Louise M. Randall

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.

Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells

Breaking the long‐held paradigm that primary B cells are not phagocytic, several studies have demonstrated recently that B cells from fish, amphibians, and reptilians have a significant phagocytic capacity. Whether such capacity has remained conserved in certain mammalian B cell subsets is presently an enigma. Here, we report a previously unrecognized ability of PerC B‐1a and B‐1b lymphocytes to phagocytose latex beads and bacteria. In contrast, B‐2 lymphocytes had an almost negligible ability to internalize these particles. Upon phagocytosis, B‐1a and B‐1b cells were able to mature their phagosomes into phagolysosomes and displayed the ability to kill internalized bacteria. Importantly, B‐1a and B‐1b cells effectively present antigen recovered from phagocytosed particles to CD4+ T cells. However, these cells showed a much lower competence to present soluble antigen or antigen from large, noninternalized particles. B‐1 B cells presented particulate and soluble antigen to CD4+ T cells more efficiently than macrophages, whereas DCs were the most potent APCs. The novel phagocytic and microbicidal abilities identified in B‐1 B lymphocytes strengthen the innate nature that has long been attributed to these cells. In the context of adaptive immunity, we show that these innate immune processes are relevant, as they enable B‐1 B cells to present phagocytosable particulate antigen. These capacities position these cells at the crossroads that link innate with adaptive immune processes. In a broader context, these newly identified capacities of B‐1 B cells further support the previously recognized functional, developmental, and evolutionary relationships between these cells and macrophages.

Cutting Edge: Conventional Dendritic Cells Are the Critical APC Required for the Induction of Experimental Cerebral Malaria

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4+ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.

Activation of Invariant NKT Cells Exacerbates Experimental Visceral Leishmaniasis

We report that natural killer T (NKT) cells play only a minor physiological role in protection from Leishmania donovani infection in C57BL/6 mice. Furthermore, attempts at therapeutic activation of invariant NKT (iNKT) cells with α-galactosylceramide (α-GalCer) during L. donovani infection exacerbated, rather than ameliorated, experimental visceral leishmaniasis. The inability of α-GalCer to promote anti-parasitic immunity did not result from inefficient antigen presentation caused by infection because α-GalCer–loaded bone marrow–derived dendritic cells were also unable to improve disease resolution. The immune-dampening affect of α-GalCer correlated with a bias towards increased IL-4 production by iNKT cells following α-GalCer stimulation in infected mice compared to naïve controls. However, studies in IL-4–deficient mice, and IL-4 neutralisation in cytokine-sufficient mice revealed that α-GalCer–induced IL-4 production during infection had only a minor role in impaired parasite control. Analysis of liver cell composition following α-GalCer stimulation during an established L. donovani infection revealed important differences, predominantly a decrease in IFNγ+ CD8+ T cells, compared with control-treated mice. Our data clearly illustrate the double-edged sword of NKT cell–based therapy, showing that in some circumstances, such as when sub-clinical or chronic infections exist, iNKT cell activation can have adverse outcomes.

The Aryl Hydrocarbon Receptor Promotes IL-10 Production by NK Cells

The cytokine IL-10 has an important role in limiting inflammation in many settings, including toxoplasmosis. In the present studies, an IL-10 reporter mouse was used to identify the sources of this cytokine following challenge with Toxoplasma gondii. During infection, multiple cell types expressed the IL-10 reporter but NK cells were a major early source of this cytokine. These IL-10 reporter+ NK cells expressed high levels of the IL-12 target genes T-bet, KLRG1, and IFN-γ, and IL-12 depletion abrogated reporter expression. However, IL-12 signaling alone was not sufficient to promote NK cell IL-10, and activation of the aryl hydrocarbon receptor (AHR) was also required for maximal IL-10 production. NK cells basally expressed the AHR, relevant chaperone proteins, and the AHR nuclear translocator, which heterodimerizes with the AHR to form a competent transcription factor. In vitro studies revealed that IL-12 stimulation increased NK cell AHR levels, and the AHR and AHR nuclear translocator were required for optimal production of IL-10. Additionally, NK cells isolated from T. gondii–infected Ahr−/− mice had impaired expression of IL-10, which was associated with increased resistance to this infection. Taken together, these data identify the AHR as a critical cofactor involved in NK cell production of IL-10.

Common Strategies To Prevent and Modulate Experimental Cerebral Malaria in Mouse Strains with Different Susceptibilities

ABSTRACT Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children and nonimmune adults, which results in significant mortality and long-term sequelae. Previous studies have reported distinct susceptibility gene loci in CBA/CaH (CBA) and C57BL/6 (B6) mice with experimental CM (ECM) caused by infection with Plasmodium berghei ANKA. Here we present an analysis of genome-wide expression profiles in brain tissue taken from B6 and CBA mice with ECM and report significant heterogeneity between the two mouse strains. Upon comparison of the leukocyte composition of ECM brain tissue, microglia were expanded in B6 mice but not CBA mice. Furthermore, circulating levels of gamma interferon, interleukin-10, and interleukin-6 were significantly higher in the serum of B6 mice than in that of CBA mice with ECM. Two therapeutic strategies were applied to B6 and CBA mice, i.e., (i) depletion of regulatory T (Treg) cells prior to infection and (ii) depletion of CD8+ T cells after the establishment of ECM. Despite the described differences between susceptible mouse strains, depletion of Treg cells before infection attenuated ECM in both B6 and CBA mice. In addition, the depletion of CD8+ T cells when ECM symptoms are apparent leads to abrogation of ECM in B6 mice and a lack of progression of ECM in CBA mice. These results may have important implications for the development of effective treatments for human CM.

Critical roles for LIGHT and its receptors in generating T cell-mediated immunity during Leishmania donovani infection.

LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTβR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTβR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4+ T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.

Analysis of Behavior and Trafficking of Dendritic Cells within the Brain during Toxoplasmic Encephalitis

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo

Vitiligo is a common autoimmune disease of the skin that results in disfiguring white spots. There are no FDA-approved treatments, and current treatments are time-consuming, expensive, and have low efficacy. We sought to identify new treatments for vitiligo, and first considered repurposed medications because of the availability of safety data and expedited regulatory approval. We previously reported that the IFN-γ-induced chemokine CXCL10 is expressed in lesional skin from vitiligo patients, and that it is critical for the progression and maintenance of depigmentation in our mouse model of vitiligo. We hypothesized that targeting IFN-γ signaling might be an effective new treatment strategy. STAT1 activation is required for IFN-γ signaling and recent studies revealed that simvastatin, an FDA-approved cholesterol-lowering medication, inhibited STAT1 activation in vitro. Therefore, we hypothesized that simvastatin may be an effective treatment for vitiligo. We found that simvastatin both prevented and reversed depigmentation in our mouse model of vitiligo, and reduced the number of infiltrating autoreactive CD8+ T cells in the skin. Treatment of melanocyte-specific, CD8+ T cells in vitro decreased proliferation and IFN-γ production, suggesting additional effects of simvastatin directly on T cells. Based on these data, simvastatin may be a safe, targeted treatment option for patients with vitiligo.

Soluble lymphotoxin is an important effector molecule in GVHD and GVL

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTalpha3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTalpha have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTalpha3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTalpha is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTalpha- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta(-/-) donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTalpha were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTalpha3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.