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The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro.

Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svärd SG - BMC Microbiol. (2013)

Bottom Line: In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro.Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell- and Molecular Biology, Uppsala University, BMC, Box 596, Uppsala SE-751 24, Sweden. staffan.svard@icm.uu.se.

ABSTRACT

Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections.

Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.

Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

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Giardia reduces host cell nitric oxide (NO) production. A, Expression changes of inducible nitric oxide synthase (nos2) in differentiated Caco-2 cells in medium with (+ arginine) and without (- arginine) arginine as assessed by qPCR in technical quadruplicates. Data is expressed as fold change expression compared to the 0 h timepoint. Significant expression changes compared to 0 h are indicated by asterisks. B, Expression changes of nos2 upon host cell (HCT-8) stimulation with cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)) and Giardia infection 40 h later. Data is expressed as fold change expression compared to the 0 h unstimulated control (squares). C, NO production of host cells (HCT-8) stimulated with cytokines 5 h after infection with Giardia trophozoites of 3 different isolates (WB, GS, P15). This experiment was repeated two times independently and lead to similar results. D, Giardia (isolate WB) infected host cells (HCT-8) were stimulated by cytokines to produce NO after 5 h of infection. Subsequently arginine (Arg) or citrulline (Citr) were added to 0.4 mM after 1 h of interaction. NO production was measured 40 h later. The described experiment was repeated two times independently and lead to similar results. Significant differences in the figure are indicated by asterisks (*for p < 0.5 and **for p < 0.01).
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Figure 3: Giardia reduces host cell nitric oxide (NO) production. A, Expression changes of inducible nitric oxide synthase (nos2) in differentiated Caco-2 cells in medium with (+ arginine) and without (- arginine) arginine as assessed by qPCR in technical quadruplicates. Data is expressed as fold change expression compared to the 0 h timepoint. Significant expression changes compared to 0 h are indicated by asterisks. B, Expression changes of nos2 upon host cell (HCT-8) stimulation with cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)) and Giardia infection 40 h later. Data is expressed as fold change expression compared to the 0 h unstimulated control (squares). C, NO production of host cells (HCT-8) stimulated with cytokines 5 h after infection with Giardia trophozoites of 3 different isolates (WB, GS, P15). This experiment was repeated two times independently and lead to similar results. D, Giardia (isolate WB) infected host cells (HCT-8) were stimulated by cytokines to produce NO after 5 h of infection. Subsequently arginine (Arg) or citrulline (Citr) were added to 0.4 mM after 1 h of interaction. NO production was measured 40 h later. The described experiment was repeated two times independently and lead to similar results. Significant differences in the figure are indicated by asterisks (*for p < 0.5 and **for p < 0.01).

Mentions: Inducible nitric oxide, iNOS, encoded by nos2, is a key enzyme in NO production during infections [10,18]. To further investigate the observed effects on the nos2 expression and iNOS activity in host cells upon Giardia infection, effects of different arginine levels were assessed. The growth of IECs in low-arginine medium compared to growth with extra arginine (0.4 mM arginine added to the low-arginine medium) surprisingly showed that nos2 was highly induced on the RNA level under low-arginine conditions (Figure 3a). The profile of nos2 induction in low-arginine medium was similar to the profile induced by Giardia infection with a peak of expression after 6 h (Figure 2). Strikingly, the level of expression upon parasite-interaction was lower than in the low-arginine medium. We therefore tested the hypothesis that Giardia can induce expression of nos2 via arginine depletion, but at the same time also down-regulate its expression. To test this hypothesis an alternative model was used, where nos2 expression was first induced in HCT-8 cells by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL, IFN-γ (500 ng/mL) prior to Giardia infection (40 h later). Parasite addition clearly and strongly down-regulated the expression of nos2 (Figure 3b). Thus, Giardia can both induce and down-regulate expression of iNOS.


The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro.

Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svärd SG - BMC Microbiol. (2013)

Giardia reduces host cell nitric oxide (NO) production. A, Expression changes of inducible nitric oxide synthase (nos2) in differentiated Caco-2 cells in medium with (+ arginine) and without (- arginine) arginine as assessed by qPCR in technical quadruplicates. Data is expressed as fold change expression compared to the 0 h timepoint. Significant expression changes compared to 0 h are indicated by asterisks. B, Expression changes of nos2 upon host cell (HCT-8) stimulation with cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)) and Giardia infection 40 h later. Data is expressed as fold change expression compared to the 0 h unstimulated control (squares). C, NO production of host cells (HCT-8) stimulated with cytokines 5 h after infection with Giardia trophozoites of 3 different isolates (WB, GS, P15). This experiment was repeated two times independently and lead to similar results. D, Giardia (isolate WB) infected host cells (HCT-8) were stimulated by cytokines to produce NO after 5 h of infection. Subsequently arginine (Arg) or citrulline (Citr) were added to 0.4 mM after 1 h of interaction. NO production was measured 40 h later. The described experiment was repeated two times independently and lead to similar results. Significant differences in the figure are indicated by asterisks (*for p < 0.5 and **for p < 0.01).
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Figure 3: Giardia reduces host cell nitric oxide (NO) production. A, Expression changes of inducible nitric oxide synthase (nos2) in differentiated Caco-2 cells in medium with (+ arginine) and without (- arginine) arginine as assessed by qPCR in technical quadruplicates. Data is expressed as fold change expression compared to the 0 h timepoint. Significant expression changes compared to 0 h are indicated by asterisks. B, Expression changes of nos2 upon host cell (HCT-8) stimulation with cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)) and Giardia infection 40 h later. Data is expressed as fold change expression compared to the 0 h unstimulated control (squares). C, NO production of host cells (HCT-8) stimulated with cytokines 5 h after infection with Giardia trophozoites of 3 different isolates (WB, GS, P15). This experiment was repeated two times independently and lead to similar results. D, Giardia (isolate WB) infected host cells (HCT-8) were stimulated by cytokines to produce NO after 5 h of infection. Subsequently arginine (Arg) or citrulline (Citr) were added to 0.4 mM after 1 h of interaction. NO production was measured 40 h later. The described experiment was repeated two times independently and lead to similar results. Significant differences in the figure are indicated by asterisks (*for p < 0.5 and **for p < 0.01).
Mentions: Inducible nitric oxide, iNOS, encoded by nos2, is a key enzyme in NO production during infections [10,18]. To further investigate the observed effects on the nos2 expression and iNOS activity in host cells upon Giardia infection, effects of different arginine levels were assessed. The growth of IECs in low-arginine medium compared to growth with extra arginine (0.4 mM arginine added to the low-arginine medium) surprisingly showed that nos2 was highly induced on the RNA level under low-arginine conditions (Figure 3a). The profile of nos2 induction in low-arginine medium was similar to the profile induced by Giardia infection with a peak of expression after 6 h (Figure 2). Strikingly, the level of expression upon parasite-interaction was lower than in the low-arginine medium. We therefore tested the hypothesis that Giardia can induce expression of nos2 via arginine depletion, but at the same time also down-regulate its expression. To test this hypothesis an alternative model was used, where nos2 expression was first induced in HCT-8 cells by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL, IFN-γ (500 ng/mL) prior to Giardia infection (40 h later). Parasite addition clearly and strongly down-regulated the expression of nos2 (Figure 3b). Thus, Giardia can both induce and down-regulate expression of iNOS.

Bottom Line: In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro.Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell- and Molecular Biology, Uppsala University, BMC, Box 596, Uppsala SE-751 24, Sweden. staffan.svard@icm.uu.se.

ABSTRACT

Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections.

Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.

Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

Show MeSH
Related in: MedlinePlus