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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Nov 20, 2008.
Published in final edited form as:
PMCID: PMC2585514
NIHMSID: NIHMS53600

Neuronal Nitric Oxide Synthase is Necessary for Elimination of Giardia lamblia Infections in Mice1

Abstract

Nitric oxide (NO) produced by inducible nitric oxide synthase (NOS2) is important for 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 following infection compared to 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 non-specific 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 elimination of an infection.

Keywords: Parasitic- protozoan, nitric oxide, mucosa, neuroimmunology

Introduction

Infections with Giardia lamblia are one of the most common intestinal maladies in the world (1). Infections can lead to acute diarrhea, cramps and nausea, although asymptomatic infections are the most common. While most infections are controlled by an effective immune response, some individuals develop chronic disease. It is unclear what immune mechanisms are responsible for effective control of infections (2, 3). While IgA has been shown to have a role in controlling infections (4), it is clear that IgA independent mechanisms are also able to eliminate Giardia and that IL-6 appears to pivotal for these pathways (5-7).

In addition to antibodies, a number of different mechanisms have been proposed which might control Giardia infections. These include anti-microbial peptides, NO, and mast cell products (8-12). We have recently found that mice that are unable to produce a mast cell response are deficient in elimination of Giardia infections (13). In contrast, Eckmann et al. (3) reported that mice lacking functional α-defensins due to deletion of the gene for matrilysin control G. muris infections as well as wild-type mice.

NO has been recognized as playing a pivotal role in the control of infections with numerous microbes, including Leishamania spp., Toxoplasma gondii, Salmonella typhimurium and Mycobacterium tuberculosis (14, 15). Since NO can also inhibit G. lamblia replication and differentiation in vitro we decided to examine the role of NO during infections of mice with G. lamblia. Using both gene-targeted mice and inhibitors of nitric oxide synthase (NOS3) we show that it is not the inducible isoform of NOS (NOS2), but instead the neuronal isoform (NOS1) that is responsible for contributing to elimination of this infection.

Materials and Methods

Mice, Parasites and Infections- C57BL/6J, B6.129S2-Il6tm1Kopf/J, B6.129P2-Nos2tm1Lau/J, B6;129S4-Nos1tm1Plh/J, B6129SF2/J and B6.CB17-Prkdcscid/SzJ mice were all obtained from the Jackson Laboratories (Bar Harbor, ME). Females between five and eight weeks of age were used for all experiments. The GS/H7 clone of G. lamblia was propagated in vitro and used for infections as previously described (5). Briefly, mice were gavaged with 106 trophozoites in PBS on day 0 and sacrificed on different days post-infection. Parasite numbers in the distal duodenum and proximal jejunum were counted by mincing tissue in ice-cold PBS, chilling for 15’, and counting with a hemacytometer. All animal experiments were approved by the Georgetown University Animal Care and Use Committee.

Enzyme Inhibitors- All inhibitors were obtained from Sigma-Alrdrich Co., (St. Louis, MO) The non-selective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was administered in drinking water at a concentration of 0.5 mg / ml (16). Water was replaced every other day. The NOS1 selective inhibitor 7-nitroindazole (7-NI) was dissolved in DMSO and injected i.p. at a dose of 25 mg / kg (16). The NOS2 selective inhibitor N-iminoethyl-L-lysine (L-NIL) was dissolved in PBS and injected i.p. at a dose of 10 mg / kg (16). The peristalsis inhibitor loperamide was dissolved in water and administered p.o. at a dose of 25 mg / kg (17). Inhibitors were given starting on day 2 post-infection and every other day thereafter.

RT-PCR- Total RNA was isolated from the jejunum, immediately distal to the segment used for quantifying parasite numbers using RNA-STAT-60 (Tel-Test Inc. Friendswood, TX). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and cDNA was amplified with specific primers. Primer and probe sequences for TaqMan real-time PCR of GAPDH, NOS2, TNFα, IL-4 and IFN-γ (18) and NOS1 (19) have been previously described. Reactions were performed using TaqMan reagents and an ABI7000 (Perkin-Elmer Corp., Boston, MA). Normalization with GAPDH was performed using the ΔΔCt method as described by Perkin-Elmer.

