Accumulating experimental evidence suggests that
Giardia infections are also capable of modulating pro-inflammatory responses to other stimuli via several mechanisms. Observations that
Giardia infections can protect against the development of diarrheal disease are consistent with the immunomodulatory capabilities of the parasite. Indeed, acute GI inflammatory responses represent a collection of cellular and humoral effector responses and involve a variety of different cell types and mediators; several of these have been shown to contribute to the development of diarrheal disease. For example, infection with enterohaemorrhagic
E. coli causes chloride hypersecretion, a major driving force for diarrheal disease, via mechanisms that require PMN infiltration [
75]. Research has demonstrated that certain
Giardia infections are capable of attenuating recruitment of pro-inflammatory leukocytes and decreasing nitric oxide (NO) production (as referenced below). In addition, evidence is accumulating that
Giardia infections may modulate other pro-inflammatory events. However, these mechanisms have not been fully characterized. The following sections will describe the immunomodulatory mechanisms of
Giardia and describe how this may result in the attenuation of diarrheal disease during GI co-infection.
4.1. Giardia and the Intestinal Mucus Layer
The entire GI tract is lined with a layer of mucus of varying thickness with a structural backbone comprised of mucin glycoproteins dissolved in luminal water. In the colon, this layer can be further subdivided into two separate layers: a dense, inner mucus layer largely devoid of bacterial populations and an outer, loosely packed outer layer containing various bacterial populations [
76,
77]. In the intestinal tract, the primary mucus constituent is the mucin-2 (MUC2) protein [
76,
77,
78]. Preliminary research in our lab has demonstrated
in vivo Giardia assemblage B GS/M isolate infections in mice damages the small intestinal mucus layer by degrading the MUC2 protein and inducing the hypersecretion of mucus in the small intestine and colon, resultantly leading to mucin depletion from goblet cells; this culminated in a weakened mucus layer and facilitated disease (unpublished data). Furthermore, studies monitoring mucus disruption during
in vivo Giardia GS/M infections have observed an increase in bacterial translocation across the epithelial barrier, but this was not associated with an increase in pro-inflammatory markers at the point of acute infection [
52]. Separate
in vivo studies have demonstrated that pro-inflammatory enteropathogens, such as
H. pylori,
Entamoeba histolytica, and
Trichuris muris, alter the mucus layer and this contributes to the initiation or exacerbation of GI disease [
79,
80,
81,
82]. Similarly, modulation or aberrant assembly of the mucus layer is often associated with intestinal inflammation and increased expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-4, IL-6, CXCL8 IL-13, and TNF-α [
83,
84,
85,
86]. Finally,
in vivo studies using mice devoid of Muc2 have revealed that the mucus layer plays an important role in protection against GI infection from pro-inflammatory enteropathogens, such as
E. histolytica and
T. muris, and deletion of this gene results in exacerbated intestinal inflammatory responses [
87,
88,
89]. Similarly, disruption or aberrant expression of MUC2 has been observed in patients with chronic intestinal inflammatory disorders, such as ulcerative colitis [
90,
91,
92]. Collectively, these results demonstrate that disruption of the intestinal mucus layer is largely associated with GI inflammation. It remains to be determined why disruption of the mucus layer during
Giardia infections fails to elicit pro-inflammatory intestinal responses. Moreover, it remains to be seen how
Giardia co-infections may alter host pro-inflammatory responses and/or alter susceptibility to co-infecting GI pathogens.
4.2. Giardia and Neutrophil Recruitment
The tissue accumulation of polymorphonuclear leukocytes or neutrophils (PMNs) is a hallmark of numerous bacterial, viral, and parasitic GI infections. PMNs are myeloid-derived innate immune cells essential to host defence against a variety of bacterial and fungal pathogens, and they possess various anti-microbial mechanisms, including the ability to phagocytose infectious agents, secrete anti-microbial proteases, and release neutrophil extracellular traps (NETs) (reviewed in [
93]). In the absence of pro-inflammatory stimuli, PMNs are kept in a non-activated state within the bone marrow and circulation. During an acute inflammatory response, increased expression and production of PMN chemoattractants promotes PMN activation and recruitment into tissues, including the GI tract (reviewed in [
94,
95]). Certain PMN chemoattractants are capable of inducing the transepithelial migration of PMNs; this process occurs following PMN contact with the basolateral surface of the intestinal epithelium and results in functional changes to both PMNs and intestinal epithelial cells (IECs) [
96]. Importantly, PMN infiltration can induce pathophysiological responses that result in water and solute loss and, hence, diarrheal disease, and
in vivo and
in vitro experiments have suggested this may involve PMN-mediated intestinal barrier dysfunction and/or anion secretion [
75,
97,
98,
99]. Collectively, these results demonstrate the importance PMNs have in contributing to diarrheal disease.
