Steven M. Singer

T-Cell-Dependent Control of Acute Giardia lamblia Infections in Mice

ABSTRACT We have studied immune mechanisms responsible for control of acuteGiardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice withG. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to controlG. lamblia infections, as has been shown previously forG. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either αβ- or γδ-T-cell receptor (TCR)-expressing T cells, we show that the αβ-TCR-expressing T cells are required to control parasites but that the γδ-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection fromG. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

Mast Cell-Dependent Control of Giardia lamblia Infections in Mice

ABSTRACT Mast cells are important for protective immunity to intestinal helminth infections and as mediators of allergic disease. Their role in protozoan infections is less well described. We have therefore analyzed mast cell responses and parasite control in mice infected with the protozoan Giardia lamblia. We also measured immunoglobulin A (IgA) responses to the parasite, as IgA can have a protective role in this model. c-kitw/wv mice failed to make parasite-specific IgA, mount a mast cell response, or eliminate the infection. Anti-c-kit-treated C57BL/6 mice had normal IgA responses, lacked mast cell responses, had reduced interleukin-6 (IL-6) mRNA in the small intestine, and failed to control the infection within 10 days. IL-9-deficient mice had a significant but reduced mast cell response and still controlled the infection within 2 weeks. Interestingly, IL-6-deficient mice had enhanced mast cell responses yet failed to rapidly control the infection. However, prevention of mast cell responses in IL-6-deficient mice by anti-c-kit treatment did not lead to parasite elimination. Both IL-6- and IL-9-deficient mice had normal IgA production. IL-6-deficient mice had significant serum levels of mast cell mediators, histamine and mast cell protease 1, following infection. Together, these results show that mast cells are important for the rapid control of Giardia infections in mice. Furthermore, they show that IL-6 is not necessary for these mast cell responses. Instead, they suggest that mast cell production of IL-6 appears to be important for control of this infection.

Initiator and upstream elements in the α2-tubulin promoter of Giardia lamblia

Abstract Giardia lamblia , one of the earliest diverging eukaryotes and a major cause of diarrhea world-wide, has unusually short intergenic regions, raising questions concerning its regulation of gene expression. We have approached this issue through examination of the α2-tubulin promoter and in particular investigated the function of an AT-rich element surrounding the transcription start site. Its placement and the ability of this sequence to direct transcription initiation in the absence of any other promoter elements is similar to the initiator element in higher eukaryotes. However, the sequence diversity of extremely short (8–10 bp) initiator elements is surprising, as is their ability to independently direct substantial levels of transcription. We also identified a large AT-rich element located between −64 and −29 bp upstream of the transcriptional start site and show using both deletions and site-specific mutations of this region that sequences between −60 and the start of transcription are important for promoter strength; interestingly this AT-rich sequence is not highly conserved among different Giardia promoters. These data suggest that while the overall structure of the core promoter has been conserved throughout eukaryotic evolution, significant variation and flexibility is allowed in element consensus sequences and roles in transcription. In particular, the short and diverse sequences that function in transcription initiation in Giardia suggest the potential for relaxed transcriptional regulation.

Role of Interleukin-6 in the Control of Acute and Chronic Giardia lamblia Infections in Mice

ABSTRACT In this study, we investigated the role of interleukin-6 (IL-6) in Giardia lamblia infections in mice. Elevated IL-6 expression was found in wild-type mice 15 days postinfection. Furthermore, IL-6-deficient mice controlled infections only slowly although normal immunoglobulin A production was observed. Thus, IL-6 is necessary for early control of acute G. lamblia infections.

Neuronal Nitric Oxide Synthase Is Necessary for Elimination of Giardia lamblia Infections in Mice

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-l-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-l-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-l-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.

A meta-analysis of the effectiveness of albendazole compared with metronidazole as treatments for infections with Giardia duodenalis.

Background Metronidazole is the most commonly used drug for the treatment of giardiasis in humans. In spite of its therapeutic efficacy for giardiasis, low patient compliance, especially in children, side effects, and the emergence of metronidazole-resistant strains may restrict its use. Albendazole has been used to treat Giardia duodenalis infections in recent years. However, efficacy studies in vivo and in vitro have produced diverse results as to its effectiveness. A moderately benign side effect profile, combined with established efficacy against many helminths, renders it promising for treatment of giardiasis in humans. Methodology and Principal Findings We performed a search in the PubMed, Scopus, EMBASE, the ISI Web of Science, LILIACS, and Cochrane Controlled Trials Register for trials published before February 2010 as well as in references of relevant research and review articles. Eight randomized clinical trials (including 900 patients) comparing the effectiveness of albendazole with that of metronidazole were included in meta-analysis. After extracting and validating the data, the pooled risk ratio (RR) was calculated using an inverse-variance random-effects model. Albendazole was found to be equally as effective as metronidazole in the treatment of giardiasis in humans (RR 0.97; 95% CI, 0.93, 1.01). In addition, safety analysis suggested that patients treated with albendazole had a lower risk of adverse effects compared with those who received metronidazole (RR 0.36; 95% CI, 0.10, 1.34), but limitations of the sample size precluded a definite conclusion. Conclusions/Significance The effectiveness of albendazole, when given as a single dose of 400 mg/day for 5 days, was comparable to that of metronidazole. Patients treated with albendazole tended to have fewer side effects compared with those who took metronidazole. Given the safety, effectiveness, and low costs of albendazole, this drug could be potentially used as an alternative and/or a replacement for the existing metronidazole therapy protocols in the treatment of giardiasis in humans.