Immunohistology- Segments of distal jejunum were snap frozen and sectioned using OCT (Miles Sceintific, Naperville, IL). 8 μ sections were thawed on glass slides, fixed with cold acetone for 5’ and stained with rabbit anti NOS1 or rabbit anti-NOS2 antisera (Santa Cruz Biotechnologies, Santa Cruz, CA). Staining was revealed with goat anti-rabbit FITC (Santa Cruz Biotechnologies) and visualized with a Zeiss Axiophot epifluorescence microscope equipped with a CoolSnap FX CCD camera (Roper Scientific, Trenton, NJ) and connected to a Macintosh G3 computer running OpenLab 3.0 (Improvision Inc., Cambridge, MA). Imagers were further processed using Photoshop (Adobe Systems, San Jose, CA).

Gastrointestinal Motility- Mice were fasted overnight prior to measuring motility. Water was freely available during this period. Mice were gavaged with 0.2 ml 10% charcoal in 5% gum acacia (both from Sigma-Aldrich, Inc.). Thirty minutes later mice were euthanized and the intestinal tracts were carefully removed. The total length from the pylorus to the cecum was measured as was the distance from the pylorus to the leading edge of the charcoal. Motility is expressed as the percentage of the total intestinal length traversed by the charcoal.

Statistics- Numbers of parasites, PCR results and gastrointestinal motility were compared between groups using students t-test statistics (Prism 3.0, GraphPad Inc.). The non-parametric Mann-Whitney test was used to compare parasite number data that included animals with undetectable parasites.

Results

Because IL-6 deficient mice have a defect in control of Giardia infections and because NO can inhibit parasite replication and differentiation in vitro, we began by investigating whether inducible NOS (NOS2) was upregulated during infections of wild-type mice but not IL-6 deficient mice. Figure 1A shows that NOS2 mRNA levels were ~3-fold higher in wild-type mice five days post-infection. In this model, parasite numbers typically remain high until between five and seven days post-infection. By day twelve, when most parasites have typically been eliminated, a 17-fold increase in NOS2 mRNA was seen. Uninfected IL-6 deficient mice had ~70% less NOS2 mRNA than did wild-type mice. Consistent with their inability to eliminate the infection, NOS2 mRNA only increased 3–fold by day twelve post-infection and remained lower than in uninfected wild-type mice.

Figure 1
Expression of NOS isoforms and TNFα following Giardia infection. Intestinal RNA from wild-type (filled bars) and IL-6 deficient (open bars) C57BL/6 mice was collected from uninfected (Day 0) or infected mice on the indicated days post-infection. ...

TNFα is a key inducer of NOS2 expression (20), while IL-4 has been shown to inhibit NOS2 induction (21). We therefore examined TNFα and IL-4 mRNA levels in the small intestine of these mice. Similar to the results previously seen in mesenteric lymph nodes by Bienz et al. (7), IL-4 mRNA levels were increased in IL-6 deficient mice infected for twelve days compared to twelve-day infected wild-type mice or uninfected IL-6 deficient mice (data not shown). No significant differences in IFN-γ mRNA levels were observed (data not shown). Similar to NOS2, TNF-α mRNA levels were also decreased in the Giardia infected IL-6 deficient mice compared with wild-type mice (Fig. 1C). However, these differences were only 3-fold at day five and 2-fold at day twelve. Differences in TNFα and IL-4 may account for the reduced NOS2 expression in IL-6 deficient mice following Giardia infection.

