Eric N. Villegas

IL-10 mediates susceptibility to Leishmania donovani infection

Human visceral leishmaniasis (VL) results in a severe and potentially fatal systemic disease, accompanied by cellular immune depression. The production of IL‐10 correlates with ongoing diseaseand it has been suggested that the cellular immune depression that accompanies active disease may be due to a predominance of IL‐10 production rather than a lack of IFN‐γ production, which is essential for optimal macrophage activation and parasite elimination. To examine the role of IL‐10 in resistance during L. donovani infection (a causative agent of VL), the course of infection was examined in mice lacking the gene for IL‐10. BALB/c IL‐10‐/‐, as well as C57BL/6 IL‐10‐/‐ mice, were highly resistant to L. donovani infection, asevidenced by liver parasite burdens which were tenfold lower than those in control mice after 14 days of infection. Enhanced resistance was accompanied by increased production of IFN‐γ and nitric oxide in BALB/c IL‐10‐/‐ mice. Susceptibility to infection in BALB/c IL‐10‐/‐ mice was enhanced following in vivo treatment with a neutralizing antibody to IFN‐γ or IL‐12. Together these studies demonstrate for the first time that IL‐10 is a critical component of the immune response that inhibits resistance to L. donovani.

The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis

ABSTRACT Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/− mice infected withT. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/− mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication ofT. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.

Identification of STAT4-Dependent and Independent Mechanisms of Resistance to Toxoplasma gondii

The capacity of IL-12 to stimulate T and NK cell production of IFN-γ is required for resistance to Toxoplasma gondii. To identify the transcription factors involved in this mechanism of resistance, mice deficient in STAT4, a protein involved in IL-12 signaling, were infected with T. gondii and their immune responses were analyzed. STAT4−/− mice were unable to control parasite replication and died during the acute phase of infection, whereas wild-type mice controlled parasite replication and survived this challenge. The susceptibility of STAT4−/− mice to toxoplasmosis correlated with a defect in their ability to produce IFN-γ in response to infection, whereas administration of IFN-γ to these mice inhibited parasite replication and delayed time to death. Interestingly, analysis of infected STAT4−/− mice revealed that these mice did produce low levels of IFN-γ during infection, and the ability of splenocytes from infected or uninfected STAT4−/− mice to produce IFN-γ was enhanced by the addition of IL-2 plus IL-18. Moreover, administration of IL-2 plus IL-18 to STAT4−/− mice resulted in elevated serum levels of IFN-γ associated with a decreased parasite burden and delayed time to death. In vivo depletion studies demonstrated that the ability of IL-2 plus IL-18 to mediate STAT4-independent resistance to T. gondii is dependent on NK cell production of IFN-γ. Together, these studies identify STAT4 as an important transcription factor required for development of the innate NK and adaptive T cell responses necessary for resistance to T. gondii. However, other signaling pathways can be used to bypass STAT4-dependent production of IFN-γ and enhance innate resistance to T. gondii.

Identification of a Role for NF-κB2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii

The NF-κB family of transcription factors are involved in the regulation of innate and adaptive immune functions associated with resistance to infection. To assess the role of NF-κB2 in the regulation of cell-mediated immunity, mice deficient in the NF-κB2 gene (NF-κB2−/−) were challenged with the intracellular parasite Toxoplasma gondii. Resistance to this opportunistic pathogen is dependent on the production of IL-12, which is required for the development of innate NK cell and adaptive T cell responses dominated by the production of IFN-γ necessary to control replication of this parasite. Although wild-type controls were resistant to T. gondii, NF-κB2−/− mice developed severe toxoplasmic encephalitis and succumbed to disease between 3 and 10 wk following infection. However, NF-κB2 was not required for the ability of macrophages to produce IL-12 or to inhibit parasite replication and during the acute stage of infection, NF-κB2−/− mice had no defect in their ability to produce IL-12 or IFN-γ and infection-induced NK cell responses appeared normal. In contrast, during the chronic phase of the infection, susceptibility of NF-κB2−/− mice to toxoplasmic encephalitis was associated with a reduced capacity of their splenocytes to produce IFN-γ associated with a loss of CD4+ and CD8+ T cells. This loss of T cells correlated with increased levels of apoptosis and with elevated expression of the pro-apoptotic molecule Fas by T cells from infected NF-κB2−/− mice. Together, these results suggest a role for NF-κB2 in the regulation of lymphocyte apoptosis and a unique role for this transcription factor in maintenance of T cell responses required for long-term resistance to T. gondii.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

ABSTRACT Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Cryptosporidium source tracking in the Potomac River watershed.

ABSTRACT To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii.

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-γ. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28−/− mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-γ suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28−/− mice with T. gondii resulted in the increased expression of ICOS by activated CD4+ and CD8+ T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-γ by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-γ production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28−/− mice to T. gondi associated with reduced serum levels of IFN-γ, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.

Comparison of filters for concentrating microbial indicators and pathogens in lake-water samples

ABSTRACT Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Blockade of costimulation prevents infection-induced immunopathology in interleukin-10-deficient mice.

ABSTRACT Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-γ) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-γ or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-γ and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-γ but not IL-12. Further reduction of IFN-γ production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

Interleukin‐10 does not contribute to the pathogenesis of a virulent strain of Toxoplasma gondii

Interleukin (IL)‐10 is an inhibitor of cell mediated immunity and an antagonist of the development of protective immune responses associated with resistance to T. gondii. These observations led to the hypothesis that the production of IL‐10 could contribute to the ability of T. gondii to replicate and survive in an immune competent host. To determine whether the production of IL‐10 affects the ability of the RH strain of T. gondii to cause a lethal infection in mice, we compared the immune response to RH in IL‐10+/+ and IL‐10−/– BALB/c mice. Both groups of mice produced comparable amounts of IL‐12 and interferon (IFN)‐γ and had similar mortality curves and parasite burdens. The use of green fluorescent protein‐labelled parasites allowed us to infect IL‐10+/+ and IL‐10−/− mice and use a fluorescence‐activated cell sorter to distinguish infected and uninfected populations of macrophages and compare their expression of CD80, CD86 and major histocompatibility complex (MHC) class II. Although infected cells expressed higher overall levels of these molecules than uninfected cells, there were no differences between cells isolated from IL‐10+/+ and IL‐10−/– mice. Taken together, these results indicate that IL‐10 is not required for the virulence of the RH strain of T. gondii, nor is it involved in the regulation of the CD80, CD86 and MHC class II molecules during RH‐infection.