Phosphoinositide 3-Kinase-Dependent Inhibition of Dendritic Cell Interleukin-12 Production by Giardia lamblia

ABSTRACT Dendritic cell interactions with pathogenic microbes initiate and direct the development of subsequent adaptive responses. The protozoan pathogen Giardia lamblia infects the mammalian small intestine, leading to nutrient malabsorption and diarrhea but rarely causing inflammation. In order to begin to understand how the innate immune system responds to this parasite and shapes the eventual adaptive response, we examined the interaction between parasites and murine bone marrow-derived dendritic cells (DCs). DCs incubated with live parasites or parasite extracts displayed enhanced levels of CD40. The expression of CD80 and CD86 also increased, but less than was seen with lipopolysaccharide-activated DCs. Small amounts of interleukin-6 (IL-6) and tumor necrosis factor alpha were secreted by these DCs, whereas no IL-10 or IL-12 could be detected. Coincubation of DCs with parasite extracts along with known Toll-like receptor (TLR) ligands resulted in enhanced secretion of IL-10 and reduced secretion of IL-12. The levels of major histocompatibility complex class II, CD80, and CD86 were also reduced compared to DCs stimulated with TLR ligands alone. Finally, studies with an extracellular signal-regulated kinase 1/2 pathway inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, and anti-IL-10 receptor antibody revealed that the PI3K pathway is the dominant mechanism of inhibition in DCs incubated with both lipopolysaccharide and Giardia. These data suggest that this parasite actively interferes with host innate immunity, resulting in an immune response able to control the infection but devoid of strong inflammatory signals.

Giardia duodenalis: The double-edged sword of immune responses in giardiasis

Giardiasis is one of the most common intestinal protozoan infections worldwide. The etiological agent, Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), is a flagellated, binucleated protozoan parasite which infects a wide array of mammalian hosts (Adam, 2001). The symptoms of giardiasis include abdominal cramps, nausea, and acute or chronic diarrhea, with malabsorption and failure of children to thrive occurring in both sub-clinical and symptomatic disease (Thompson et al., 1993). Infections are transmitted by cysts which are excreted in the feces of infected humans and animals. Human giardiasis is distributed worldwide, with rates of detection between 2-5% in the developed world and 20-30% in the developing nations (Farthing, 1994). There is significant variation in the outcome of Giardia infections. Most infections are self-limiting, although re-infection is common in endemic areas and chronic infections also occur. Moreover, some individuals suffer from severe cramps, nausea and diarrhea while others escape these overt symptoms. This review will describe recent advances in parasite genetics and host immunity that are helping to shed light on this variability.

Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation

Giardia lamblia, an intestinal dwelling protozoan parasite, undergoes surface antigenic variation where only one of an estimated 150 variant‐specific surface proteins (VSPs) is expressed and present on the surface at any one time. Transcriptional switching between VSPs results in replacement of one VSP by another. The mechanisms that control antigenic variation are poorly understood and difficult to study because there are multiple copies of each VSP and strong similarity with other VSPs. In order to study transcriptional regulation of one specific vsp, a haemagglutinin (HA) epitope‐tagged h7 was integrated into the G. lamblia GS genome. We show that HA‐tagged H7 undergoes antigenic variation in the same manner as native H7, also present in the GS genome. Control of expression of both HA‐tagged H7 and native H7 is independent of each other even though the genes and their surrounding 5′ and 3′ flanking sequences are virtually identical. Analysis of expressing and non‐expressing clones revealed an absence of HA‐tagged h7 gene rearrangements upon switching and acetylation of histone lysine residues within the 167 nucleotides 5′ to the expressed HA‐tagged h7 gene. Lack of vsp rearrangements and acetylation of expressed immediate upstream regions implicates involvement of epigenetic mechanisms in antigenic variation.

Host Immunity and Pathogen Strain Contribute to Intestinal Disaccharidase Impairment following Gut Infection

Infection or other inflammatory insults in the small intestine often result in reduced disaccharidase enzyme levels. Using a mouse model of giardiasis, we examined the role of host immunity and pathogen virulence in mediating disaccharidase deficiency postinfection (p.i.). C57BL/6J mice were infected with two strains, WB and GS, of the human parasite Giardia duodenalis. The levels of sucrase, maltase, and lactase decreased in wild-type mice p.i. with the GS strain but not with the WB strain. Both CD4-deficient and SCID mice failed to eliminate the infection and did not exhibit disaccharidase deficiency. β2-Microglobulin knockout animals controlled infections similar to wild-type mice but exhibited no decrease in disaccharidase activity. Analysis of cytokine production by spleen and mesenteric lymph node cells showed production of IL-4, IL-10, IL-13, IL-17, IL-22, TNF-α, and IFN-γ p.i. with both WB and GS, with IFN-γ being the dominant cytokine for both parasite strains. Mesenteric lymph node cells produced lower levels of cytokines compared with splenocytes in response to parasite extract, although the overall pattern was similar. These data suggest that T cell responses mediate parasite clearance whereas also contributing to pathogenesis. They also demonstrate that differences in pathogen strain can also determine the outcome of infection and further our understanding of the clinical variation seen in human giardiasis.