Christian R. Engwerda

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.

Balancing immunity and pathology in visceral leishmaniasis.

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ‐specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell‐mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)‐10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38+ stromal cells, and IL‐10 promotes impaired DC migration into T‐cell areas with consequent ineffective T‐cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL‐10‐producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.

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.

Defective CCR7 expression on dendritic cells contributes to the development of visceral leishmaniasis.

Interaction between dendritic cells (DCs) and T cells is essential for the generation of cell-mediated immunity. Here we show that DCs from mice with chronic Leishmania donovani infection fail to migrate from the marginal zone to the periarteriolar region of the spleen. Stromal cells were fewer, which was associated with loss of CCL21 and CCL19 expression. The residual stromal cells and endothelium produced sufficient CCL21 to direct the migration of DCs transferred from naïve mice. However, DCs from infected mice had impaired migration both in naïve recipients and in vitro, in response to CCL21 and CCL19. Defective localization was attributable to tumor necrosis factor-α–dependent, interleukin 10–mediated inhibition of CCR7 expression. Effective immunotherapy was achieved with CCR7-expressing DCs, without the need to identify protective Leishmania antigens. Thus defective DC migration plays a major role in the pathogenesis of this disease and the immunosuppression is mediated, at least in part, through the spatial segregation of DCs and T cells.

Parasite-dependent expansion of TNF receptor II-positive regulatory T cells with enhanced suppressive activity in adults with severe malaria.

Severe Plasmodium falciparum malaria is a major cause of global mortality, yet the immunological factors underlying progression to severe disease remain unclear. CD4+CD25+ regulatory T cells (Treg cells) are associated with impaired T cell control of Plasmodium spp infection. We investigated the relationship between Treg cells, parasite biomass, and P. falciparum malaria disease severity in adults living in a malaria-endemic region of Indonesia. CD4+CD25+Foxp3+CD127lo Treg cells were significantly elevated in patients with uncomplicated (UM; n = 17) and severe malaria (SM; n = 16) relative to exposed asymptomatic controls (AC; n = 10). In patients with SM, Treg cell frequency correlated positively with parasitemia (r = 0.79, p = 0.0003) and total parasite biomass (r = 0.87, p<0.001), both major determinants for the development of severe and fatal malaria, and Treg cells were significantly increased in hyperparasitemia. There was a further significant correlation between Treg cell frequency and plasma concentrations of soluble tumor necrosis factor receptor II (TNFRII) in SM. A subset of TNFRII+ Treg cells with high expression of Foxp3 was increased in severe relative to uncomplicated malaria. In vitro, P. falciparum–infected red blood cells dose dependently induced TNFRII+Foxp3hi Treg cells in PBMC from malaria-unexposed donors which showed greater suppressive activity than TNFRII− Treg cells. The selective enrichment of the Treg cell compartment for a maximally suppressive TNFRII+Foxp3hi Treg subset in severe malaria provides a potential link between immune suppression, increased parasite biomass, and malaria disease severity. The findings caution against the induction of TNFRII+Foxp3hi Treg cells when developing effective malaria vaccines.

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-γ and lymphotoxin α, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4+ and CD8+ T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4+ T cell responses by helminth coinfection amplified CD4+ T cell-mediated parasite sequestration, whereas vaccination could generate CD4+ T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100–1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.