Recent studies have shown that
Giardia infections may attenuate intestinal PMN recruitment. Notably, these observations have been recorded with assemblage A, the genotype that has been postulated not to induce overt intestinal pro-inflammatory responses (see above). For example,
Giardia assemblage A decreased granulocyte infiltration and cytokines and chemokines involved in PMN recruitment after intra-rectal instillation of pro-inflammatory
Clostridium difficile toxin A/B; these effects were not observed with
in vivo Giardia assemblage B GS/M infections [
100]. This study was also the first to demonstrate that co-incubation of
Giardia trophozoites with inflamed colonic mucosal biopsy tissues from patients with active Crohn’s disease decreased supernatant levels of numerous pro-inflammatory mediators, including those involved in PMN recruitment [
100]. Further studies went on to identify potential immunomodulatory molecules involved in this process. The findings demonstrated that assemblage A
Giardia cathepsin B (catB) cysteine proteases degraded CXCL8 induced by pro-inflammatory interleukin-1β, or by
Salmonella enterica serovar Typhimurium, and attenuated CXCL8-induced PMN chemotaxis; these effects were not observed with assemblage B GS/M trophozoites at early time points and, potentially, occur via different mechanisms [
101]. These studies highlight a hitherto unidentified anti-inflammatory capability for
Giardia infections and, more specifically,
Giardia catB proteases. Another recent study shows that these catB cysteine proteases may also be implicated in the degradation of epithelial villin [
102]. Otherwise, very little is known about the function of
Giardia cathepsin cysteine proteases (
Box 1).
Box 1. Giardia cathepsin cysteine proteases.
The term cathepsin was initially used to describe proteases active in a lightly acidic environment. However, as genome sequencing of different species has progressed, it has become evident that not all cathepsin-like proteases are active at an acidic pH. Cathepsin cysteine proteases consist of a catalytic diad of a cysteine and a histidine residue, whereby the histidine residue donates an electron to the cysteine residue to make it a stronger nucleophile (reviewed in [
103]). Cathepsin cysteine proteases are divided down into two superfamilies: the cathepsin-L(catL)-like and the cathepsin-B-like superfamilies. Cathepsin B-like cysteine proteases contain a unique ~20-amino-acid insertion referred to as the occluding loop; this structure allows the protease to function as an endo- and exopeptidase. The
Giardia genome contains genes for numerous catB and catL proteases [
104]. Interestingly, the Giardia catB protein appears to lack the occluding loop present in human catB [
105]. Prior to an immunomodulatory role for
Giardia catB proteases, very little was known about
Giardia cathepsin cysteine proteases. Indeed, it was demonstrated that these factors were upregulated following exposure to
in vitro intestinal epithelial monolayers and they played a role in parasite encystation and excystation [
106]. Ongoing research has demonstrated that
Giardia cathepsin-like cysteine proteases induce the myosin light chain kinase (MLCK)-mediated breakdown of cytoskeletal villin [
102]. Future studies should continue to elucidate the role of
Giardia cathepsin cysteine proteases in disease and their regulation within the parasite For example,
Toxoplasma gondii catB proteases actually require catalytic activation via the parasite's catL proteases [
107]. It remains to be seen whether similar effects are observed with
Giardia catB proteases.
