Najmeeyah Brown

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8+ T cells.

Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Innate killing of Leishmania donovani by macrophages of the splenic marginal zone requires IRF-7.

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.

Therapeutic Vaccination With Recombinant Adenovirus Reduces Splenic Parasite Burden in Experimental Visceral Leishmaniasis

Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani–infected BALB/c mice, HASPB- and KMP11-specific CD8+ T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ+CD8+ T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.

IL-10-producing Th1 cells and disease progression are regulated by distinct CD11c⁺ cell populations during visceral leishmaniasis.

IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4+ IFNγ+ T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11chi DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11chi as well as CD11cint/lo cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c+ cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c+ cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

Graphical abstract

Expression of vFLIP in a Lentiviral Vaccine Vector Activates NF-κB, Matures Dendritic Cells, and Increases CD8+ T-Cell Responses

ABSTRACT Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-κB, vFLIP from Kaposis sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-κB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8+ T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8+ T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-κB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-κB activator can improve the efficacy of a vaccine vector.

Inhibition of receptor tyrosine kinases restores immunocompetence and improves immune-dependent chemotherapy against experimental leishmaniasis in mice

Receptor tyrosine kinases are involved in multiple cellular processes, and drugs that inhibit their action are used in the clinic to treat several types of cancer. However, the value of receptor tyrosine kinase inhibitors (RTKIs) for treating infectious disease has yet to be explored. Here, we have shown in mice that administration of the broad-spectrum RTKI sunitinib maleate (Sm) blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis. Furthermore, Sm treatment restored the integrity of the splenic microarchitecture. Although restoration of splenic architecture was accompanied by an increase in the frequency of IFN-gamma+CD4+ T cells, Sm treatment alone was insufficient to cause a reduction in tissue parasite burden. However, preconditioning by short-term Sm treatment proved to be successful as an adjunct therapy, increasing the frequency of IFN-gamma+ and IFN-gamma+TNF+CD4+ T cells, enhancing NO production by splenic macrophages, and providing dose-sparing effects when combined with a first-line immune-dependent anti-leishmanial drug. We propose, therefore, that RTKIs may prove clinically useful as agents to restore immune competence before the administration of chemo- or immunotherapeutic drugs in the treatment of visceral leishmaniasis or other diseases involving lymphoid tissue remodeling, including cancer.

Compartment-Specific Remodeling of Splenic Micro-Architecture during Experimental Visceral Leishmaniasis

Progressive splenomegaly is a hallmark of visceral leishmaniasis in humans, canids, and rodents. In experimental murine visceral leishmaniasis, splenomegaly is accompanied by pronounced changes in microarchitecture, including expansion of the red pulp vascular system, neovascularization of the white pulp, and remodeling of the stromal cell populations that define the B-cell and T-cell compartments. Here, we show that Ly6C/G+ (Gr-1+) cells, including neutrophils and inflammatory monocytes, accumulate in the splenic red pulp during infection. Cell depletion using monoclonal antibody against either Ly6C/G+ (Gr-1; RB6) or Ly6G+ (1A8) cells increased parasite burden. In contrast, depletion of Ly6C/G+ cells, but not Ly6G+ cells, halted the progressive remodeling of Meca-32+ and CD31+ red pulp vasculature. Strikingly, neither treatment affected white pulp neovascularization or the remodeling of the fibroblastic reticular cell and follicular dendritic cell networks. These findings demonstrate a previously unrecognized compartment-dependent selectivity to the process of splenic vascular remodeling during experimental murine visceral leishmaniasis, attributable to Ly6C+ inflammatory monocytes.

A transcriptomic network identified in uninfected macrophages responding to inflammation controls intracellular pathogen survival.

Summary Intracellular pathogens modulate host cell function to promote their survival. However, in vitro infection studies do not account for the impact of host-derived inflammatory signals. Examining the response of liver-resident macrophages (Kupffer cells) in mice infected with the parasite Leishmania donovani, we identified a transcriptomic network operating in uninfected Kupffer cells exposed to inflammation but absent from Kupffer cells from the same animal that contained intracellular Leishmania. To test the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival, we pharmacologically perturbed the activity of retinoid X receptor alpha (RXRα), a key hub within this network, and showed that this intervention enhanced the innate resistance of Kupffer cells to Leishmania infection. Our results illustrate a broadly applicable strategy for understanding the host response to infection in vivo and identify Rxra as the hub of a gene network controlling antileishmanial resistance.

Interferon Regulatory Factor 7 Contributes to the Control of Leishmania donovani in the Mouse Liver

ABSTRACT Optimal hepatic resistance to Leishmania donovani in mice requires the coordinated effort of a variety of leukocyte populations that together induce activation of local macrophages to a leishmanicidal state. Although nitric oxide and reactive oxygen intermediates are potent leishmanicidal effector molecules operating in the acquired phase of immunity, there have long been suggestions that other mechanisms of leishmanicidal activity exist. We recently discovered that Irf-7 regulates a novel innate leishmanicidal response in resident splenic macrophages that line the marginal zone. Here, we tested whether this mechanism also operates in Kupffer cells, the resident macrophage population of the liver and the major target for hepatic infection by L. donovani. Comparing the Kupffer cell responses in situ in B6 and B6.Irf-7 −/− mice, we found no evidence that Irf-7 affected amastigote uptake or early survival. However, we did find that Irf-7-deficient mice had impaired acquired resistance to hepatic L. donovani infection. This phenotype was attributable to a reduction in the capacity of hepatic CD4+ T cells, NK cells, and NKT cells to produce gamma interferon (IFN-γ) and also to defective induction of NOS2 in infected Kupffer cells. Our data therefore add interferon regulatory factor 7 (IRF-7) to the growing list of interferon regulatory factors that have effects on downstream events in the acquired cellular immune response to nonviral pathogens.