To test whether NOS2 production of NO was necessary for control of infections we infected NOS2 deficient mice (22) with G. lamblia. We also treated wild-type mice with the isoform non-specific NOS inhibitor, L-NAME (16). Figure 2 shows that five days post-infection, L-NAME treated mice had 3−4 times more parasites in the small intestine than untreated mice, and almost as many parasites as IL-6 deficient mice. In contrast, NOS2 deficient mice had 8−10 times fewer parasites than wild-type controls five days post-infection. By ten days post-infection the untreated wild-type mice and NOS2 deficient mice no longer had detectable parasites, whereas all the L-NAME treated mice still had measurable parasite loads. This difference between NOS2 deficient mice and L-NAME treated mice could represent compensatory mechanisms at work in the genetically deficient mice or the inhibition of additional isoforms of NOS, e.g., the neuronal isoform NOS1 or the endothelial isoform NOS3, by L-NAME. To determine if NOS deficient or L-NAME treated mice had markedly altered immune responses we analyzed production of anti-parasite IgA and cytokine responses in infected mice. No differences were seen in either IgA or cytokine responses (data not shown) suggesting that manipulation of NOS2 did not have widespread effects in this system. Nevertheless, we can conclude that NOS2 is not absolutely required to control infections with G. lamblia in mice. Indeed, the ability of both IFN-γ and IL-4 deficient mice to control infections (5) suggested that there are likely redundant mechanisms able to control this infection.

Figure 2
Inhibition of NOS activity, but not deletion of the NOS2 gene, delays parasite elimination. Mice were infected with Giardia on day 0 and parasite numbers in the small intestine were determined on either day 5 (filled symbols) or day 11 (open symbols) ...

In order to determine if inhibition of NOS1 or NOS3 by L-NAME was effecting elimination of Giardia, we infected NOS2 deficient mice and treated them with L-NAME. If the observed effect of L-NAME was due to inhibition of NOS2, we reasoned that treatment of the NOS2 mutant mice should have no effect in these mice. Figure 3A shows that L-NAME had a similar effect when given to NOS2 deficient mice as when given to wild-type mice, however. Thus, L-NAME inhibition of either NOS1 or NOS3 must be involved in elimination of Giardia. Wild-type mice were therefore also treated with 7-nitroindazole (7-NI), a NOS inhibitor that shows selective activity against NOS1 (16). Treatment with 7-NI resulted in extended infections similar to treatment with L-NAME (Fig. 3A), supporting the idea that NOS1 is indeed important for elimination of Giardia.

Figure 3
NOS1 rather than NOS2 activity is necessary for elimination of Giardia. A. Groups of four mice were infected with Giardia and parasite numbers were determined on day 10 post-infection. Wild-type C57BL/6 mice were treated with the non-specific NOS inhibitor ...

To further confirm a role for NOS1 but not NOS2, we treated wild-type mice with L-NIL (data not shown), an inhibitor that has a preference for NOS2 over NOS1 (16). As expected from results with NOS2 deficient mice, C57BL/6 mice treated with L-NIL had no detectable parasites ten days post-infection, similar to untreated mice (n= 4 / group). We also infected NOS1 deficient mice in order to confirm the results seen with 7-NI. While both wild-type B6X129 F2 mice and NOS1 deficient mice had readily detectable infections five days after inoculation with parasites, the number of parasites / mouse was significantly higher in NOS1 deficient mice at this time (Fig. 3B). Moreover, by twelve days post-infection the wild-type mice had eliminated all detectable parasites while, the NOS1 deficient mice were still heavily infected. These results confirmed that NOS1 plays an essential role in the elimination of Giardia infections.

NOS1 positive neurons have been described in the intestinal tract and are known to play a role in regulating intestinal motility (23, 24). Figure 4 shows that NOS2 in the small intestine of both uninfected and infected mice is predominantly expressed just under the epithelial cell layer of the villi (Fig. 4C and D). Consistent with the results from real-time PCR (Fig. 1), more NOS2 appears in infected mice (Fig. 4C) than in uninfected mice (Fig. 4A). Importantly, no NOS2 staining was seen using NOS2 deficient mice (Fig. 4B), confirming the specificity of the reagents used for these studies.

Figure 4
Localization of NOS2 in the small intestine of Giardia infected mice. Frozen sections of uninfected (A) and 10-day Giardia-infected (C, D) wild-type mice were stained with antibodies to NOS2 as described in Methods. The absence of staining for NOS2 in ...

