophages preferentially induced gamma interferon and interleukin-2 secretion by syngeneic Leishmania-specific T cells, whereas B cells were more efficient in activating interleukin 4 production. B cells from susceptible BALB/c mice were better in inducing TH2 responses than B cells from resistant C57BL/6 mice, whereas macrophages from C57BL/6 mice were superior to BALB/c macrophages in inducing TH1 responses.     

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.     

Comparative study of American cutaneous leishmaniasis and diffuse cutaneous leishmaniasis in two strains of inbred mice.

Two Leishmania strains, AZV (isolated from a typical case of American cutaneous leishmaniasis) and AMP (from a case of diffuse cutaneous leishmaniasis), were studied in C57BL/6 and BALB/c mice. After infection with 10(4) amastigotes of either strain, C57BL/6 mice developed self-resolving lesions lasting 20 to 23 weeks and showed both delayed hypersensitivity response to leishmanial antigen and specific agglutinating antibodies. On the other hand, BALB/c mice infected with 10(4) AZV or AMP amastigotes developed chronic, large, ulcerated lesions and showed impaired cellular and humoral responses to the parasite. When BALB/c and C57BL/6 mice received 10(2) AMP amastigotes, patterns of infection were similar to those observed after inoculation of 10(4) amastigotes. In vitro studies revealed that spleen cells from AZV- or AMP-infected C57BL/6 mice showed an increased DNA-synthetic response to leishmanial antigen, concanavalin A, and phytohemagglutinin. Spleen cells from AZV- or AMP-infected BALB/c mice showed an increased response to concanavalin A and diminished responses to leishmanial antigen, phytohemagglutinin, and lipopolysaccharide.     

Cutaneous leishmaniasis: co-ordinate expression of granzyme A and lymphokines by CD4+ T cells from susceptible mice.

We have recently demonstrated that the frequency of T cells expressing granzyme A is significantly higher in skin lesions and spleens of susceptible BALB/c mice compared with resistant C57BL/6 mice infected with Leishmania major, a cause of human cutaneous leishmaniasis. In the present study, we have performed in vitro studies to characterize the subpopulation, the antigen responsiveness and the lymphokine production pattern of granzyme A-expressing T cells in L. major-infected mice. Using a limiting dilution system for functional analysis of selected T cells at the clonal level, we could show that granzyme A activity in infected BALB/c mice can be assigned to L. major-reactive CD4+ T cells secreting interleukin-2 (IL-2) and IL-4. Granzyme A production was most pronounced in the early phase of infection. On the other hand, granzyme A expression could not be detected in C57BL/6-derived T cells responding to L. major. The data support the suggestion that granzyme A is produced by L. major-responsive CD4+ T cells facilitating lesion formation and the dissemination of infection.     

Differential susceptibility to acute Trypanosoma cruzi infection in BALB/c and C57BL/6 mice is not associated with a distinct parasite load but cytokine abnormalities

Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome-related cytokines, the in vitro features of T. cruzi infection in peritoneal macrophages and the immunophenotype of thymic cells. The greater disease severity of T. cruzi-infected C57BL/6 mice was not linked to an increased parasite load, as parasitaemia, myocardial parasite nests and amastigote counts in peritoneal macrophages were not different from those in BALB/c mice. Cortical thymocyte loss was accompanied by the presence of apoptotic bodies and fragmented nuclear DNA, whereas fluorocytometric analysis at 17 days postinfection (p.i.) revealed a more pronounced loss of CD4+ CD8+ cells in C57BL/6 mice. This group displayed higher levels of TNF-alpha on days 14 and 21 p.i., in the presence of lower IL-1beta and IL-10 concentrations by days 14 and 21, and days 7 and 14 p.i., respectively. Day-21 evaluation showed higher concentrations of nitrate and TNF-alpha soluble receptors in C57BL/6 mice with no differences in IFN-gamma levels, with respect to the BALB/c group. Increased morbidity of C57BL/6 T. cruzi-infected mice does not seem to result from an aggravated infection but from an unbalanced relationship between pro- and anti-inflammatory mediators.     

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

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

The site of Leishmania major infection determines disease severity and immune responses

Inbred strains of mice infected with Leishmania major have been classified as genetically resistant or susceptible on the basis of their ability to cure their lesions, the parasite burden in the draining lymph nodes, and their type of T helper cell immune responses to the parasite. Using the intradermal infection at the base of the tail and the ear pinna, we compared for the first time the above-mentioned parameters in six strains of mice infected with metacyclic promastigotes, and we show that the severity of disease depends greatly on the site of infection. Although the well-documented pattern of disease susceptibility of BALB/c and C57BL/6 mice described for the footpad and base-of-the-tail models of leishmaniasis were confirmed, C3H/HeN and DBA/2 mice, which are intermediate and susceptible, respectively, in the tail and other models, were resistant to ear infection. Moreover, in the CBA/H, C3H/HeN, C57BL/6J, and DBA/2 mouse strains, there was little correlation between the pattern of cytokines produced and the disease phenotype observed at the ear and tail sites. We conclude that the definition of susceptibility and the immune mechanisms leading to susceptibility or resistance to infection may differ substantially depending on the route of infection.     