The construction of preliminary phylogenetic trees using ClustalW [
108] for
Giardia cathepsin B (catB) (
Figure 1) and cathepsin L (catL) (
Figure 2) cysteine proteases of sequenced parasite isolates used in our above study (see Cotton
et al [
101]) suggests certain parasite isolates may contain unique catB proteases; this may explain differences in the ability of parasites to degrade CXCL8. However, it should be noted that sequencing of
Giardia genomes is incomplete and, therefore, construction of these phylogenetic trees requires re-analysis following their completion. These data may lend credence to the hypothesis that certain
Giardia isolates may possess unique immunomodulatory cathepsin cysteine proteases. The utilization of these phylogenetic trees in association with the Cre/loxP system in
Giardia trophozoites [
109] may aid in the identification of immunomodulatory functions of cathepsin cysteine proteases. In addition, it remains to be determined whether
Giardia catB proteases modulate or degrade other cytokines or chemokines, as indicated by the above observations that
G. duodenalis trophozoites reduce tissue concentrations of numerous cytokines and chemokines released from inflamed colonic mucosal biopsy tissues [
100]. Indeed, other parasites use cathepsin-like cysteine proteases to modulate host immune responses via the alteration of cytokines or chemokines (reviewed in [
105,
110]). For example,
Entamoeba histolytica cysteine proteases alter interleukin-18 [
111] and the end-target PMN chemokine C5a [
112]. As a result, future studies could investigate other pro-inflammatory mediators targeted by
Giardia cathepsin cysteine proteases.
Figure 1.
Phylogenetic tree and bootstrap values of Giardia WB and Giardia GS/M cathepsin B cysteine proteases. Proteases were compared against human, bovine, mouse, rat, Schisotoma mansonii, Leishmania major, L. donovani, L. chagasi, Trypanosoma brucei, and T. cruzi catB cysteine proteases. Alignment and phylogenetic trees of cathepsin B cysteine proteases were assembled using ClustalW and CLC Sequence Viewer (Qiagen). These observations indicate that Giardia isolates may contain unique catB cysteine proteases.
Figure 1.
Phylogenetic tree and bootstrap values of Giardia WB and Giardia GS/M cathepsin B cysteine proteases. Proteases were compared against human, bovine, mouse, rat, Schisotoma mansonii, Leishmania major, L. donovani, L. chagasi, Trypanosoma brucei, and T. cruzi catB cysteine proteases. Alignment and phylogenetic trees of cathepsin B cysteine proteases were assembled using ClustalW and CLC Sequence Viewer (Qiagen). These observations indicate that Giardia isolates may contain unique catB cysteine proteases.
Figure 2.
Phylogenetic tree and bootstrap values of Giardia WB and Giardia GS/M cathepsin L cysteine proteases. Proteases were compared against human, bovine, mouse, rat, Schisotoma mansonii, Leishmania major, and L. chagasi catL cysteine proteases. Alignment and phylogenetic trees of cathepsin L cysteine proteases were assembled using ClustalW and CLC Sequence Viewer (Qiagen).
Figure 2.
Phylogenetic tree and bootstrap values of Giardia WB and Giardia GS/M cathepsin L cysteine proteases. Proteases were compared against human, bovine, mouse, rat, Schisotoma mansonii, Leishmania major, and L. chagasi catL cysteine proteases. Alignment and phylogenetic trees of cathepsin L cysteine proteases were assembled using ClustalW and CLC Sequence Viewer (Qiagen).
CXCL8 is primarily secreted basolaterally by IECs to recruit extravasated PMNs to the basolateral membrane of the intestinal epithelium so subsequent signals can, if necessary, promote PMN transepithelial migration [
113,
114,
115]. Therefore, the immunomodulatory capability of
Giardia cathepsin cysteine proteases implies that these must be delivered to the basolateral surface of the intestinal epithelium. In the studies discussed above, apical-to-basolateral translocation of cysteine proteases occurred when
Giardia trophozoites and Caco-2 monolayers were co-incubated with
Salmonella enterica serovar Typhimurium [
101]. These results may also suggest that delivery of immunomodulatory cathepsin cysteine proteases can be facilitated by the presence of
S. Typhimurium. Another study demonstrated that co-incubation of
Giardia trophozoites, intestinal epithelial monolayers, and macrophage-like IC-21 cells
in vitro resulted in basolateral attenuation of CXCL8 [
116]; this study did not investigate causal mechanisms. As macrophages are known to increase intestinal epithelial permeability [
117], more research is needed to assess whether and how the interaction between parasites, IECs, and immune cells may facilitate the apical-to-basolateral migration of immunomodulatory
Giardia catB proteases.