NOS1 expression was seen in long processes within the intestinal muscle layers and in patches of cells in the sub-mucosa (Fig. 5A and B). Thus, it is unlikely that NO produced by NOS1 could directly effect Giardia within the lumen of the small intestine. nNOS expressing neurons and Interstital Cells of Cajal (ICC) have been described previously in the myenteric and sub-mucosal plexuses of the enteric nervous system (25-27). Some nNOS immunoreactivity was also seen within the villi, possibly reflecting neuronal processes into this tissue. Little change was observed in the level of NOS1 staining between uninfected and infected mice. This is in agreement with the real time PCR data presented in Fig. 1B which shows that the level of NOS1 mRNA is essentially identical among uninfected and infected, wild-type and IL-6 deficient C57BL/6 mice.

Figure 5
Localization of NOS1 in the small intestine of Giardia infected mice. Frozen sections from wild-type mice not infected (A) or infected for 10 days with Giardia (B) were stained with antibodies to NOS1 as described in Methods. The absence of staining for ...

Since NOS1 expressing neurons in the enteric nervous system are involved in regulating gastrointestinal motility and since Giardia infected gerbils have increased intestinal transit rates we asked whether changes in motility were involved in the elimination of Giardia. We measured gastrointestinal motility by feeding mice charcoal meals and determining the distance traveled by the charcoal in a fixed amount of time (17). This method measures both gastric emptying and intestinal transit together. We treated infected mice with either L-NAME or loperamide as a positive control for inhibiting motility. Loperamide is a μ-opiate receptor agonist commonly used to inhibit gastrointestinal motility and to treat diarrhea (28, 29). It can also inhibit Ca++ channel function in neurons (28). Treatment with loperamide extended Giardia infections in wild-type mice similar to treatment with L-NAME (Fig. 6A). As expected from results on intestinal transit in Giardia infected gerbils (30), infection significantly increased the rate of motility in infected C57BL/6 mice (Fig. 6B, C and D). Importantly, blocking NOS activity with L-NAME partially reversed this increase in transit (Fig. 6B), as did treatment with loperamide (Fig. 6C). Finally, no increase in motility was seen after infecting SCID mice with Giardia (Fig. 6D), despite the presence of high parasite loads in these mice (5). Thus, since both loperamide and L-NAME block the increase in motility and prolong Giardia infections, we conclude that the increase in intestinal motility caused by Giardia infection is important for elimination of the parasites from the intestinal tract. Furthermore, this increase in motility depends on adaptive immune responses since no changes were observed in SCID mice.

Figure 6
Inhibition of gastrointestinal motility delays elimination of Giardia infections. A. Wild-type C57BL/6 mice were infected with Giardia and not treated (squares) or treated daily, beginning on day 2 post-infection. either with L-NAME (triangles) or with ...

Discussion

Control of intestinal infections requires an effective immune response that must be balanced against destructive pathology. In mice, as in most humans, infected with Giardia lamblia, the parasites are effectively eliminated by the immune system without causing severe inflammation or diarrhea. Multiple mechanisms have been shown to be able to inhibit or kill Giardia in vitro. However, none has been found so far to be essential for controlling this infection in vivo. For example, while IgA against the parasite is clearly toxic in vitro, B cell and IgA deficient mice eliminate 90% or more of the parasites between one and two weeks post-infection (4, 5). This contrasts with SCID mice and c-kitw/wv that maintain infections for at least several months, and IL-6 deficient mice which eliminate infections between four and eight weeks post-infection (5, 6, 13). This likely reflects the fact that elimination of Giardia occurs through several redundant mechanisms, and it is only in mouse mutants deficient in multiple or non-redundant mechanisms that a clear phenotype is observed.

We have shown that IL-6 deficient mice infected with G. lamblia have decreased levels of TNFα and NOS2 mRNA in the small intestine compared to wild-type mice. Although production of NOS2 by intestinal epithelial cells can prevent parasite replication in vitro (9), treatment of wild-type mice with L-NIL which specifically inhibit this isoform of NOS and infections in mice lacking the NOS2 gene clearly show that this pathway is not necessary for elimination of infections in vivo. However, these data do not rule out a non-redundant role for NOS2 in parasite elimination. In contrast, the isoform non-specific inhibitor L-NAME and the NOS1-specific inhibitor 7-NI both resulted in inhibition of parasite elimination, indicating that the neuronal enzyme NOS1 is necessary for control of Giardia infection. Enhanced infections in NOS1 deficient mice confirmed the assignment of NOS1 as the essential NOS isoform for eliminating this infection.