MURINE CUTANEOUS LEISHMANIASIS: RESISTANCE IN RECONSTITUTED NUDE MICE AND SEVERAL F1 HYBRIDS INFECTED WITH LEISHMANIA TROPICA MAJOR

After cutaneous injection of promastigotes of an isolate of the intramacrophage protozoan parasite, Leishmania tropica major, mouse strains develop chronic cutaneous lesions or show a resolving pattern of disease. On this basis, they can be classified as resistant (e.g. CBA/H and C57BL/6) or susceptible (e.g. BALB/c, BALB/c.H-2^b and BALB/c.H-2^k). Hypothymic nude (nu/nu) mice of either BALB/c, CBA/H or C57BL/6 genotype are susceptible to chronic disease. However, nude mice of these genotypes, including BALB/c, are resistant to chronic cutaneous leishmaniasis when injected at the time of parasite challenge with small numbers of H-2 compatible lymphoid cells from normal mice. Nude mice remain susceptible when injected with fully H-2 incompatible cells. Using cells from H-2 mutant mice for reconstitution of resistance in C57BL/6.nu/nu mice, evidence was obtained that I region compatibility is necessary for cells to mediate host-protective effects. Cells from chronically-diseased BALB/c mice do not have protective effects in BALB/c.nu/nu mice at any cell dose and will abrogate the resistance-promoting effect of lymphoid cell populations from chronically-diseased BALB/c.H-2^k and BALB/c.H-2^b mice can be demonstrated when assayed at certain cell doses in H-2 compatible CBA/H.nu/nu and C57BL/6.nu/nu mice, respectively. The data suggest that chronically-diseased (genetically-susceptible) mice contain a mixture of resistance-promoting and disease-promoting T cells in their peripheral lymphoid organs and that expression of the resistance-promoting subset can occur in nude mice of resistant genotype. Previous data have indicated that Lyl⁺2^? T cells are efficient mediators of both T cell-dependent activities. No evidence for the operation of disease-promoting or resistance-promoting antibodies in perpetuation or resolution of disease has been obtained in extensive serum transfer experiments. Some discrepancies exist in the literature on the question of the dominance of susceptibility or resistance in F₁ hybrid mice. A re-examination of susceptibility/resistance in F₁ hybrids between BALB/c and several other parental strains was undertaken using cloned pathogenic promastigotes derived from a heterogeneous L. t. major isolate in order to reduce effects of parasite heterogeneity in the analysis. Resistance was dominant in some but not all F₁ hybrids, with most showing a delayed healing pattern of disease relative to the resistant parental strain. Despite the use of genetically-homogeneous parasites, the analysis was complicated by variability within groups of F₁ hybrid mice as well as between males and females and between F₁ hybrids of reciprocal crosses. A hypothesis based on antigen and H-2 expression on infected macrophages is advanced to account for the balance between the effects of resistance-promoting and disease-promoting Lyl⁺2^? T cells in mice of various genotypes.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

Amastigote load and cell surface phenotype of infected cells from lesions and lymph nodes of susceptible and resistant mice infected with Leishmania major

Cells of the dendritic cell (DC) lineage, by their unique ability to stimulate naive T cells, may be of crucial importance in the development of protective immune responses to Leishmania parasites. The aim of this study was to compare the impact of L. major infection on DCs in BALB/c (susceptible, developing Th2 responses), C57BL/6 (resistant, developing Th1 responses), and tumor necrosis factor (TNF)(-/-) C57BL/6 mice (susceptible, developing delayed and reduced Th1 responses). We analyzed by immunohistochemistry the phenotype of infected cells in vivo. Granulocytes (GR1(+)) and macrophages (CD11b(+)) appear as the mainly infected cells in primary lesions. In contrast, cells expressing CD11c, a DC specific marker, are the most frequently infected cells in draining lymph nodes of all mice tested. These infected CD11c(+) cells harbored a particular morphology and cell surface phenotype in infected C57BL/6 and BALB/c mice. CD11c(+) infected cells from C57BL/6 and TNF(-/-) C57BL/6 mice displayed a weak parasitic load and a dendritic morphology and frequently expressed CD11b or F4/80 myeloid differentiation markers. In contrast, some CD11c(+) infected cells from BALB/c mice were multinucleated giant cells. Giant cells presented a dramatic accumulation of parasites and differentiation markers were not detectable at their surface. In all mice, lymph node CD11c(+) infected cells expressed a low major histocompatibility complex II level and no detectable CD86 expression. Our results suggest that infected CD11c(+) DC-like cells might constitute a reservoir of parasites in lymph nodes.     

Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes

Using a metacyclic promastigote ear infection model of cutaneous leishmaniasis, we examined the phenotype, parasite load, and cytokine production of dendritic cells in the skin and draining lymph nodes of resistant C57BL/6J and susceptible BALB/c mice. Five dendritic cell populations were isolated from the skin and lymph nodes, and the main difference between the groups of mice was an increased number of plasmacytoid dendritic cells in the lymph nodes of the susceptible mice. Although similar cell types were present in the skin emigrants of both strains, there was a 10-fold larger number of cells in BALB/c mouse skin early in infection than in C57BL/6J mouse skin. None of the dendritic cells in the lymph nodes harbored parasites until 3 weeks after infection, with the Langerhans cells having the largest load and the plasmacytoid dendritic cells having the smallest load but the longest lasting infection. Although parasites could be detected in the lymph nodes a few hours after infection, none of the skin emigrants harbored parasites, indicating that they are not the vehicle that ferries the parasites from the skin to the lymph nodes. The presence of larger numbers of plasmacytoid cells in infected BALB/c mice, the more protracted infection of these cells, and their production of alpha interferon point to a complex and important role for the plasmacytoid cells in leishmaniasis.     

Induction of tumor necrosis factor alpha by Leishmania infantum in murine macrophages from different inbred mice strains.

The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor alpha (TNF alpha) in murine macrophages. Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNF alpha production with the ability to control parasite growth and replication. Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lshr), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8:1. No significant differences in the percentages of infected peritoneal cells of Lshs versus Lshr mice were observed until 72 h of in vitro culture. On the contrary, Kupffer cells from Lshr mice inhibited Leishmania replication. Peritoneal macrophages of resistant mice produced significantly higher amounts of TNF alpha as compared to susceptible mice. TNF alpha production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite. No TNF alpha was found in supernatants of infected Kupffer cells from all the strains tested. The ability of macrophages from susceptible or resistant mice strains to produce TNF alpha after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro.     

Macrophage-T cell interaction in experimental visceral leishmaniasis: failure to express costimulatory molecules on Leishmania-infected macrophages and its implication in the suppression of cell-mediated immunity

The most important immunopathological consequence of infection with Leishmania seen in murine and human hosts is the suppression of T cell-mediated immune responses to both mitogens and leishmanial antigens. It has been suggested that this suppression is mediated by macrophages, either by defective antigen processing and presentation or by the elaboration of suppressive mediators like prostaglandins. Optimum activation of T helper cells requires not only T cell receptor occupancy by the antigen-Ia complex, but also costimulatory signals provided by the antigen-presenting cells. We investigated the status of several costimulatory molecules on infected macrophages from both genetically susceptible BALB/c and resistant C57BL/6 mice. Our results demonstrate that upon parasitization, the macrophages become unable to deliver costimulatory signals to T helper cells, and that this effects is mediated by prostaglandins, as the inhibition of its synthesis by indomethacin recovered the defect. Upon infection with L. donovani, B7-1 expression was decreased, while ICAM-1 was marginally increased in BALB/c macrophages and there was no significant change in the expression of B7-1 and ICAM-1 in Leishmania-infected C57BL/6 macrophages. Expression of VCAM-1 did not change during infection. This selective alteration in the expression of costimulatory molecules on L. donovani-infected BALB/c macrophages was caused by the living parasite, as shown by the fact that killing of the parasites by stibogluconate led to no alteration in the levels of costimulatory molecules. We found that the change in B7-1 expression on the surface of infected macrophages resulted in the inhibition of delayed-type hypersensitivity-mediating functions of T helper cells from BALB/c mice. The results described in this study not only throw light on the possible mechanism of leishmanial pathogenesis, but also open up the possibility of immunotherapy of leishmaniasis by selective manipulation of costimulatory molecules.     

Experimental cutaneous leishmaniasis: transmission electron microscopy of the inoculation site

Tissue response against inoculation of Leishmania (Leishmania) amazonensis promastigotes in the hind footpad was quite different between two strains of mice: in BALB/c animals there was parasitism of perineurial cells by the 8th week post inoculation (WPI) and heavy parasitism of macrophages, as well as degenerated extracellular parasites close to collagen fibers at the 39th WPI, whereas in C57Bl/6j mice there was heavy parasitism of macrophages at 6th WPI, dermal vessels with high endothelial cell at 21st WPI and well preserved intracellular amastigote forms by 51st WPI. In both animals there was no parasitism of keratinocytes or Langerhans cells. Thus BALB/c mice were useful as an experimental model for diffuse cutaneous leishmaniasis and showing a new feature, parasitism of perineurial cells, whereas C57Bl/6j animals show hypersensitivity signs, together with a few preserved parasites, only late in the course of infection. From a morphological point of view, there were no differences in macrophages, or in the interaction between this target cell and the parasite, between the animal models studied. This suggests that the difference in the response of the hosts towards the parasite could depend on the way in which they activate a cellular, i.e. lymphocyte mediated immune, response.