Modulation of Neutrophil Recruitment and Co-Infections
It is well established that individuals with genetic mutations resulting in defective PMN function are highly susceptible to bacterial and fungal infection [
118,
119,
120], and similar events have been observed during certain experimental GI infections. For example,
in vivo depletion of PMNs increases mortality due to
C. difficile infection [
121,
122], while intestinal PMN influx reduces pathogen burdens from the attaching and effacing pathogen
Citrobacter rodentium and protects against pathogen-induced diarrheal disease [
123]. In contrast, other reports indicate that GI inflammatory responses and PMN infiltration may increase susceptibility to GI infection, and it has been postulated that the development of GI inflammatory responses disrupts resident microbiota populations, which in turn aides pathogen colonization [
124]. Research has shown that
S. Typhimurium outcompetes the host’s resident microbiota during intestinal inflammatory responses to facilitate its colonization [
125,
126,
127,
128]. Similarly, attenuated PMN recruitment
in vivo reduces colonization by
Campylobacter jejuni [
129] and
C. rodentium [
130]. In addition, PMN recruitment has been shown to aggravate experimental colitis [
131]; this may occur via the PMN's ability to induce protective responses within IECs via the induction of hypoxia-inducible factor (HIF) [
132] or the secretion of interleukin-22 (IL-22) [
133]. In this context, the above observations that certain
Giardia infections modulate PMN recruitment require further investigation in the context of GI co-infection. Specifically, experiments need to ascertain whether
Giardia-mediated modulation of PMN recruitment into intestinal tissues is of benefit or detriment to a host co-infected with another GI pathogen. This may, ultimately, be dependent upon the co-infecting GI pathogen.
4.4. Intestinal Epithelial Cell Death
Changes in intestinal epithelial cellular proliferation are essential responses to GI infection and facilitate the removal of damaged and/or pathogen-infected cells; however, GI pathogens can alter the kinetics of epithelial cell death and turnover to facilitate their colonization and subsequent invasion (reviewed in [
158,
159,
160,
161]). For example, enteroinvasive
Escherichia coli,
Salmonella sp., and
Shigella sp. initially suppress and, subsequently, induce various forms of intestinal epithelial cell death to facilitate replication and dissemination within their host, respectively; this can be associated with the activation of pro-survival pathways such as the NF-κB pathway [
162,
163,
164,
165,
166].
Giardia infections can inhibit intestinal epithelial proliferation and, subsequently, induce intestinal epithelial apoptosis; however, the pathophysiological processes may differ from those of other GI pathogens discussed above. L-arginine is involved in cellular proliferation via its conversion into polyamines [
167], and
Giardia arginine deiminase-mediated consumption of arginine has been associated with the inhibition of
in vitro IEC proliferation [
137]; this consumption was proposed to reduce intestinal epithelial cell turnover and create a more stable environment for the parasite [
137]. Contrastingly, increases in intestinal epithelial proliferation have been reported in
in vivo G. duodenalis GS/M mouse infections [
168]; therefore, it remains to be determined whether the consumption of arginine by parasites inhibits IEC proliferation. Other reports have demonstrated that
Giardia trophozoites induce IEC apoptosis via the activation of cysteinyl asparate proteases (caspases) through mechanisms that remain incompletely understood [
35,
37,
39]. However, it remains to be determined how these pathophysiologic processes induced by
Giardia potentially modulate host immune responses and their interaction during GI co-infections. It is possible that
Giardia-mediated upregulation of intestinal epithelial cell death may increase the expulsion of co-infecting GI pathogens. It is also currently unknown whether
Giardia trophozoites modulate pro-inflammatory signaling cascades, such as the NF-κB pathway, in IECs to delay the induction of cell death. Caspase proteins inactivate or degrade various proteins associated with the NF-κB signaling cascade [
169,
170,
171]. Research needs to determine whether
Giardia may degrade pro-inflammatory transcription factors, thereby preventing bacterial pathogens from initially inhibiting cell death cascades within IECs to allow for their replication prior to dissemination into deeper host tissues.