In a previous analysis of Giardia infections in IL-6 deficient mice, Bienz et al. found elevated IL-4 mRNA in the mesenteric lymph nodes, but no NOS2 expression was detected at this site (7). They also reported that neither wild-type nor IL-6 deficient mice had detectable NOS2 mRNA in the mesenteric lymph nodes following infection. However, because Giardia replicates exclusively in the lumen of the small intestine one would expect that NO production by intestinal epithelial cells would be more relevant for eliminating the infection as suggested by Eckmann (9). Our immunolocalization of NOS2 showed that NOS2 is found in cells underlying the intestinal epithelium. This is consistent both with an absence of NOS2 expression in the mesenteric lymph nodes and with the lack of effect of inhibiting this enzyme on the outcome of infection.

Normal intestinal motility is a consequence of the coordinated contraction and relaxation of intestinal smooth muscles. This coordination is achieved by the enteric nervous system. Smooth muscle contraction is the result of cholinergic stimulation of smooth muscle, whereas relaxation is mediated, in part, through inhibitory signaling via nitric oxide. We propose that immune responses during infection increase motility and that inhibition of NOS activity with L-NAME resulted in reduced gastrointestinal motility by interfering with muscle relaxation. Our measurements of gastrointestinal motility reflect both gastric emptying as well as intestinal transit and it is therefore possible that gastric emptying and/or intestinal motility are important in elimination of this infection. Indeed, NOS1 deficient mice have been shown to have reduced gastric emptying (31). In contrast, the major action of loperamide is to reduce intestinal transit, rather than gastric emptying (32, 33). Nevertheless, while loperamide is primarily an agonist of μ-opioid receptors, it can also block Ca++ channels and inhibit calmodulin-mediated effects in neurons (28). Since NOS1 activity is regulated by calmodulin (34), we suggest that loperamide's role in prolonging Giardia infection may be mediated through reducing intestinal motility through μ-opioid receptors, and perhaps also via inhibition of NOS1 activity. Additional studies will be needed to determine if altered gastric emptying has a role in elimination of Giardia infection.

The effects of intestinal infection on motility have been investigated previously in several models. Gerbils infected with G. lamblia were shown to have increased intestinal transit rates, but roles for transit and nitric oxide in eliminating the infection were not investigated (30). One of the best studied models of infection induced changes in motility is infections with the nematode Trichinella spiralis, where infection leads to hypermotility and mastocytosis, similar to Giardia infections (35). Interestingly, treatment with L-NAME prevented hypermotility in Trichinella infected rats but led to increased expulsion of worms (36), in contrast to the effect of L-NAME on parasite numbers in Giardia infected mice. This suggests that while hypermotility is not needed for elimination of Trichinella, it is needed for elimination of Giardia.

Human infection with G. lamblia often results in severe abdominal cramps and malabsorptive diarrhea. We have now shown that Giardia infections in mice lead to increased rates of intestinal transit and that this transit is required for elimination of the infection. It is likely that similar changes in intestinal transit in humans contribute to the symptoms associated with this infection. Further investigation into the pathophysiology of Giardia infection is needed as no pathogenic mechanisms have yet been identified. Furthermore, these data indicate that signals mediated by the enzyme NOS1 play a key role in elimination of this infection, but that NO production by NOS2 is clearly not required. While NOS2 has been implicated in elimination of numerous infections, this is the first example of which we are aware where activity of NOS1 is required.

Acknowledgements

The authors gratefully acknowledge Dr. Heidi Elmendorf for suggesting the loperamide experiments and Dr. Terez Shea-Donohue for helpful discussions. The authors have no conflicting financial interests.

Footnotes

1This work was supported by an NIH grant, AI-49565 to S.M.S. These studies were conducted using the animal core facility supported by NCI-CCSG and the histopathology and tissue shared resource facility constructed with support from the Research Facilities Improvement Grant C06 RR14567 from the National Center for Research Resources, National Institutes of Health.

3Abbreviations: NOS, nitric oxide sythase. 7-NI, 7-nitroindazole. L-NAME, NG-nitro-L-arginine methyl ester. L-NIL, N-iminoethyl-L-lysine.

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