Paul M. Kaye

Leishmaniasis: complexity at the host–pathogen interface

Leishmania is a genus of protozoan parasites that are transmitted by the bite of phlebotomine sandflies and give rise to a range of diseases (collectively known as leishmaniases) that affect over 150 million people worldwide. Cellular immune mechanisms have a major role in the control of infections with all Leishmania spp. However, as discussed in this Review, recent evidence suggests that each host–pathogen combination evokes different solutions to the problems of parasite establishment, survival and persistence. Understanding the extent of this diversity will be increasingly important in ensuring the development of broadly applicable vaccines, drugs and immunotherapeutic interventions.

The role of dendritic cells in the induction and regulation of immunity to microbial infection.

Dendritic cells (DCs) play a critical role in the initiation and regulation of immune responses. Recent advances have begun to uncover the nature and diversity of DC-pathogen interactions and the modulation of DC function by microbial stimuli. Antigen pulsed DCs have also been shown in several infection models to induce high levels of protective immunity and to display immunotherapeutic potential. The study of the function of DCs in the response to infection is thus an exciting and rapidly expanding field with important implications for both fundamental and clinical immunology.

Leishmaniasis: new approaches to disease control

Leishmaniasis is one of the major infectious diseases affecting the poorest regions of the world, but new developments in diagnosis, treatment, and control offer some fresh hope

DENDRITIC CELLS, BUT NOT MACROPHAGES, PRODUCE IL-12 IMMEDIATELY FOLLOWING LEISHMANIA DONOVANI INFECTION

Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN‐γ from NK cells, via a pathway which is dependent upon IL‐12 and IL‐2. IL‐12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL‐12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL‐12‐inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL‐12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL‐12 production following Leishmania infection. IL‐12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL‐12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF‐α production is induced by day 3 post infection. Not only do these data satisfactorily explain the dfferences between in vivo and in vitro data by identifying the cellular source of IL‐12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL‐12 production by macrophages, DC‐T cell clusters provide the microenvironment for initial NK cell activation.

Locally Up-regulated Lymphotoxin α, Not Systemic Tumor Necrosis Factor α, Is the Principle Mediator of Murine Cerebral Malaria

Cerebral malaria (CM) causes death in children and nonimmune adults. TNF-α has been thought to play a key role in the development of CM. In contrast, the role of the related cyto-kine lymphotoxin α (LTα) in CM has been overlooked. Here we show that LTα, not TNFα, is the principal mediator of murine CM. Mice deficient in TNFα (B6.TNFα−/−) were as susceptible to CM caused by Plasmodium berghei (ANKA) as C57BL/6 mice, and died 6 to 8 d after infection after developing neurological signs of CM, associated with perivascular brain hemorrhage. Significantly, the development of CM in B6.TNFα−/− mice was not associated with increased intracellular adhesion molecule (ICAM)-1 expression on cerebral vasculature and the intraluminal accumulation of complement receptor 3 (CR3)-positive leukocytes was moderate. In contrast, mice deficient in LTα (B6.LTα−/−) were completely resistant to CM and died 11 to 14 d after infection with severe anemia and hyperparasitemia. No difference in blood parasite burden was found between C57BL/6, B6.TNFα−/−, and B6.LTα−/− mice at the onset of CM symptoms in the two susceptible strains. In addition, studies in bone marrow (BM) chimeric mice showed the persistence of cerebral LTα mRNA after irradiation and engraftment of LTα-deficient BM, indicating that LTα originated from a radiation-resistant cell population.

Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8 + T-cell responses

CD8+ T cells are essential for long-term, vaccine-induced resistance against intracellular pathogens. Here we show that natural antibodies, acting in concert with complement, are endogenous adjuvants for the generation of protective CD8+ T cells after vaccination against visceral leishmaniasis. IL-4 was crucial for the priming of vaccine-specific CD8+ T cells, and we defined the primary source of IL-4 as a CD11b+CD11clo phagocyte. IL-4 secretion was not observed in antibody-deficient mice and could be reconstituted with serum from normal, but not Btk immune-deficient, mice. Similarly, no IL-4 response or CD8+ T-cell priming was seen in C1qa−/− mice. These results identify a new pathway by which immune complex–mediated complement activation can regulate T-cell-mediated immunity. We propose that this function of natural antibodies could be exploited when developing new vaccines for infectious diseases.

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis.

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4+ T cells producing IFN-γ, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-γ-producing CD8+ T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.

