Priming of CD8+ and CD4+ T cells in experimental leishmaniasis is initiated by different dendritic cell subtypes.

The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin- dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

Human primary dendritic cell subsets differ in their IL-12 release in response to Leishmania major infection.

Immunity against leishmaniasis has primarily been studied in experimental infections of mice. It was shown that infected skin dendritic cells (DC) are critical for the induction of protection against this pathogen, and targeting skin DC in vaccination approaches in mice has proven to be successful. However, little is known about the contribution of human DC subsets from the skin to primary immunity against this pathogen. In this study, we have analysed the interaction between different human DC subsets and Leishmania major. Primary human myeloid and monocyte-derived DC ingested the parasite comparable to that of murine skin DC, and this resulted in DC activation and IL-12 release, a cytokine essential for the induction of Th1/Tc1-dependent protection. Interestingly, both Langerhans cells and plasmacytoid DC did not appear to contribute to protection in humans. Thus, in leishmaniasis, both murine and human data suggest that dermal inflammatory DC appear to be superior in promoting protection.

Modulatory Effect of Trypanosoma cruzi Infective Stages in Different Dendritic Cell Populations in vitro.

Trypanosoma cruzi is a protozoan parasite that infects at least 7 million persons in the world (OMS, 2019). In endemic areas, infection normally occurs by vectorial transmission; however, outside, it normally happens by blood and includes congenital transmission. The persistence of T. cruzi during infection suggests the presence of immune evasion mechanisms and the modulation of the anti-parasite response to a profile incapable of eradicating the parasite. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) that patrol tissues with a key role in mediating the interface between the innate and adaptive immune response. Previous results from our lab and other groups have demonstrated that T. cruzi modulates the functional properties of DCs, in vitro and in vivo. During vectorial transmission, metacyclic (m) trypomastigotes (Tps) eliminated along with the insect feces reach the mucous membranes or injured skin. When transmission occurs by the hematic route, the parasite stage involved in the infection is the circulating or blood (b) Tp. Here, we studied in vitro the effect of both infective mTp and bTp in two different populations of DCs, bone marrow-derived DCs (BMDCs) and XS106, a cell line derived from epidermal DCs. Results demonstrated that the interaction of both Tps imparts a different effect in the functionality of these two populations of DCs, suggesting that the stage of T. cruzi and DC maturation status could define the immune response from the beginning of the ingress of the parasite, conditioning the course of the infection.

Epidermal compromise in American cutaneous leishmaniasis.

In American cutaneous leishmaniasis (ACL), Leishmania parasites enter the epidermis of the host via the bite of infected sandflies. Immune responses against the parasite vary from "effective" in localized (LCL) to a state of "selective anergy" in diffuse (DCL) cutaneous leishmaniasis, whereas the intermediate muco-cutaneous form (MCL) is characterized by an exacerbated cell-mediated immunity. We have shown that in LCL epidermis, Langerhans cells (LC) are increased, HLA-DR is universally expressed and intercellular adhesion molecule-1 (ICAM-1) immunoreactivity is distributed in patches. In addition, mRNA for IL-1 beta, IL-8, TNF alpha, TNF beta, and INF gamma may be detected in epidermal sheets by reverse transcriptase followed by polymerase chain reaction (RT-PCR). In contrast, DCL epidermis shows fewer LC than LCL epidermis, and expression of ICAM-1, HLA-DR, and IL-1 beta mRNA cannot be detected. MCL lesions show a mucosal epithelium lacking LC, but ICAM-1 is universally expressed. The clinical manifestations of ACL can be reproduced experimentally in different strains of inbred mice. In healthy mice, we have shown a positive correlation between LC and dendritic epidermal T cells (DETC) numbers. This correlation was not, however, observed in L. mexicana-infected mice, suggesting that infection alters the balance between the two cell types. In addition, agents that modulate LC and DETC cell densities change the development of experimental leishmaniasis. These results suggest that the epidermis is essential in determining the type of immune response that is developed against the Leishmania parasites.

Skin dendritic cells in murine cutaneous leishmaniasis.

