The fact that Giardia consumes arginine as an energy source is well-known [8, 24]. However, possible roles of arginine in the pathophysiology of the host have only recently caught attention [2, 7]. Within the present study we therefore assessed the effects of Giardia-infection of human IECs on the expression of arginine-metabolizing enzymes. Since gene expression changes during the very first hours of infection can only be studied in vitro, we used the in vitro interaction system described in [2, 7]. We focused on changes on the RNA level since we earlier identified large changes in host cell gene expression already after 1.5 h  and early changes of gene expression are best detectable on the RNA level. As shown in Figure 2, most of the host arginine-metabolizing genes were unaffected or slightly down-regulated upon Giardia-infection. nos2, the inducible form of the nitric oxide synthases (iNOS), was induced after 3 and 6 h of parasite interaction, but down-regulated after 24 h to levels slightly lower than before interaction. We detected a similar induction of nos2 expression in IECs cultivated without arginine as compared to cells grown with arginine, peaking at 6 h (Figure 3). When we induced iNOS expression in host IECs by addition of cytokines, Giardia trophozoites immediately down-regulated this expression (Figure 3), which is not in accordance with earlier results , however, fewer parasites per IEC, a different cell line (HT-29), different cytokine concentrations and another experimental approach with measurements after 18 h was used in that study. Thus, Giardia infection on one hand immediately induces iNOS by arginine-depletion, but at the same time there are also iNOS down-regulating mechanisms in the parasite. Accordingly, iNOS expression was down-regulated in Giardia-infected calves in vivo on RNA and protein level after several weeks of infection [25, 26].
As shown in Figure 2, the host’s cationic amino acid transporter 1 (CAT1), used for arginine-uptake into host cells, was down-regulated in an early response (1.5-3 h), but up-regulated after 6 h of interaction. This response of co-induction of nos2 and cat1, combined with a down-regulation of arginases, ensures that the host cells take up sufficient arginine for NO synthesis (Figure 1). Such a co-induction has been shown in murine macrophages  though it was claimed that CAT2 and not CAT1 is up-regulated together with iNOS . Within our study we could not detect expression of cat2 in IECs.
A variety of microbes are known to affect the host’s immune response by down-regulating host NO production, either via an up-regulation of host arginases or expression of their own arginases [18, 19] that compete for consumption of arginine with iNOS. As shown in Figure 2, host arginases were not up-regulated upon IEC-Giardia interaction in vitro. However, later time points than 24 h were not included due to limitations of the setup. Whether arginase expression is up-regulated at later time points in vivo is, to the best of our knowledge, unknown. Interestingly however, the expression of ODC, a downstream enzyme of arginase, was highly up-regulated at all times (Figure 2). This might lead to a shift of the arginine-flux away from iNOS into polyamine synthesis .
Giardia infection leads to an increased expression of odc, inos and cat1 during the first hours of interaction, whereas other arginine-consuming enzymes are down-regulated or constant. We therefore studied how the parasite can defend itself against this initial response. As shown in Figure 3, we were able to see a NO reduction similar to Giardia-infection of IECs  and addition of Giardia ADI expressed in E. coli. Moreover, this effect was observed for parasites of 3 different isolates (from humans (WB and GS) and pigs (P15)). Interestingly, the observed effect could be reverted by addition of arginine and also by its metabolite citrulline. This finding is interesting with regards to use of citrulline as a supplement in rehydration therapy, as discussed below.
In addition to actively taking up arginine, Giardia consumes arginine also indirectly via the secretion of the enzymes ADI and OCT that degrade arginine to ornithine via citrulline . Ornithine, secreted as a final product of arginine fermentation via an arginine-ornithine antiporter , has been shown to block arginine transport into IECs  (Figure 1). Upon interaction with host cells, the expression of arginine-consuming enzymes ADI, OCT and CK was down-regulated already after 1.5 h on the RNA level (Figure 4), which is in accordance to Ringqvist et al . As suggested, the expression of these enzymes might be increased shortly after secretion (15 minutes after host-parasite interaction), but is down-regulated at later time points due to depletion of arginine in the medium and due to a possible switch to glucose as main energy source .
It is not known to date, whether Giardia leads to a systemic arginine-deficiency in patients, this needs to be followed up. However, the local reduction of arginine levels by G. intestinalis could have additional consequences on the host response, the immune response in particular, since replication and infiltration of immune cells in the intestine might be blocked. It was recently shown that the phenotype and cytokine production of dendritic cells can be affected by the arginine-metabolizing enzyme ADI from Giardia. Even though a variety of cytokines are induced upon Giardia-host cell interaction, there is no strong intestinal inflammatory response exerted. Nevertheless, a role of T cells in elimination of Giardia infection has been shown by Singer and Nash in mice . A specific T cell proliferative response to Giardia proteins in humans has been reported  and it has been suggested that ADI can inhibit this response . Indeed, we could show that the secreted Giardia protein ADI is capable of reducing the human PBMC proliferative response after T cell specific stimulation (Figure 6) and thereby probably inhibit a strong immune response in vivo. Maximum effects were gained with a concentration of 5 μg/mL GiADI or above. This amount of GiADI is reasonable for mimicking the in vivo situation, since Giardia produces and releases ADI constantly. This finding is also in accordance with the decreased proliferation shown for T cells cultured without L-arginine  that was shown to be due to down-regulation of the CD3zeta chain of the T cell receptor. Furthermore, we were able to completely revert the observed reduction in T cell specific stimulated PBMC proliferation by addition of arginine to physiological levels (Figure 6).
Arginine is part of certain oral rehydration formulations used for treating diarrhea. However, adverse reactions such as osmotic diarrhea and excessive liver urea production [35, 36] are not in favor of such a therapy. In addition, arginine supplementation therapy might also be beneficial for the growth of Giardia itself, since the parasite uses arginine as an energy source. For these reasons we also tested the arginine-metabolite citrulline as an alternative supplementary therapy within this study. Citrulline can be reverted into arginine by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), which were both expressed in the IECs used for this study, but not in Giardia. It is not clear up to now if citrulline can also be reconverted into arginine in vivo by human cells such as IECs, dendritic cells and T cells. However, in children up to 3 years the arginine-reconverting enzymes ASS and ASL are actively expressed in IECs . In addition, ASS and ASL were detected in the canine intestine  and it was shown that citrulline supplementation leads to increased arginine levels also in IECs in adult mice . Thus it is likely, that citrulline conversion into arginine is possible in the intestine of human adults. In accordance to this, we could show that citrulline is capable of reversing all the described arginine-dependent effects on NO-production and T cell proliferation that Giardia is exerting (Figures 3d and Figure 6). Interestingly, the arginine-dependent block of proliferation that was shown to be induced in IECs upon Giardia infection, could also be reverted by citrulline . Therefore citrulline, an abundant component of watermelon, should gain more attention in the future to be used as a supplement in oral rehydration solutions. It could help generating a proper immune response against Giardia and inhibiting pathophysiological effects in the intestinal epithelium that are caused by arginine-consumption of Giardia.