4.5. Dendritic Cells
Dendritic cells (DCs) are essential to the induction of adaptive immune responses and/or tolerance. Following their activation, DCs become immunogenic antigen-presenting cells capable of promoting the expansion and differentiation of naïve T-cells into effector T-cells via a three-step process. DCs consume and process antigen, couple it to major histocompatibility complexes (MHC), and, subsequently, present this to naïve T-cell populations; in addition, DCs also use co-stimulatory molecules, such as CD80 and CD86, and produce mediators, such as cytokines, to influence the differentiation of naïve T cells in various subsets (reviewed in [
172,
173]). Within the GI tract, especially in the distal small intestine, DCs directly sample luminal contents via the extension of dendrites between adjacent IECs [
174,
175]. Research to date has produced conflicting results on how
Giardia trophozoites affect DC activation and their ability to induce and/or modulate effector immune responses. The co-incubation of
Giardia assemblage B GS/M trophozoite extracts and murine bone marrow-derived DCs
in vitro resulted in the upregulation of co-stimulatory CD40, and to a lesser extent, CD80 and CD86; moreover, these extracts altered DC responses to toll-like receptor (TLR) ligands, whereby parasites reduced the expression of MHC Class II, CD80, and C86, decreased the secretion of IL-12, and enhanced IL-10 production via activation of the PI3K pathway [
176]. In contrast, separate experiments found that the
Giardia homolog of immunoglobulin protein (BiP) triggered the expression of MHC Class II molecules and concomitantly resulted in the secretion of TNFα, IL-12, and IL-6 via several pro-inflammatory signaling cascades in
in vitro murine dendritic cells [
177]. Experiments using assemblage A
Giardia WB trophozoites demonstrated that the parasite decreases the production of IL-12p40, IL-12p70, and IL-23 by human DC
in vitro and the expression of co-stimulatory molecules and human leukocyte antigen (HLA) DR (HLA-DR) while enhancing the production of anti-inflammatory IL-10; interestingly, DCs incubated with these parasites and concurrently exposed to TLR2 ligands enhanced IL-12p40, IL-23, and IL-10 production [
178]. Separately, arginine depletion induced by the same parasite isolate was shown to reduce the surface expression of CD83 and CD86, decrease the secretion of IL-10 and IL-12p40, and enhance TNFα production in
in vitro human DCs [
179]. In other experiments, the
in vitro co-incubation of bovine DCs and a mixture of
Giardia assemblage A and E trophozoites resulted in elevated MHC Class II molecules, TGF-β, TNFα, IL-10, and IL-4; these DCs are able to induce T-cell proliferation [
180]. Collectively, these results demonstrate that
Giardia trophozoites are capable of modulating DC cell function. Future studies should compare and contrast DC function and activation following exposure to different
Giardia isolates and assemblages.
4.6. Macrophages
Ongoing research has demonstrated these macrophages change their function based on endogenous and exogenous stimuli within the local tissue environment; this also results in the altered expression of several surface markers and leads to their classification into subgroups, commonly known as M1 and M2 macrophages [
181,
182]. M1 macrophages have been labelled as “inflammatory macrophages” that produce various inflammatory mediators and molecules, such as TNFα, whereas the M2 macrophage phenotype is often thought to antagonize host pro-inflammatory responses, including the production of nitric oxide, and can result in the expression of Arginase-1 [
183]. However, current research suggests these cell types do not exist as distinct entities, but rather as a continuum of differing phenotypes [
184]. To date, very little research has examined how
Giardia infections modulate macrophage phenotypes during infection, and only one study has shown that
in vivo Giardia assemblage B infections result in the accumulation of macrophages positive for both NOS2 and Arginase-1 [
185]. Additional research is required in order to investigate how
Giardia infections induce this unique macrophage phenotype, and, moreover, whether these macrophages are observed during human
Giardia infections or
in vivo assemblage A or
Giardia muris infections. Future studies are also required to assess whether a
Giardia-induced switch to macrophage phenotype, if present, may alter susceptibility to GI co-infection. As individuals with mutations in cytokines associated with M1 macrophage polarization are more susceptible to infection by numerous microorganisms [
186,
187],
Giardia-induced changes to macrophage phenotypes may significantly affect susceptibility to a variety of infections. For example, the intracellular replication of
S. Typhimurium is greatly impaired in monocyte-derived macrophages with an M1 phenotype [
188]. Moreover, macrophage Arginase-1 expression has been found to limit helper Th2-mediated immune responses and fibrosis during
in vivo Schistosoma mansonii infection [
189]. Collectively, these studies highlight the need for additional research examining the interaction between
Giardia and host macrophages.