Studies of the immunopathogenesis of Leishmania major-induced murine cutaneous leishmaniasis provide a framework for understanding the evolution of L. major infection of skin in humans and the foundation for rationale vaccine design. Experiments in which infection is initiated with "suprap hysiologic" numbers of parasites clearly identify Th-derived type I cytokines as essential participants in macrophage activation and macrophage nitric oxide production as prerequisite for parasite control. Dendritic cells, rather than macrophages, appear to be responsible for L. major-specific Th priming in these studies. Recent studies of murine cutaneous leishmaniasis in a model system in which infection is initiated with lower, more physiologic numbers of parasites confirm many of the important findings obtained in "high dose" inoculation models, but important differences have been noted. The low dose inoculation model should ultimately provide insights into mechanisms that are responsible for dendritic cell recruitment into leishmania lesions, mechanisms that facilitate parasite acquisition by skin dendritic cells and cellular interactions that eventuate in T cell priming and lesion involution.

HIV target cells in Schistosoma haematobium-infected female genital mucosa.

The parasite Schistosoma haematobium frequently causes genital lesions in women and could increase the risk of human immunodeficiency virus (HIV) transmission. This study quantifies the HIV target cells in schistosome-infected female genital mucosa. Cervicovaginal biopsies with and without schistosomiasis were immunostained for quantification of CD4(+) T lymphocytes (CD3, CD8), macrophages (CD68), and dendritic Langerhans cells (S100 protein). We found significantly higher densities of genital mucosal CD4(+) T lymphocytes and macrophages surrounding schistosome ova compared with cervicovaginal mucosa without ova (P = 0.034 and P = 0.018, respectively). We found no increased density of Langerhans cells (P = 0.25). This study indicates that S. haematobium may significantly increase the density of HIV target cells (CD4(+) T lymphocytes and macrophages) in the female genitals, creating a beneficial setting for HIV transmission. Further studies are needed to confirm these findings and to evaluate the effect of anti-schistosomal treatment on female genital schistosomiasis.

Epidermal Langerhans cells are critical for immunoregulation of cutaneous leishmaniasis.

In leishmaniasis, macrophages are known to play a central role as modulators of the specific immune activity. In this article, Heidrun Moll presents evidence for the critical involvement of another component of the skin immune system, the epidermal Langerhans cell. She proposes that Langerhans cells take up parasites in the skin and transport them to the draining lymph node for presentation to T cells and initiation of the specific immune response.

Antigen presentation by epidermal Langerhans cells in experimental cutaneous leishmaniasis.

Cutaneous leishmaniasis is a disease induced by intradermal injection of leishmania promastigotes. Since the first cells the parasite encounters are those of the skin, the involvement of this organ in the early immune response might be relevant to the outcome of the disease. In this study we examined the ability of epidermal langerhans cells (LC) to become infected in vivo and to function as antigen presenting cells during the early hours of infection with Leishmania major. Our experiments showed that LC from mice injected with parasites can present antigen to a leishmania-specific T cell line when LC are obtained as early as four h after infection. The stimulation was specific, since LC from leishmania injected mice did not present antigen to an ovalbumin-specific T cell line nor did LC from ovalbumin-injected mice present antigen to the leishmania specific T cell line. Despite the ability of epidermal LC cells to present antigen, no parasites were detected in the epidermis, suggesting that these cells are not directly involved in establishing an infection.

Increased interleukin 4 (IL-4) receptor expression and IL-4-induced decrease in IL-12 production by Langerhans cells infected with Leishmania major.

Langerhans cells (LC) take up Leishmania major and are critical for the induction of the parasite-specific T-cell response. Their functional activities are regulated by cytokines. We analyzed whether infection of LC with L. major modulates the expression of their cytokine receptors. The expression of the interleukin 4 (IL-4) receptor was increased on infected LC from susceptible mice but not on those from resistant mice. Moreover, IL-4 treatment strongly decreased the lipopolysaccharide-induced IL-12 response of infected LC from susceptible mice. This modulation of IL-4 receptor expression and IL-12 production by infection of LC with Leishmania may contribute to the development of Th2 cells and to susceptibility to infection.

[Leishmania major lipophosphoglycan modulates the expression of receptors involved in parasite internalization in skin Langerhans cells]. / El lipofosfoglicano de Leishmania major modula la expresion de receptores involucrados en la internalización del parasito en celulas de Langerhans de la piel.