B Cell-Deficient Mice Are Highly Resistant to Leishmania donovani Infection, but Develop Neutrophil-Mediated Tissue Pathology

Resolution of Leishmania infection is T cell-dependent, and B lymphocytes have been considered to play a minimal role in host defense. In this study, the contribution of B lymphocytes to the response against Leishmania donovani was investigated using genetically modified IgM transmembrane domain (μMT) mutant mice, which lack mature B lymphocytes. When compared with wild-type mice, μMT mice cleared parasites more rapidly from the liver, and infection failed to establish in the spleen. The rapid clearance of parasites in μMT mice was associated with accelerated and more extensive hepatic granuloma formation compared with wild-type mice. However, the liver of infected μMT mice also showed signs of destructive pathology, associated with the presence of increased numbers of neutrophils. The role of neutrophils in controlling parasite growth in the viscera was determined by depletion with the mAb RB6-8C5. This treatment led to a dramatic enhancement of parasite growth in both the liver and spleen of μMT and wild-type mice. As assessed by transfer of both normal and chronic-infection serum, Ig protects μMT mice from destructive hepatic pathology, but minimally alters their resistance compared with wild-type mice. However, adoptive transfer of CD4+ and CD8+ T cells into recombinase activating gene 1 (RAG1−/−) recipients, suggested that T cell function was not altered by maturation in a B cell-deficient environment. Taken together, these data suggest an inhibitory role for B lymphocytes in resistance to L. donovani unrelated to the presence or absence of Ig. However, Ig protects μMT mice from the exaggerated pathology that occurs during infection.

The immunopathology of experimental visceral leishmaniasis

Summary:  Experimental murine infection with the parasites that cause human visceral leishmaniasis (VL) results in the establishment of infection in the liver, spleen, and bone marrow. In most strains of mice, parasites are eventually cleared from the liver, and hepatic resistance to infection results from a coordinated host response involving a broad range of effector and regulatory pathways targeted within defined tissue structures called granulomas. In contrast, parasites persist in the spleen and bone marrow by mechanisms that are less well understood. Parasite persistence is accompanied by the failure of granuloma formation and by a variety of pathologic changes, including splenomegaly, disruption of lymphoid tissue microarchitecture, and enhanced hematopoietic activity. Here, we review the salient features of these distinct tissue responses and highlight the varied roles that cytokines of the tumor necrosis factor family play in immunity to this infection. In addition, we also discuss recent studies aimed at understanding how splenomegaly affects the survival and function of memory cells specific for heterologous antigens, an issue of considerable importance for our understanding of the disease‐associated increase in secondary infections characteristic of human VL.

NEUTRALIZATION OF IL-12 DEMONSTRATES THE EXISTENCE OF DISCRETE ORGAN-SPECIFIC PHASES IN THE CONTROL OF LEISHMANIA DONOVANI

IL‐12 plays a key role in stimulating both innate and antigen‐specific immune responses against a number of intracellular pathogens. A neutralizing anti‐IL‐12 monoclonal antibody (mAb) was used to define and compare the role of endogenous IL‐12 in the liver and spleen of mice infected with Leishmania donovani. IL‐12 neutralization both early and late in infection caused delayed resolution of parasite load, a transient decrease in IFN‐γ, IL‐4, TNF‐α and inducible nitric oxide synthase (NOS‐2) production, and suppressed tissue granuloma formation in the liver of genetically susceptible BALB/c mice. In contrast to the liver of BALB/c mice, neutralization of IL‐12 had no effect on parasite burden in the spleen over the first 28 days of infection. However, IL‐12 appeared to be critical for the development of mechanisms which subsequently contain the growth of persistent parasites in this organ in that neutralization of IL‐12 dramatically enhanced parasite growth after day 28 of infection. Following IL‐12 neutralization, the later unchecked growth of parasites in the spleen was coincident with an extensive breakdown of the tissue microarchitecture. Immunohistochemical studies revealed that IL‐12 was largely produced by uninfected cells in L. donovani‐infected BALB/c mice. In contrast, the course of infection in the liver and spleen of genetically resistant CBA/n mice was unaffected by the administration of anti‐IL‐12 mAb. These results suggest that the liver and spleen in susceptible BALB/c mice have different temporal requirements for IL‐12 in controlling L. donovani infection, whereas IL‐12 plays little role in either organ in resistant CBA/n mice. In addition, IL‐12 appears to be involved in the generation of both Th1 and Th2 responses during L. donovani infection in BALB/c mice.