Despite the immunological changes recognized to be produced during Leishmania infection and the central role played by Langerhans cells, it is not known whether Leishmaina lipophosphoglycan, the most abundant glycolipid on the parasite surface, affects the functions of Langerhans cells. Here, we provide evidence that exposure of Langerhans cells to Leishmaina (L.) major lipophosphoglycan has consequences for the expression of surface receptors. Down-regulation of receptors involved in host cell-parasite interaction are observed after 4 h exposure of Langerhans cells to lipophosphoglycan. Many of the changes are also induced in Langerhans cells incubated with L. major-conditioned medium, indicating that the observed effects may be mediated by soluble factors released by the parasite into the culture, as it is the case for the carbohydrate moiety of lipophosphoglycan. Taken together, these results indicate that the changes in surface molecule expression induced by the exposure of Langerhans cells to lipophosphoglycan might reflect changes in their signalling functions from the infected skin.

Kinetics of Trypanosoma cruzi infection in guinea-pigs, with special reference to the involvement of epidermal Langerhans' cells in the induction of immunity.

Several studies have confirmed that epidermal Langerhans' cells (LC) play a central role in the induction of skin-related immunological events. In order to assess the role of LC in Chagas' disease, guinea-pigs were infected intradermally with Trypanosoma cruzi, sacrificed at different time-points, and their tissues were processed for routine histology, electron microscopy and immunohistochemistry. Parasitaemia was observed earliest at day 6 p.i. with 2 peaks at days 9 and 28, and disappeared on day 56 p.i. Parasite-specific serum IgG and IgM were first detected on day 12 p.i. The level of IgG gradually increased by day 84 p.i. All the infected guinea-pigs showed significant alterations in the distribution and morphology of epidermal LC during parasitacmia. The number of LC had significantly decreased in the epidermis by day 3 p.i., only returning to normal levels by day 56 p.i., although the number of LC in the underlying dermis increased concomitantly. Parasites were carried to the regional lymph node, where clustering of parasite-laden dendritic cells (DC) with lymphocytes was seen by electron microscopy. This evidence suggests that LC might be involved in antigen presentation in Chagas' disease.

Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major.

Murine epidermal Langerhans cells (LC) have been demonstrated to stimulate a vigorous T cell response to Leishmania major, a cause of human cutaneous leishmaniasis. It was therefore of interest to analyze whether LC can take up viable parasites. Epidermal cells were obtained from mouse ear skin for incubation with L. major and subsequent detection of intracellular parasites by cytochemistry. Freshly isolated LC, but not cultured LC, phagocytosed L. major and the uptake was inhibited by antibodies to the complement receptor type 3. Electron microscopic studies revealed the presence of viable amastigotes within LC. Moreover, with double-labeling techniques, L. major-containing LC could also be detected in infected skin. The results demonstrate that LC can internalize L. major. Since the number of organisms per infected LC remained consistently low, the prime task of LC may not be the promotion of parasite spreading but the presentation of L. major antigen to T cells and, thus, the regulation of the cellular immunity during cutaneous leishmaniasis.

Murine epidermal Langerhans cells do not express inducible nitric oxide synthase.

In Leishmania-infected macrophages (M phi), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to M phi, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and M phi display the same leishmanicidal effector mechanism. Unlike M phi, stimulation of unselected epidermal cells with interferon-gamma/lipopolysaccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in M phi as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine-activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated M phi and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO-independent mechanism appears to be responsible for the control of parasite replication in LC.

Dendritic cell (DC)-based protection against an intracellular pathogen is dependent upon DC-derived IL-12 and can be induced by molecularly defined antigens.

Upon loading with microbial Ag and adoptive transfer, dendritic cells (DC) are able to induce immunity to infections. This offers encouragement for the development of DC-based vaccination strategies. However, the mechanisms underlying the adjuvant effect of DC are not fully understood, and there is a need to identify Ag with which to arm DC. In the present study, we analyzed the role of DC-derived IL-12 in the induction of resistance to Leishmania major, and we evaluated the protective efficacy of DC loaded with individual Leishmania Ag. Using Ag-pulsed Langerhans cells (LC) from IL-12-deficient or wild-type mice for immunization of susceptible animals, we showed that the inability to release IL-12 completely abrogated the capacity of LC to mediate protection against leishmaniasis. This suggests that the availability of donor LC-derived IL-12 is a requirement for the development of protective immunity. In addition, we tested the protective effect of LC loaded with Leishmania homolog of receptor for activated C kinase, gp63, promastigote surface Ag, kinetoplastid membrane protein-11, or Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a. The results show that mice vaccinated with LC that had been pulsed with selected molecularly defined parasite proteins are capable of controlling infection with L. major. Moreover, the protective potential of DC pulsed with a given Leishmania Ag correlated with the level of their IL-12 expression. Analysis of the cytokine profile of mice after DC-based vaccination revealed that protection was associated with a shift toward a Th1-type response. Together, these findings emphasize the critical role of IL-12 produced by the sensitizing DC and suggest that the development of a DC-based subunit vaccine is feasible.

Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface.

To assess the physiologic interactions between the infective stage of Brugia malayi--one of the extracellular parasites responsible for lymphatic filariasis in humans--and the APC with which they come in contact during their development and routes of travel, we have investigated the interaction between the infective stage (L3) of B. malayi and human Langerhans cells (LC) in the skin. Our data indicate that live L3 result in increased migration of LC from the epidermis without affecting the viability of these cells and up-regulation of the IL-18 cytokine involved in LC migration. Live L3 also result in down-regulation of MHC class I and II on the LC cell surface. Additionally, microarray data indicate that live L3 significantly down-regulated expression of IL-8 as well as of multiple genes involved in Ag presentation, reducing the capacity of LC to induce CD4(+) T cells in allogeneic MLR, and thus resulting in a decreased ability of LC to promote CD4(+) T cell proliferation and production of IFN-gamma and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4(+) T cells.

Treatment of two patients with diffuse cutaneous leishmaniasis caused by Leishmania mexicana modifies the immunohistological profile but not the disease outcome.

Two patients with diffuse cutaneous leishmaniasis caused by Leishmania mexicana were treated with two leishmanicidal drugs (pentamidine and allopurinol) combined with recombinant interferon-gamma restoring Th-1 favouring conditions in the patients. Parasites decreased dramatically in the lesions and macrophages diminished concomitantly, while IL-12-producing Langerhans cells and interferon-gamma- producing NK and CD8 + lymphocytes increased in a reciprocal manner. The CD4+/CD8 + ratio in the peripheral blood normalized. During exogenous administration of interferon-gamma the parasites' capacity to inhibit the oxidative burst of the patients' monocytes was abolished. Even though Th-1-favouring conditions were restored, both patients relapsed two months after therapy was discontinued. We conclude that the tendency to develop a disease-promoting Th-2 response in DCL patients is unaffected by, and independent of, parasite numbers. Even though intensive treatment in DCL patients induced Th-1 disease restricting conditions, the disease-promoting immunomodulation of few persistent Leishmania sufficed to revert the immune response.

CD8 alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis.

In the early phase of leishmaniasis three types of potential antigen-presenting cells, including epidermal Langerhans cells (LC), dermal dendritic cells (DC) and inflammatory DC, are localized at the site of infection. Therefore, it has been a central question which cell type is responsible for the initiation of a protective immune response. In the early stage of an anti-Leishmania immune response, detectable Leishmania major antigen was localized in the paracortex of the draining lymph nodes (LN). Characterization of antigen-positive cells showed that L. major co-localized with DC of a CD11c(+) CD8 alpha(-) Langerin(-) phenotype. To determine the area of antigen uptake, dermis or epidermis, and to further define the type of antigen-transporting cells, L. major was inoculated subcutaneously and concurrently LC were mobilized with fluorescein isothiocyanate (FITC). After 3 days, DC carrying L. major antigen were always FITC(-), indicating a dermal and not an epidermal origin. Moreover, addition of L. major antigen to ex vivo isolated CD8 alpha(-) and CD8 alpha(+) DC from the draining LN of L. major-infected C57BL/6 mice demonstrated that both DC subpopulations were able to stimulate antigen-specific T cell proliferation in vitro. Without addition of exogenous antigen only the CD8 alpha(-) Langerin(-) DC were capable of stimulating antigen-specific T cell proliferation. Thus, we demonstrate that CD8 alpha(-) Langerin(-) DC and not LC are the basis of the protective immune response to intracellular L. major parasites in vivo.